The Relationship between Cell Number, Division Behavior and Developmental Potential of Cleavage Stage Human Embryos: A Time-Lapse Study.

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Xiangyi Kong

Full Text Available Day 3 cleavage embryo transfer is routine in many assisted reproductive technology centers today. Embryos are usually selected according to cell number, cell symmetry and fragmentation for transfer. Many studies have showed the relationship between cell number and embryo developmental potential. However, there is limited understanding of embryo division behavior and their association with embryo cell number and developmental potential. A retrospective and observational study was conducted to investigate how different division behaviors affect cell number and developmental potential of day 3 embryos by time-lapse imaging. Based on cell number at day 3, the embryos (from 104 IVF/intracytoplasmic sperm injection (ICSI treatment cycles, n = 799 were classified as follows: less than 5 cells (10C; n = 42. Division behavior, morphokinetic parameters and blastocyst formation rate were analyzed in 5 groups of day 3 embryos with different cell numbers. In 10C embryos increased compared to 7-8C embryos (45.8%, 33.3% vs. 11.1%, respectively. In ≥5C embryos, FR and DC significantly reduced developmental potential, whereas 10C. In NB embryos, the cell cycle elongation or shortening was the main cause for abnormally low or high cell number, respectively. After excluding embryos with abnormal division behaviors, the developmental potential, implantation rate and live birth rate of day 3 embryos increased with cell number.

Urochordate ascidians possess a single isoform of Aurora kinase that localizes to the midbody via TPX2 in eggs and cleavage stage embryos.

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Celine Hebras

Full Text Available Aurora kinases are key proteins found throughout the eukaryotes that control mitotic progression. Vertebrate Aurora-A and B kinases are thought to have evolved from a single Aurora-kinase isoform closest to that found in present day urochordates. In urochordate ascidians Aurora binds both TPX2 (a vertebrate AURKA partner and INCENP (a vertebrate AURKB partner and localizes to centrosomes and spindle microtubules as well as chromosomes and midbody during both meiosis and mitosis. Ascidian Aurora also displays this localization pattern during mitosis in echinoderms, strengthening the idea that non-vertebrate deuterostomes such as the urochordates and echinoderms possess a single form of Aurora kinase that has properties of vertebrate Aurora-kinase A and B. In the ascidian, TPX2 localizes to the centrosome and the spindle poles also as in vertebrates. However, we were surprised to find that TPX2 also localized strongly to the midbody in ascidian eggs and embryos. We thus examined more closely Aurora localization to the midbody by creating two separate point mutations of ascidian Aurora predicted to perturb binding to TPX2. Both forms of mutated Aurora behaved as predicted: neither localized to spindle poles where TPX2 is enriched. Interestingly, neither form of mutated Aurora localized to the midbody where TPX2 is also enriched, suggesting that ascidian Aurora midbody localization required TPX2 binding in ascidians. Functional analysis revealed that inhibition of Aurora kinase with a pharmacological inhibitor or with a dominant negative kinase dead form of Aurora caused cytokinesis failure and perturbed midbody formation during polar body extrusion. Our data support the view that vertebrate Aurora-A and B kinases evolved from a single non-vertebrate deuterostome ancestor. Moreover, since TPX2 localizes to the midbody in ascidian eggs and cleavage stage embryos it may be worthwhile re-assessing whether Aurora A kinase or TPX2 localize to the midbody

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

DEFF Research Database (Denmark)

Grøndahl, Marie Louise; Christiansen, Sofie Lindgren; Kesmodel, Ulrik Schiøler

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.......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...

Children born after cryopreservation of embryos or oocytes: a systematic review of outcome data

DEFF Research Database (Denmark)

Wennerholm, U-B; Söderström-Anttila, V; Bergh, C

2009-01-01

embryos, blastocysts and oocytes. METHODS: A systematic review was performed. We searched the PubMed, Cochrane and Embase databases from 1984 to September 2008. Inclusion criteria for slow freezing of early cleavage stage embryos were controlled studies reporting perinatal or child outcomes. For slow...... freezing and vitrification of blastocysts and oocytes, and vitrification of early cleavage stage embryos, case reports on perinatal or child outcomes were also included. Three reviewers independently read and evaluated all selected studies. RESULTS: For early cleavage embryos, data from controlled studies...... of blastocysts and for vitrification of early cleavage stage embryos, blastocysts and oocytes, limited neonatal data was reported. We found no long-term child follow-up data for any cryopreservation technique. CONCLUSION: Data concerning infant outcome after slow freezing of embryos was reassuring. Properly...

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

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Wong, Christopher Yee; Mills, James K

2017-03-01

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

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

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Slaby, Sylvain [Univ. Lille Nord de France, EA 4515 – LGCgE – Laboratoire Génie Civil et géo-Environnement, Université de Lille 1, Cité scientifique, SN3, F-59655 Villeneuve d’Ascq (France); Univ. Lille, CNRS, INRA, UMR 8576 – UGSF – Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille (France); Lemière, Sébastien [Univ. Lille Nord de France, EA 4515 – LGCgE – Laboratoire Génie Civil et géo-Environnement, Université de Lille 1, Cité scientifique, SN3, F-59655 Villeneuve d’Ascq (France); Hanotel, Julie; Lescuyer, Arlette [Univ. Lille, CNRS, INRA, UMR 8576 – UGSF – Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille (France); Demuynck, Sylvain [Univ. Lille Nord de France, EA 4515 – LGCgE – Laboratoire Génie Civil et géo-Environnement, Université de Lille 1, Cité scientifique, SN3, F-59655 Villeneuve d’Ascq (France); Bodart, Jean-François [Univ. Lille, CNRS, INRA, UMR 8576 – UGSF – Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille (France); and others

2016-08-15

Highlights: • First embryonic steps were studied. • Fertilization success was impacted by cadmium exposures. • Oocytes were most affected instead of spermatozoa by cadmium exposures. • First embryonic cleavages were slown down or stopped by cadmium exposures. • Lead exposures did not affected fertilization and segmentation. - Abstract: Among the toxicological and ecotoxicological studies, few have investigated the effects on germ cells, gametes or embryos, while an impact at these stages will result in serious damage at a population level. Thus, it appeared essential to characterize consequences of environmental contaminant exposures at these stages. Therefore, we proposed to assess the effects of exposure to cadmium and lead ions, alone or in a binary mixture, on early stages of Xenopus laevis life cycle. Fertilization and cell division during segmentation were the studied endpoints. Cadmium ion exposures decreased in the fertilization rates in a concentration-dependent manner, targeting mainly the oocytes. Exposure to this metal ions induced also delays or blockages in the embryonic development. For lead ion exposure, no such effect was observed. For the exposure to the mixture of the two metal ions, concerning the fertilization success, we observed results similar to those obtained with the highest cadmium ion concentration.

Effects of 60Co gamma radiation on Biomphalaria glabrata (Say, 1818) Embryo, I. Mortality

International Nuclear Information System (INIS)

Okazaki, K.; Kawano, T.

1990-01-01

A study was conducted on the radiosensitivity of Biomphalaria glabrata embryos submitted to doses of 5, 10,15,20 and 25 Gy of 60 Co during the cleavage, blastula, gastrula, young trochophore and trochophore stages. Mortality was the parameter used to evaluated the damage induced by ionizing radiation. Susceptibility decreased with increasing embryo age and with decreased radiation dose. Estimated LD 50 values (15 days) showed that the cleavage stage (4.3 Gy) was approximately four times more radiosensitive than the trochophore stage (17.0 Gy). The survival curves obtained for each embryo stage are discussed on the basis of the multitarget theory. (author) [pt

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

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

2017-08-01

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

Do donor oocyte cycles comply with ASRM/SART embryo transfer guidelines? An analysis of 13,393 donor cycles from the SART registry.

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Acharya, Kelly S; Keyhan, Sanaz; Acharya, Chaitanya R; Yeh, Jason S; Provost, Meredith P; Goldfarb, James M; Muasher, Suheil J

2016-09-01

To analyze donor oocyte cycles in the Society for Assisted Reproductive Technology (SART) registry to determine: 1) how many cycles complied with the 2009 American Society for Reproductive Medicine/SART embryo transfer guidelines; and 2) cycle outcomes according to the number of embryos transferred. For donor oocyte IVF with donor age cycles from 2011 to 2012. Embryos transferred in donor IVF cycles. Percentage of compliant cycles, multiple pregnancy rate. There were 3,157 donor cleavage-stage transfers and 10,236 donor blastocyst transfers. In the cleavage-stage cycles, 88% met compliance criteria. The multiple pregnancy rate (MPR) was significantly higher in the noncompliant cycles. In a subanalysis of compliant cleavage-stage cycles, 91% transferred two embryos and only 9% single embryos. In those patients transferring two embryos, the MPR was significantly higher (33% vs. 1%). In blastocyst transfers, only 28% of the cycles met compliance criteria. The MPR was significantly higher in the noncompliant blastocyst cohort at 53% (compared with 2% in compliant cycles). The majority of donor cleavage-stage transfers are compliant with current guidelines, but the transfer of two embryos results in a significantly higher MPR compared with single-embryo transfer. The majority of donor blastocyst cycles are noncompliant, which appears to be driving an unacceptably high MPR in these cycles. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Effects of Co60 gamma radiation on Biomphalaria glabrata (Say, 1818) Embryo. II. Malformations

International Nuclear Information System (INIS)

Okazaki, K.; Kawano, T.

1990-01-01

The morphogenetic effects of ionizing radiation were investigated in Biomphalaria glabrata embryos irradiated in the cleavage, blastula, gastrula, young trochophore and trochophore stages with 5 to 25 Gy doses of 60 CO gamma radiation. The number of malformed embryos rapidly increased with increasing radiation dose, reaching a maximum between 5th to 8th day after irradiation in all stages analyzed. Susceptibility to malformation induction was higher the younger than the age of the irradiated embryo. However, for the cleavage stage the frequency of malformed embryos was inversely proportional to radiation dose for the same radiation dose. Several types of morphogenetic malformations were obtained, among then cephalic malformations, exogastrula, shell malformations and embryos with everted stomodeum, unspecific malformations being the most frequent. The results show that the types of malformation induced by radiation probably are not radiation-specific and do not depend on the dose applied [pt

Silver nanoparticles induce developmental stage-specific embryonic phenotypes in zebrafish

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

2013-11-01

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

Transfer of human frozen-thawed embryos with further cleavage during culture increases pregnancy rates

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Bharat V Joshi

2010-01-01

Full Text Available Aim: To compare the pregnancy rate following transfer of frozen-thawed embryos with or without overnight culture after thawing. Settings and Design: This is a retrospective analysis of frozen-thawed embryo transfer (FET cycles performed between January 2006 and December 2008. Materials and Methods: Out of 518 thaw cycles, 504 resulted in embryo transfers (ETs. Of the total FET cycles, 415 were performed after an overnight culture of embryos (group A; and in 89 cycles, ET was performed within 2 hours of embryo thawing (group B. Statistical Analysis: The data were statistically analyzed using chi-square test. Results: We observed that with FET, women ≤30 years of age had a significantly higher (P=0.003 pregnancy rate (PR=28.9% as compared to women >30 years of age (17.5%. A significantly higher (P<0.001FNx08 pregnancy rate was also observed in women receiving 3 frozen-thawed embryos (29% as compared to those who received less than 3 embryos (10.7%. The difference in PR between group A (PR=24.3% and group B (PR=20.3% was not statistically significant. However, within group A, ET with cleaved embryos showed significantly ( P≤0.01 higher pregnancy rate compared to the uncleaved embryos, depending on the number of cleaved embryos transferred. Conclusion: No significant difference was noticed between FETs made with transfer of embryos with overnight culture and those without culture. However, within the cultured group, transfer of embryos cleaved during overnight culture gave significantly higher PR than transfers without any cleavage.

Radiation effects on cultured mouse embryos in relation to cell division cycle

International Nuclear Information System (INIS)

Domon, M.

1982-01-01

The authors have worked with mouse embryos in vitro asking first, what are the suitable parameters to define the radiation sensitivity of embryos, and second what is a major factor determining it. The LD 50 was adopted as a parameter of the radiation sensitivity of a population in a mouse embryo system in culture. The fertilized ova were collected into Whitten's medium at various times during the pronuclear and 2-cell stages of development. They were irradiated in chambers with X-rays at doses of 0 to 800 rads. After the embryos were cultured, a set of the lethal fractions for various X-ray doses were obtained. Regarding the radiation sensitivity variation of the embryos, the LD 50 varied from 100 to 200 rads during the pronuclear stage and from 100 to 600 rads during the 2-cell stage. The embryos during the pronuclear stage were most radioresistant at early G 2 phase, followed by an increase in the sensitivity. The embryos during the 2-cell stage were also most radioresistant at early G 2 phase and were more sensitive when they got close to either the first or the second cleavage division. Furthermore, it seems that the factor 6 of the large variation was due to the extremely long G 2 period, 14 hrs for the 2-cell embryos. That is, the pooled 2-cell embryos were in a relative sense well synchronized with G 2 phase. In contrast, the synchrony was poor during the pronuclear stage, which led to less variation of the LD 50 for the pronuclear embryos. It is concluded that during the early cleavage stages of mice, radiosensitivity is mainly governed by the content of cells of various cell cycle ages in the embryo. (Namekawa, K.)

Analysis of embryo morphokinetics, multinucleation and cleavage anomalies using continuous time-lapse monitoring in blastocyst transfer cycles.

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Desai, Nina; Ploskonka, Stephanie; Goodman, Linnea R; Austin, Cynthia; Goldberg, Jeffrey; Falcone, Tommaso

2014-06-20

Time-lapse imaging combined with embryo morphokinetics may offer a non-invasive means for improving embryo selection. Data from clinics worldwide are necessary to compare and ultimately develop embryo classifications models using kinetic data. The primary objective of this study was to determine if there were kinetic differences between embryos with limited potential and those more often associated with in vitro blastocyst formation and/or implantation. We also wanted to compare putative kinetic markers for embryo selection as proposed by other laboratories to what we were observing in our own laboratory setting. Kinetic data and cycle outcomes were retrospectively analyzed in patients age 39 and younger with 7 or more zygotes cultured in the Embryoscope. Timing of specific events from the point of insemination were determined using time-lapse (TL) imaging. The following kinetic markers were assessed: time to syngamy (tPNf), t2, time to two cells (c), 3c (t3), 4c ( t4), 5c (t5), 8c (t8), morula (tMor), start of blastulation (tSB); tBL, blastocyst (tBL); expanded blastocyst (tEBL). Durations of the second (cc2) and third (cc3) cell cycles, the t5-t2 interval as well as time to complete synchronous divisions s1, s2 and s3 were calculated. Incidence and impact on development of nuclear and cleavage anomalies were also assessed. A total of 648 embryos transferred on day 5 were analyzed. The clinical pregnancy and implantation rate were 72% and 50%, respectively. Morphokinetic data showed that tPNf, t2,t4, t8, s1, s2,s3 and cc2 were significantly different in embryos forming blastocysts (ET or frozen) versus those with limited potential either failing to blastulate or else forming poor quality blastocysts ,ultimately discarded. Comparison of embryo kinetics in cycles with all embryos implanting (KID+) versus no implantation (KID-) suggested that markers of embryo competence to implant may be different from ability to form a blastocyst. The incidence of multinucleation

Silver nanoparticles incite size- and dose-dependent developmental phenotypes and nanotoxicity in zebrafish embryos.

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

2013-10-21

Nanomaterials possess distinctive physicochemical properties and promise a wide range of applications, from advanced technology to leading-edge medicine. However, their effects on living organisms remain largely unknown. Here we report that the purified silver nanoparticles (Ag NPs) (97 ± 13 nm) incite specific developmental stage embryonic phenotypes and nanotoxicity in a dose-dependent manner, upon acute exposure of given stage embryos to the NPs (0-24 pM) for only 2 h. The critical concentrations of the NPs that cause 50% of embryos to develop normally for cleavage, early gastrula, early segmentation, late segmentation, and hatching stage zebrafish embryos are 3.5, 4, 6, 6, and 8 pM, respectively, showing that the earlier developmental stage embryos are much more sensitive to the effects of the NPs than the later stage embryos. Interestingly, distinctive phenotypes (head abnormality and no eyes) are observed only in cleavage and early gastrula stage embryos treated with the NPs, showing the stage-specific effects of the NPs. By comparing these Ag NPs with smaller Ag NPs (13.1 ± 2.5 nm), we found that the embryonic phenotypes strikingly depend upon the sizes of Ag NPs and embryonic developmental stages. These notable findings suggest that the Ag NPs are unlike any conventional chemicals or ions. They can potentially enable target-specific study and therapy for early embryonic development in size-, stage-, dose-, and exposure duration-dependent manners.

Increased cleavage and blastocyst rate in ewes treated with bovine somatotropin 5 days before the end of progestin-based estrous synchronization.

Science.gov (United States)

Montero-Pardo, A; Hernández-Cerón, J; Rojas-Maya, S; Valencia, J; Rodríguez-Cortez, A; Gutiérrez, C G

2011-05-01

Treatment with bovine somatotropin (bST) during estrous synchronization increased fertility and prolificacy in sheep. In the present study, a single dose of bST 5 days before the end of progestin treatment improved cleavage and embryo development. Stage of estrous cycle was synchronized in ewes (n=32) with progestin and superovulation was induced by use of FSH. Five days before the end of progestin treatment, ewes were randomly assigned to two groups: bST group (n=16) received a depot injection of 125 mg of bST sc (Lactotropina, Elanco, México) and the control group (n=16) received saline solution. Estrous was detected with rams fitted with an apron every 2 h and estrous sheep were mated every 8 h whilst in estrous. Embryos were recovered on Day 7 post mating, assessed microscopically and fixed in 4% paraformaldehyde. Cell number in blastocysts was counted after Hoechst 33342 staining. Plasma concentrations of IGF-I, insulin and progesterone were determined in eight sheep per group from the day of bST treatment to the day of embryo recovery. Cleavage rate, percentage of transferable embryos (transferable embryos/cleaved) and percentage of embryos reaching the blastocyst stage (blastocyst/cleaved) were compared between groups by logistic regression. IGF-I, insulin and progesterone plasma concentrations were analyzed by ANOVA for repeated measurements and cell number by ANOVA. Cleavage rate was greater (Psheep. Plasma concentrations of IGF-I and insulin were greater (Pprogesterone concentrations (P=0.5). It is concluded that bST injection 5 days before progestin removal increases cleavage rate and the proportion of embryos that reach the blastocyst stage. These effects are associated with an increase in IGF-I and insulin concentrations. Copyright © 2011 Elsevier B.V. All rights reserved.

Synthetic profiles of polypeptides of human oocytes and normal and abnormal preimplantation embryos.

Science.gov (United States)

Capmany, G; Bolton, V N

1999-09-01

There is considerable variation in the rate of development in vitro of individual preimplantation human embryos. The relationship between the rate of development and patterns of polypeptide synthesis in individual embryos was examined using SDS-PAGE and autoradiography. After incubation in [35S]methionine, 19 polypeptide bands were identified that change between fertilization and the morula stage. Although changes in two of the bands occurred in embryos that were developing normally and in ageing oocytes, and are thus independent of fertilization, the changes identified in the remaining 17 bands occurred only after fertilization. In embryos that were developing abnormally, as assessed by delayed cleavage, cleavage arrest or extensive fragmentation, the alteration in polypeptide synthetic profiles increased with increasing abnormality.

Silver Nanoparticles Incite Size and Dose-Dependent Developmental Phenotypes and Nanotoxicity in Zebrafish Embryos

Science.gov (United States)

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

2013-01-01

Nanomaterials possess distinctive physicochemical properties and promise a wide range of applications, from advanced technology to leading-edge medicine. However, their effects on living organisms remain largely unknown. Here we report that the purified silver nanoparticles (Ag NPs, 97 ± 13 nm) incite specific developmental stage embryonic phenotypes and nanotoxicity in a dose-dependent manner, upon acute exposure of given-stage embryos to the NPs (0–24 pM) for only 2 h. The critical concentrations of the NPs that cause 50% of embryos develop normally for cleavage, early-gastrula, early-segmentation, late-segmentation, and hatching stage zebrafish embryos are 3.5, 4, 6, 6, and 8 pM, respectively, showing that the earlier developmental stage embryos are much more sensitive to the effects of the NPs than the later stage. Interestingly, distinctive phenotypes (head abnormality and no eyes) are observed only in cleavage and early-gastrula stage embryos treated with the NPs, showing the stage-specific effects of the NPs. By comparing with our study of the smaller Ag NPs (13.1 ± 2.5 nm), we found that the embryonic phenotypes strikingly depend upon the sizes of Ag NPs and embryonic developmental stages. These notable findings suggest that the Ag NPs are unlike any conventional chemicals or ions. They can potentially enable target specific study and therapy for early embryonic development in size, stage, dose, and exposure-duration dependent manners. PMID:24024906

Vertebrate Embryonic Cleavage Pattern Determination.

Science.gov (United States)

Hasley, Andrew; Chavez, Shawn; Danilchik, Michael; Wühr, Martin; Pelegri, Francisco

2017-01-01

The pattern of the earliest cell divisions in a vertebrate embryo lays the groundwork for later developmental events such as gastrulation, organogenesis, and overall body plan establishment. Understanding these early cleavage patterns and the mechanisms that create them is thus crucial for the study of vertebrate development. This chapter describes the early cleavage stages for species representing ray-finned fish, amphibians, birds, reptiles, mammals, and proto-vertebrate ascidians and summarizes current understanding of the mechanisms that govern these patterns. The nearly universal influence of cell shape on orientation and positioning of spindles and cleavage furrows and the mechanisms that mediate this influence are discussed. We discuss in particular models of aster and spindle centering and orientation in large embryonic blastomeres that rely on asymmetric internal pulling forces generated by the cleavage furrow for the previous cell cycle. Also explored are mechanisms that integrate cell division given the limited supply of cellular building blocks in the egg and several-fold changes of cell size during early development, as well as cytoskeletal specializations specific to early blastomeres including processes leading to blastomere cohesion. Finally, we discuss evolutionary conclusions beginning to emerge from the contemporary analysis of the phylogenetic distributions of cleavage patterns. In sum, this chapter seeks to summarize our current understanding of vertebrate early embryonic cleavage patterns and their control and evolution.

Preimplantation development of embryos in women of advanced maternal age

Directory of Open Access Journals (Sweden)

O. V. Chaplia

2014-04-01

Full Text Available In order to reveal the influence of genetic component on the early embryo development, the retrospective study of morphokinetic characteristics of 717 embryos subjected to preimplantation genetic testing was conducted. Blastomere biopsy for FISH-based preimplantation genetic screening of 7 chromosomes was performed on the third day of culture, while embryo developmental potential and morphological features at the cleavage and blastulation stage were studied regarding maternal age particularly in the group of younger women and patients older than 36. Results of genetic testing revealed that euploid embryos rate gradually decreased with maternal age comprising 39.9% in young women group and 25.3% of specimen belonging to elder patients. At the cleavage stage, morphological characteristics of aneuploid and euploid embryos didn’t differ significantly regardless of the age of patients that could be accounted for the transcriptional silence of embryo genome till the third day of its development. However, in case of prolonged culture chromosomally balanced embryos rarely faced developmental arrest (in 7.9% and formed blastocysts half more frequently compared to aberrant embryos (respectively 75.6 versus 49.8%. Nevertheless, no substantial difference was found between blastocyst formation rate among embryos with similar genetic component regardless of the maternal age. Taking into consideration high rate of chromosomally unbalanced embryos specific to patients of advanced maternal age, the relative proportion of aneuplouid blastocysts was significantly higher in this group of embryos. Thus, without genetic screening there is a possibility of inaccurate selection of embryos for women of advanced reproductive age for transfer procedure even in case of prolonged culture. Consequently, increase of aneuploid embryos frequency associated with permanent preimplantation natural selection effectiveness along with the postimplantation natural selection failure

Live embryo imaging to follow cell cycle and chromosomes stability after nuclear transfer.

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Balbach, Sebastian T; Boiani, Michele

2015-01-01

Nuclear transfer (NT) into mouse oocytes yields a transcriptionally and functionally heterogeneous population of cloned embryos. Most studies of NT embryos consider only embryos at predefined key stages (e.g., morula or blastocyst), that is, after the bulk of reprogramming has taken place. These retrospective approaches are of limited use to elucidate mechanisms of reprogramming and to predict developmental success. Observing cloned embryo development using live embryo cinematography has the potential to reveal otherwise undetectable embryo features. However, light exposure necessary for live cell cinematography is highly toxic to cloned embryos. Here we describe a protocol for combined bright-field and fluorescence live-cell imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This protocol, which can be adapted to observe other reporters such as Oct4-GFP or Nanog-GFP, allowed us to quantitatively analyze cleavage kinetics of cloned embryos.

Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

International Nuclear Information System (INIS)

Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun

2014-01-01

Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further

Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

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Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun, E-mail: yinxj33@msn.com

2014-02-21

Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

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

Science.gov (United States)

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

2015-02-01

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

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

Science.gov (United States)

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

2013-06-15

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

Tripolar chromosome segregation drives the association between maternal genotype at variants spanning PLK4 and aneuploidy in human preimplantation embryos.

Science.gov (United States)

McCoy, Rajiv C; Newnham, Louise J; Ottolini, Christian S; Hoffmann, Eva R; Chatzimeletiou, Katerina; Cornejo, Omar E; Zhan, Qiansheng; Zaninovic, Nikica; Rosenwaks, Zev; Petrov, Dmitri A; Demko, Zachary P; Sigurjonsson, Styrmir; Handyside, Alan H

2018-04-24

Aneuploidy is prevalent in human embryos and is the leading cause of pregnancy loss. Many aneuploidies arise during oogenesis, increasing with maternal age. Superimposed on these meiotic aneuploidies are frequent errors occurring during early mitotic divisions, contributing to widespread chromosomal mosaicism. Here we reanalyzed a published dataset comprising preimplantation genetic testing for aneuploidy in 24,653 blastomere biopsies from day-3 cleavage-stage embryos, as well as 17,051 trophectoderm biopsies from day-5 blastocysts. We focused on complex abnormalities that affected multiple chromosomes simultaneously, seeking insights into their formation. In addition to well-described patterns such as triploidy and haploidy, we identified 4.7% of blastomeres possessing characteristic hypodiploid karyotypes. We inferred this signature to have arisen from tripolar chromosome segregation in normally-fertilized diploid zygotes or their descendant diploid cells. This could occur via segregation on a tripolar mitotic spindle or by rapid sequential bipolar mitoses without an intervening S-phase. Both models are consistent with time-lapse data from an intersecting set of 77 cleavage-stage embryos, which were enriched for the tripolar signature among embryos exhibiting abnormal cleavage. The tripolar signature was strongly associated with common maternal genetic variants spanning the centrosomal regulator PLK4, driving the association we previously reported with overall mitotic errors. Our findings are consistent with the known capacity of PLK4 to induce tripolar mitosis or precocious M-phase upon dysregulation. Together, our data support tripolar chromosome segregation as a key mechanism generating complex aneuploidy in cleavage-stage embryos and implicate maternal genotype at a quantitative trait locus spanning PLK4 as a factor influencing its occurrence.

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

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

2015-10-15

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

Long-distance transportation of primate embryos developing in culture: a preliminary study.

Science.gov (United States)

Nichols, Stephanie; Harvey, Alexandra; Gierbolini, Lynette; Gonzalez-Martinez, Janis; Brenner, Carol; Bavister, Barry

2010-03-01

Non-human primate embryos are invaluable for conducting research relevant to human infertility and stem cells, but their availability is restricted. In this preliminary study, rhesus monkey embryos were produced by IVF at the Caribbean Primate Research Centre and shipped in tubes of gassed culture medium within a battery-powered transport incubator by overnight courier to Wayne State University in Michigan. Upon arrival, the embryos were incubated in fresh culture medium to evaluate further development. In 11 shipments comprising 98 cleavage-stage embryos developing from oocytes that were mature (MII) upon collection, 51 (52%) reached advanced preimplantation stages (morula to hatched blastocyst) during prolonged culture following transportation. However, most embryos produced from oocytes that were immature (MI) at collection arrested and only 5/51 (10%) reached advanced stages of development. This study demonstrates that non-cryopreserved primate embryos can be routinely transported between distant sites without loss of developmental ability. In this way, the processes of production and study of non-cryopreserved primate embryos need not be restricted to the same or nearby laboratories. This will expand the use of these embryos for research and facilitate generation of translationally relevant information. Published by Elsevier Ltd.

The effect of hepatocyte growth factor on mouse oocyte in vitro maturation and subsequent fertilization and embryo development

Directory of Open Access Journals (Sweden)

Mohammad H. Bahadori

2011-05-01

Full Text Available Background: Oocyte invitro maturation is an enormously promising technology for the treatment of infertility, yet its clinical application remains limited owing to poor success rates. Therefore, this study was devised to evaluate the effect of hepatocyte growth factor (HGF on in vitro maturation of immature mouse oocytes and resulting embryos development. Materials and Method: Cumulus – oocyte complex and germinal vesicle were obtained from eighteen 6-8 weeks-old female NMRI mice 46-48 hours after administration of an injection of 5 IU PMSG (Pregnant Mares’ Serum Gonadotrophin. Oocytes were culture in TCM199 (Tissue culture medium-199 supplemented with dosages of 0, 10, 20, 50 and 100 ng/ml of HGF. After 24 hours, metaphase ІІ oocytes were co-incubated with sperms for 4-6 hours in T6 medium. Following isolation of two pronucleus embryos, cleavage of embryos was assessed in the same medium till blastocyst stage. The number of oocytes and embryos was recorded under an invert microscope and the rate of oocyte maturation, fertilization and embryos cleavage until blastocyst stage compared using of student χ2 test. Results: In all compared groups, oocytes growth and embryos development rate in the 20 ng/ml of HGF treatment group was significantly higher (p<0.05 than the control group (p<0.05.Conclusion: 20 ng/ml of HGF improved the nuclear maturation and embryo development up to blastocyst stage during culture condition

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

Science.gov (United States)

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.

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

Science.gov (United States)

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 Pembryo group were significantly higher than those in classⅡ frozen embryo group (both Pembryos of the better quality embryo are higher.

Influence of recipient cytoplasm cell stage on transcription in bovine nucleus transfer embryos

DEFF Research Database (Denmark)

Smith, Steven D.; Soloy, Eva; Kanka, Jiri

1996-01-01

Nucleus transfer for the production of multiple embryos derived from a donor embryo relies upon the reprogramming of the donor nucleus so that it behaves similar to a zygotic nucleus. One indication of nucleus reprogramming is the RNA synthetic activity. In normal bovine embryogenesis, the embryo....... NTE were produced using either a MII phase (nonactivated) cytoplasts at 32 hr of maturation or S-phase (activated) cytoplasts activated with calcium ionophore A23187 and cycloheximide treatment approximately 8 hr prior to fusion with a blastomere from an in-vitro-produced morula stage embryo at 32 hr...... of maturation. Control in-vitro-produced embryos were 3H-uridine-labelled and fixed at the 2-, 4-, early 8-, and late 8-cell stages. NTE were similarly prepared at 1, 3, and 20 hr postfusion and at the 2-, 4-, and 8-cell stages. In the control embryos, RNA synthesis was absent in the 2-, 4-, and early 8-cell...

The Impact of Biopsy on Human Embryo Developmental Potential during Preimplantation Genetic Diagnosis

Directory of Open Access Journals (Sweden)

Danilo Cimadomo

2016-01-01

Full Text Available Preimplantation Genetic Diagnosis and Screening (PGD/PGS for monogenic diseases and/or numerical/structural chromosomal abnormalities is a tool for embryo testing aimed at identifying nonaffected and/or euploid embryos in a cohort produced during an IVF cycle. A critical aspect of this technology is the potential detrimental effect that the biopsy itself can have upon the embryo. Different embryo biopsy strategies have been proposed. Cleavage stage blastomere biopsy still represents the most commonly used method in Europe nowadays, although this approach has been shown to have a negative impact on embryo viability and implantation potential. Polar body biopsy has been proposed as an alternative to embryo biopsy especially for aneuploidy testing. However, to date no sufficiently powered study has clarified the impact of this procedure on embryo reproductive competence. Blastocyst stage biopsy represents nowadays the safest approach not to impact embryo implantation potential. For this reason, as well as for the evidences of a higher consistency of the molecular analysis when performed on trophectoderm cells, blastocyst biopsy implementation is gradually increasing worldwide. The aim of this review is to present the evidences published to date on the impact of the biopsy at different stages of preimplantation development upon human embryos reproductive potential.

Cosmetic micromanipulation of vitrified-warmed cleavage stage embryos does not improve ART outcomes: An ultrastructural study of fragments.

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Safari, Somayyeh; Khalili, Mohammad Ali; Barekati, Zeinab; Halvaei, Iman; Anvari, Morteza; Nottola, Stefania A

2017-09-01

The aim was to study the ultrastructure of cytoplasmic fragments along with the effect of cosmetic micromanipulation (CM) on the morphology and development of vitrified-warmed embryos as well as assisted reproductive technology (ART) outcomes. A total of 96 frozen embryo transfer (FET) cycles were included in this prospective randomized study. They were divided into three groups of CM (n=32), sham (n=32) and control (n=32). In the CM group, the vitrified- warmed embryos were subjected to fragments and coarse granules removal (cosmetic micromanipulation) after laser assisted zona hatching (LAH); sham group subjected only to LAH and no intervention was taken for the control group. Fragmented embryo was evaluated by transmission electron microscopy (TEM). Significant improvement was observed in the morphological parameters, such as fragmentation degrees, evenness of the blastomeres and embryo grade during the subsequent development, after applying cosmetic micromanipulation, when compared to sham or control groups (P=0.00001). However, there were no differences in the clinical outcomes amongst the three studied groups e.g. the rates of clinical, ongoing and multiple pregnancies, implantation, delivery and live birth. In fine structure view, fragments exhibited uniform cytoplasmic texture containing majority of organelles that were observed in normal blastomeres including mitochondria. In conclusion, application of cosmetic micromanipulation in low-grade vitrified-warmed embryos showed significant improvement on embryo morphology parameters; however, did not result in noticeable improvements in clinical outcomes of the patients undergoing ART program. In addition, embryo vitrification had no adverse effects on fine structure of the fragments. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. 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.

Science.gov (United States)

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

Ultrastructural observations of lethal yellow (A/sup y//A/sup y/) mouse embryos

Energy Technology Data Exchange (ETDEWEB)

Calarco, P G; Pedersen, R A

1976-01-01

A/sup y//A/sup y/ embryos were identified by the presence of large excluded blastomeres (Pedersen, 1974) and examined cytologically and ultrastructurally. Cell organelles, inclusions and junctions in the excluded blastomeres were compared with those of non-excluded cells of A/sup y//A/sup y/ embryos and control embryos. Excluded blastomeres always had the fine structural characteristics of earlier developmental stages and may have arrested at the 4- to 8-cell stage or slightly later. Interior cells (inner cell mass) were observed in all mutant blastocysts. Nonexcluded cells of A/sup y//A/sup y/ embryos were normal until degenerative changes appear in the late blastocyst stage. The mode of action of the +/sup A/sup y/ gene was not determined, but evidence from this study and others indicates that the effects of +/sup A/sup y/ gene action occur over a wide range of time in early cleavage and implantation.

Optimal developmental stage for vitrification of parthenogenetically activated porcine embryos

DEFF Research Database (Denmark)

Li, Rong; Li, Juan; Kragh, Peter

2012-01-01

The objective of this experiment was to determine the optimal developmental stage to vitrify in-vitro cultured porcine parthenogenetically activated (PA) embryos. Embryos were vitrified by Cryotop on Day 4, 5 or 6 after oocyte activation (Day 0), and immediately after warming they were either time...

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

Science.gov (United States)

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

2010-02-01

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

The impact of food intake and social habits on embryo quality and the likelihood of blastocyst formation.

Science.gov (United States)

Braga, Daniela Paes Almeida Ferreira; Halpern, Gabriela; Setti, Amanda S; Figueira, Rita Cássia S; Iaconelli, Assumpto; Borges, Edson

2015-07-01

The aim of this study was to evaluate the influence of patients' lifestyle factors and eating habits on embryo development. A total of 2659 embryos recovered from 269 patients undergoing intracytoplasmic sperm injection cycles were included. The frequency of intake of food items and social habits were registered and its influences on embryo development evaluated. The consumption of cereals, vegetables and fruits positively influenced the embryo quality at the cleavage stage. The quality of the embryo at the cleavage stage was also negatively correlated with the consumption of alcoholic drinks and smoking habits. The consumption of fruits influenced the likelihood of blastocyst formation, which was also positively affected by the consumption of fish. Being on a weight-loss diet and consumption of red meat had a negative influence on the likelihood of blastocyst formation. The likelihood of blastocyst formation was also negatively influenced by the consumption of alcoholic drinks and by smoking habits. The consumption of red meat and body mass index had a negative effect on the implantation rate and the likelihood of pregnancy. In addition, being on a weight-loss diet had a negative influence on implantation rate. Our evidence suggests a possible relationship between environmental factors and ovary biology. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

RNA profiles of porcine embryos during genome activation reveal complex metabolic switch sensitive to in vitro conditions

DEFF Research Database (Denmark)

Østrup, Olga; Olbricht, Gayla; Østrup, Esben

2013-01-01

produced in vitro. Overall, our data are in good accordance with previously published, genome-wide profiling data in other species. Moreover, comparison with mouse and human embryos showed striking overlap in functional annotation of transcripts during the EGA, suggesting conserved basic mechanisms...... a handful of reports characterize changing transcriptome profiles and resulting metabolic changes in cleavage stage embryos. The aims of the current study were to investigate RNA profiles of in vivo developed (ivv) and in vitro produced (ivt) porcine embryos before (2-cell stage) and after (late 4-cell...... from oocyte and are imposed either before oocyte aspiration or during in vitro maturation. IVT embryos have altered content of apoptotic factors, cell cycle regulation factors and spindle components, and transcription factors, which all may contribute to reduced developmental competence of embryos...

Time-lapse cinematography-compatible polystyrene-based microwell culture system: a novel tool for tracking the development of individual bovine embryos.

Science.gov (United States)

Sugimura, Satoshi; Akai, Tomonori; Somfai, Tamás; Hirayama, Muneyuki; Aikawa, Yoshio; Ohtake, Masaki; Hattori, Hideshi; Kobayashi, Shuji; Hashiyada, Yutaka; Konishi, Kazuyuki; Imai, Kei

2010-12-01

We have developed a polystyrene-based well-of-the-well (WOW) system using injection molding to track individual embryos throughout culture using time-lapse cinematography (TLC). WOW culture of bovine embryos following in vitro fertilization was compared with conventional droplet culture (control). No differences between control- and WOW-cultured embryos were observed during development to the blastocyst stage. Morphological quality and inner cell mass (ICM) and trophectoderm (TE) cell numbers were not different between control- and WOW-derived blastocysts; however, apoptosis in both the ICM and TE cells was reduced in WOW culture (P < 0.01). Oxygen consumption in WOW-derived blastocysts was closer to physiological level than that of control-derived blastocysts. Moreover, WOW culture improved embryo viability, as indicated by increased pregnancy rates at Days 30 and 60 after embryo transfer (P < 0.05). TLC monitoring was performed to evaluate the cleavage pattern and the duration of the first cell cycle of embryos from oocytes collected by ovum pickup; correlations with success of pregnancy were determined. Logistic regression analysis indicated that the cleavage pattern correlated with success of pregnancy (P < 0.05), but cell cycle length did not. Higher pregnancy rates (66.7%) were observed for animals in which transferred blastocysts had undergone normal cleavage, identified by the presence of two blastomeres of the same size without fragmentation, than among those with abnormal cleavage (33.3%). These results suggest that our microwell culture system is a powerful tool for producing and selecting healthy embryos and for identifying viability biomarkers.

Ontogeny of salinity tolerance and hyper-osmoregulation by embryos of the intertidal crabs Hemigrapsus edwardsii and Hemigrapsus crenulatus (Decapoda, Grapsidae): survival of acute hyposaline exposure.

Science.gov (United States)

Taylor, H H; Seneviratna, Deepani

2005-04-01

The adults of Hemigrapsus edwardsii and Hemigrapsus crenulatus are euryhaline crabs and strong hyper-osmoregulators. Their embryos are carried externally attached to the abdominal pleopods of female crabs, where they are exposed to temporal and spatial changes in salinity associated with their intertidal and estuarine habitats. Although embryos lack the branchial and excretory organs responsible for adult osmoregulation, post-gastrula embryos were highly tolerant of exposure to hypo-osmotic sea water. Detached eggs (embryos+envelopes), of both species, at all developmental stages between gastrulation and hatching, exhibited 80-100% survival for periods up to 96 h in sea water (osmolality, 1050 mmol kg(-1)) and in dilutions to 50%, 10%, and 1%. Cleavage stages were less tolerant of dilution; H. edwardsii, <50% survived 24 h in 10% sea water; H. crenulatus <50% survived 6 h in 10% sea water. Post-gastrulation stages strongly hyper-osmoregulated but cleavage stages were hyper-osmoconformers (maintaining internal osmolality approximately 150 mmol kg(-1) above external). Osmoregulatory capacity was reduced just prior hatching, particularly in H. crenulatus, although salinity tolerance remained high. Gastrulation therefore marks a critical stage in the ontogeny of osmoregulation and salinity tolerance. Total Na+/K(+)-ATPase activity increased greatly during embryogenesis of H. crenulatus (undetectable in blastulae; gastrulae 0.31+/-0.05 pmol P(i) embryo(-1) min(-1); pre-hatching 16.4+/-1.0 pmol P(i) embryo(-1) min(-1)). Na+/K(+)-ATPase activity increased in embryos exposed to dilute sea water for 24 h implicating regulation of this transporter in a short-term acclimation response.

Pertussis toxin-catalyzed ADP-ribosylation of a G protein in mouse oocytes, eggs, and preimplantation embryos: Developmental changes and possible functional roles

Energy Technology Data Exchange (ETDEWEB)

Jones, J.; Schultz, R.M. (Univ. of Pennsylvania, Philadelphia (USA))

1990-06-01

G proteins, which in many somatic cells serve as mediators of signal transduction, were identified in preimplantation mouse embryos by their capacity to undergo pertussis toxin-catalyzed ADP-ribosylation. Two pertussis toxin (PT) substrates with Mr = 38,000 and 39,000 (alpha 38 and alpha 39) are present in approximately equal amounts. Relative to the amount in freshly isolated germinal vesicle (GV)-intact oocytes, the amount of PT-catalyzed ADP-ribosylation of alpha 38-39 falls during oocyte maturation, rises between the one- and two-cell stages, falls by the eight-cell and morula stages, and increases again by the blastocyst stage. The decrease in PT-catalyzed ADP-ribosylation of alpha 38-39 that occurs during oocyte maturation, however, does not require germinal vesicle breakdown (GVBD), since inhibiting GVBD with 3-isobutyl-1-methyl xanthine (IBMX) does not prevent the decrease in the extent of PT-catalyzed ADP-ribosylation. A biologically active phorbol diester (12-O-tetradecanoyl phorbol 13-acetate), but not an inactive one (4 alpha-phorbol 12,13-didecanoate, 4 alpha-PDD), totally inhibits the increase in PT-catalyzed ADP-ribosylation of alpha 38-39 that occurs between the one- and two-cell stage; TPA inhibits cleavage, but not transcriptional activation, which occurs in the two-cell embryo. In contrast, cytochalasin D, genistein, or aphidicolin, each of which inhibits cleavage of one-cell embryos, or alpha-amanitin or H8, each of which inhibits transcriptional activation but not cleavage of one-cell embryos, have little or inhibitory effects on the increase in PT-catalyzed ADP-ribosylation of alpha 38-39. Results of immunoblotting experiments using an antibody that is highly specific for alpha il-3 reveal the presence of a cross-reactive species of Mr = 38,000 (alpha 38) in the GV-intact oocyte, metaphase II-arrested egg, and one-, two-cell embryos.

Effect of oxygen concentration on human embryo development evaluated by time-lapse monitoring

DEFF Research Database (Denmark)

Ingerslev, Hans Jakob; Hindkjær, Johnny Juhl; Kirkegaard, Kirstine

2012-01-01

recently demonstrated to occur from first cleavage cycle in mice using time-lapse microscopy, with the largest impact on the pre-compaction stages. However, embryonic development in mice differs in many aspects from human embryonic development. The objective of this retrospective, descriptive study...... was to evaluate the influence of oxygen tension on human pre-implantation development using time-lapse monitoring. Materials and methods: Human embryos were cultured to the blastocyst stage in a time-lapse incubator (EmbryoScope™) in 20% O2 (group 1), 20% O2 for 24 hours followed by culture in 5% O2 (group 2......) or in 5% O2 (group 3). Eligible were patients with age 8 oocytes retrieved. Group 1 consisted of 120 IVF/ICSI embryos from 26 patients recruited to a study conducted to evaluate the safety of the time-lapse incubator by randomising 1:1 embryos from a patient to culture...

Routine use of next-generation sequencing for preimplantation genetic diagnosis of blastomeres obtained from embryos on day 3 in fresh in vitro fertilization cycles.

Science.gov (United States)

Łukaszuk, Krzysztof; Pukszta, Sebastian; Wells, Dagan; Cybulska, Celina; Liss, Joanna; Płóciennik, Łukasz; Kuczyński, Waldemar; Zabielska, Judyta

2015-04-01

To determine the usefulness of semiconductor-based next-generation sequencing (NGS) for cleavage-stage preimplantation genetic diagnosis (PGD) of aneuploidy. Prospective case-control study. A private center for reproductive medicine. A total of 45 patients underwent day-3 embryo biopsy with PGD and fresh cycle transfer. Additionally, 53 patients, matched according to age, anti-Müllerian hormone levels, antral follicles count, and infertility duration were selected as controls. Choice of embryos for transfer was based on the PGD NGS results. Clinical pregnancy rate (PR) per embryo transfer (ET) was the primary outcome. Secondary outcomes were implantation and miscarriage rates. The PR per transfer was higher in the NGS group (84.4% vs. 41.5%). The implantation rate (61.5% vs. 34.8%) was higher in the NGS group. The miscarriage rate was similar in the 2 groups (2.8% vs. 4.6%). We demonstrate the technical feasibility of NGS-based PGD involving cleavage-stage biopsy and fresh ETs. Encouraging data were obtained from a prospective trial using this approach, arguing that cleavage-stage NGS may represent a valuable addition to current aneuploidy screening methods. These findings require further validation in a well-designed randomized controlled trial. ACTRN12614001035617. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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.

Utilization of endogenous fatty acid stores for energy production in bovine preimplantation embryos.

Science.gov (United States)

Sutton-McDowall, Melanie L; Feil, Deanne; Robker, Rebecca L; Thompson, Jeremy G; Dunning, Kylie R

2012-05-01

Although current embryo culture media are based on carbohydrate metabolism of embryos, little is known about metabolism of endogenous lipids. L-carnitine is a β-oxidation cofactor absent in most culture media. The objective was to investigate the influence of L-carnitine supplementation on bovine embryo development. Abattoir-derived bovine cumulus oocyte complexes were cultured and fertilized. Post-fertilization, presumptive zygotes were transferred into a basic cleavage medium ± carbohydrates (glucose, lactate and pyruvate) ± 5 mm L-carnitine and cultured for 4 days in vitro. In the absence of carbohydrates during culture, embryos arrested at the 2- and 4-cell stages. Remarkably, +L-carnitine increased development to the morula stage compared to +carbohydrates alone (P levels were higher and ATP: ADP ratio were 1.9-fold lower (main effect, P < 0.05) compared to embryos cultured in -L-carnitine. Therefore, we inferred that +L-carnitine embryos were more metabolically active, with higher rates of ATP-ADP conversion. In conclusion, L-carnitine supplementation supported precompaction embryo development and there was an additive effect of +L-carnitine +carbohydrates on early embryo development, most likely through increased β-oxidation within embryos. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

Effect of oxygen concentration on human embryo development evaluated by time-lapse monitoring

DEFF Research Database (Denmark)

Ingerslev, Hans Jakob; Hindkjær, Johnny Juhl; Kirkegaard, Kirstine

2012-01-01

-points for each cell division and blastocyst stages were registered until 120 hours after oocyte retrieval. Only 2PN embryos completing the first cleavage were evaluated. The groups were compared using one-way ANOVA or Kruskall-Wallis test. Estimates are reported as medians with 95% confidence intervals. Time......Introduction: Data from a number of studies indicate -but not unequivocally- that culture of embryos in 5% O2 compared to 20% O2 improves blastocyst formation in humans and various animal species and may yield better pregnancy rates in IVF. The detrimental effects of atmospheric oxygen were...... was to evaluate the influence of oxygen tension on human pre-implantation development using time-lapse monitoring. Materials and methods: Human embryos were cultured to the blastocyst stage in a time-lapse incubator (EmbryoScope™) in 20% O2 (group 1), 20% O2 for 24 hours followed by culture in 5% O2 (group 2...

Chromosome segregation analysis in human embryos obtained from couples involving male carriers of reciprocal or Robertsonian translocation.

Directory of Open Access Journals (Sweden)

Ahmet Yilmaz

Full Text Available The objective of this study was to investigate the frequency and type of chromosome segregation patterns in cleavage stage embryos obtained from male carriers of Robertsonian (ROB and reciprocal (REC translocations undergoing preimplantation genetic diagnosis (PGD at our reproductive center. We used FISH to analyze chromosome segregation in 308 day 3 cleavage stage embryos obtained from 26 patients. The percentage of embryos consistent with normal or balanced segregation (55.1% vs. 27.1% and clinical pregnancy (62.5% vs. 19.2% rates were higher in ROB than the REC translocation carriers. Involvement of non-acrocentric chromosome(s or terminal breakpoint(s in reciprocal translocations was associated with an increase in the percent of embryos consistent with adjacent 1 but with a decrease in 3∶1 segregation. Similar results were obtained in the analysis of nontransferred embryos donated for research. 3∶1 segregation was the most frequent segregation type in both day 3 (31% and spare (35% embryos obtained from carriers of t(11;22(q23;q11, the only non-random REC with the same breakpoint reported in a large number of unrelated families mainly identified by the birth of a child with derivative chromosome 22. These results suggest that chromosome segregation patterns in day 3 and nontransferred embryos obtained from male translocation carriers vary with the type of translocation and involvement of acrocentric chromosome(s or terminal breakpoint(s. These results should be helpful in estimating reproductive success in translocation carriers undergoing PGD.

Ultrastructural and autoradiographic studies of nucleolar development and rDNA transcription in preimplantation mouse embryos

Energy Technology Data Exchange (ETDEWEB)

Geuskens, M.; Alexandre, H. (Universite Libre de Bruxelles (Belgium). Dep. de Biologie Moleculaire)

1984-06-01

The development of the nucleoli and the sites of rDNA transcription have been studies by high-resolution autoradiography during the cleavage stages of mouse embryos. The appearance of fibrillar centres at the periphery of the fibrillar primary nucleoli has been observed at the 4-cell stage. Several fibrillar centres interconnected by electron-dense fibrillar strands, form a reticulated region around the fibrillar mass at the 6- to 8-cell stage. After a 10 min pulse with (/sup 3/H)uridine, only this peripheral network is labelled. At the late morula and at the blastocyst stage, the fibrillar component (nucleolonema) of the reticulated nucleoli is labelled after 10 min (/sup 3/H)uridine incorporation. When the embryos are reincubated for 2 h in cold medium, the label is localized mainly in the granular component. Fibrillar centres are not labelled. Autoradiograms of in vitro developed embryos pulsed for 2 h with (/sup 3/H)uridine confirm that the central fibrillar core of the nucleoli of 6- to 8-cell embryos is never labelled. Thus, the fibrillar constituent of this core is not homologous to the fibrillar component of the nucleoli of later stage embryos, which is the site of active rDNA transcription. An interpretation of nucleologenesis during early mouse embryogenesis is proposed.

Ultrastructural and autoradiographic studies of nucleolar development and rDNA transcription in preimplantation mouse embryos

International Nuclear Information System (INIS)

Geuskens, M.; Alexandre, H.

1984-01-01

The development of the nucleoli and the sites of rDNA transcription have been studies by high-resolution autoradiography during the cleavage stages of mouse embryos. The appearance of fibrillar centres at the periphery of the fibrillar primary nucleoli has been observed at the 4-cell stage. Several fibrillar centres interconnected by electron-dense fibrillar strands, form a reticulated region around the fibrillar mass at the 6- to 8-cell stage. After a 10 min pulse with ( 3 H)uridine, only this peripheral network is labelled. At the late morula and at the blastocyst stage, the fibrillar component (nucleolonema) of the reticulated nucleoli is labelled after 10 min ( 3 H)uridine incorporation. When the embryos are reincubated for 2 h in cold medium, the label is localized mainly in the granular component. Fibrillar centres are not labelled. Autoradiograms of in vitro developed embryos pulsed for 2 h with ( 3 H)uridine confirm that the central fibrillar core of the nucleoli of 6- to 8-cell embryos is never labelled. Thus, the fibrillar constituent of this core is not homologous to the fibrillar component of the nucleoli of later stage embryos, which is the site of active rDNA transcription. An interpretation of nucleologenesis during early mouse embryogenesis is proposed. (author)

Syntheses of nucleic acid and protein in somatic embryos of Fritillaria ussuriensis maxim in different development stages

International Nuclear Information System (INIS)

Wang Shuyu; Tang Wei; Wang Hui

1993-09-01

After developing a procedure for somatic embryogenesis in Fritillaria ussuriensis, dynamics on the syntheses of DNA, RNA, and protein during globular, heart-shaped, torpedo-shaped, cotyledonary, and mature somatic embryo stages was demonstrated by both autoradiography and scintillation counting. The rates of syntheses of DNA, RNA, and protein gradually increase between the globular and cotyledonary somatic embryos stages. DNA, RNA, and protein synthesis rates are in peak at the cotyledonary later stage, precotyledonary stage, and cotyledonary stage, respectively. It appears that more DNA, RNA, and protein are synthesized in the cotyledonary somatic embryo stage than in other stages. All these results indicate that an increased syntheses of DNA, RNA, and protein is associated with the differentiation of embryogenic cells and organogenesis in somatic embryos

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

Science.gov (United States)

Sakatani, Miki

2017-08-19

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

Comparative and phylogenetic perspectives of the cleavage process in tailed amphibians.

Science.gov (United States)

Desnitskiy, Alexey G; Litvinchuk, Spartak N

2015-10-01

The order Caudata includes about 660 species and displays a variety of important developmental traits such as cleavage pattern and egg size. However, the cleavage process of tailed amphibians has never been analyzed within a phylogenetic framework. We use published data on the embryos of 36 species concerning the character of the third cleavage furrow (latitudinal, longitudinal or variable) and the magnitude of synchronous cleavage period (up to 3-4 synchronous cell divisions in the animal hemisphere or a considerably longer series of synchronous divisions followed by midblastula transition). Several species from basal caudate families Cryptobranchidae (Andrias davidianus and Cryptobranchus alleganiensis) and Hynobiidae (Onychodactylus japonicus) as well as several representatives from derived families Plethodontidae (Desmognathus fuscus and Ensatina eschscholtzii) and Proteidae (Necturus maculosus) are characterized by longitudinal furrows of the third cleavage and the loss of synchrony as early as the 8-cell stage. By contrast, many representatives of derived families Ambystomatidae and Salamandridae have latitudinal furrows of the third cleavage and extensive period of synchronous divisions. Our analysis of these ontogenetic characters mapped onto a phylogenetic tree shows that the cleavage pattern of large, yolky eggs with short series of synchronous divisions is an ancestral trait for the tailed amphibians, while the data on the orientation of third cleavage furrows seem to be ambiguous with respect to phylogeny. Nevertheless, the midblastula transition, which is characteristic of the model species Ambystoma mexicanum (Caudata) and Xenopus laevis (Anura), might have evolved convergently in these two amphibian orders.

Cryotop vitrification for in vitro produced bovine and buffalo (Bubalus bubalis embryos at different stages of development

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B. Gasparrini

2010-02-01

Full Text Available The aim of this study was to evaluate the possibility to vitrify in vitro produced (IVP buffalo and bovine embryos at different stages of development by an advanced version of the “minimal volume approaches”: the Cryotop method. In both experiments, the embryos were vitrified at the tight morula (TM, early blastocyst (eBl, blastocyst (Bl, expanded blastocyst (xBl and, only for buffalo, at the hatched blastocyst (hBl stage. After warming, the embryos were cultured in vitro for 24 hours. Stage of development affected the freezability of IVP embryos of both species with the highest embryo survival rates at advanced stages (xBl=76% and hBl=75% for buffalos and xBl=75% for bovine. These results suggest that Cryotop vitrification is an efficient method for buffalo and bovine IVP embryo cryopreservation.

Algorithms for automatic segmentation of bovine embryos produced in vitro

International Nuclear Information System (INIS)

Melo, D H; Oliveira, D L; Nascimento, M Z; Neves, L A; Annes, K

2014-01-01

In vitro production has been employed in bovine embryos and quantification of lipids is fundamental to understand the metabolism of these embryos. This paper presents a unsupervised segmentation method for histological images of bovine embryos. In this method, the anisotropic filter was used in the differents RGB components. After pre-processing step, the thresholding technique based on maximum entropy was applied to separate lipid droplets in the histological slides in different stages: early cleavage, morula and blastocyst. In the postprocessing step, false positives are removed using the connected components technique that identify regions with excess of dye near pellucid zone. The proposed segmentation method was applied in 30 histological images of bovine embryos. Experiments were performed with the images and statistical measures of sensitivity, specificity and accuracy were calculated based on reference images (gold standard). The value of accuracy of the proposed method was 96% with standard deviation of 3%

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

Science.gov (United States)

Cloney, Kellie; Franz-Odendaal, Tamara Anne

2015-01-24

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

Polypeptide profiles of human oocytes and preimplantation embryos.

Science.gov (United States)

Capmany, G; Bolton, V N

1993-11-01

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

Pentachlorophenol exposure causes Warburg-like effects in zebrafish embryos at gastrulation stage

International Nuclear Information System (INIS)

Xu, Ting; Zhao, Jing; Hu, Ping; Dong, Zhangji; Li, Jingyun; Zhang, Hongchang; Yin, Daqiang; Zhao, Qingshun

2014-01-01

Pentachlorophenol (PCP) is a prevalent pollutant in the environment and has been demonstrated to be a serious toxicant to humans and animals. However, little is known regarding the molecular mechanism underlying its toxic effects on vertebrate early development. To explore the impacts and underlying mechanisms of PCP on early development, zebrafish (Danio rerio) embryos were exposed to PCP at concentrations of 0, 20 and 50 μg/L, and microscopic observation and cDNA microarray analysis were subsequently conducted at gastrulation stage. The morphological observations revealed that PCP caused a developmental delay of zebrafish embryos in a concentration-dependent manner. Transcriptomic data showed that 50 μg/L PCP treatment resulted in significant changes in gene expression level, and the genes involved in energy metabolism and cell behavior were identified based on gene functional enrichment analysis. The energy production of embryos was influenced by PCP via the activation of glycolysis along with the inhibition of oxidative phosphorylation (OXPHOS). The results suggested that PCP acts as an inhibitor of OXPHOS at 8 hpf (hours postfertilization). Consistent with the activated glycolysis, the cell cycle activity of PCP-treated embryos was higher than the controls. These characteristics are similar to the Warburg effect, which occurs in human tumors. The microinjection of exogenous ATP confirmed that an additional energy supply could rescue PCP-treated embryos from the developmental delay due to the energy deficit. Taken together, our results demonstrated that PCP causes a Warburg-like effect on zebrafish embryos during gastrulation, and the affected embryos had the phenotype of developmental delay. - Highlights: • We treat zebrafish embryos with PCP at gastrula stage. • PCP acts as an oxidative phosphorylation inhibitor, not an uncoupler, in gastrulation. • Exogenous ATP injection will rescue the development of effected embryos. • The transcriptome of PCP

Pentachlorophenol exposure causes Warburg-like effects in zebrafish embryos at gastrulation stage

Energy Technology Data Exchange (ETDEWEB)

Xu, Ting; Zhao, Jing [Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Technology, Tongji University, Shanghai 200092 (China); Hu, Ping [Key Laboratory of Model Animal for Disease Study, Ministry of Education, Model Animal Research Center, Nanjing University, Nanjing 210061 (China); State Key Laboratory of Reproductive Medicine, Department of Prenatal Diagnosis, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210029 (China); Dong, Zhangji; Li, Jingyun [Key Laboratory of Model Animal for Disease Study, Ministry of Education, Model Animal Research Center, Nanjing University, Nanjing 210061 (China); Zhang, Hongchang [Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Technology, Tongji University, Shanghai 200092 (China); Yin, Daqiang, E-mail: yindq@tongji.edu.cn [Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Technology, Tongji University, Shanghai 200092 (China); Zhao, Qingshun, E-mail: qingshun@nju.edu.cn [Key Laboratory of Model Animal for Disease Study, Ministry of Education, Model Animal Research Center, Nanjing University, Nanjing 210061 (China)

2014-06-01

Pentachlorophenol (PCP) is a prevalent pollutant in the environment and has been demonstrated to be a serious toxicant to humans and animals. However, little is known regarding the molecular mechanism underlying its toxic effects on vertebrate early development. To explore the impacts and underlying mechanisms of PCP on early development, zebrafish (Danio rerio) embryos were exposed to PCP at concentrations of 0, 20 and 50 μg/L, and microscopic observation and cDNA microarray analysis were subsequently conducted at gastrulation stage. The morphological observations revealed that PCP caused a developmental delay of zebrafish embryos in a concentration-dependent manner. Transcriptomic data showed that 50 μg/L PCP treatment resulted in significant changes in gene expression level, and the genes involved in energy metabolism and cell behavior were identified based on gene functional enrichment analysis. The energy production of embryos was influenced by PCP via the activation of glycolysis along with the inhibition of oxidative phosphorylation (OXPHOS). The results suggested that PCP acts as an inhibitor of OXPHOS at 8 hpf (hours postfertilization). Consistent with the activated glycolysis, the cell cycle activity of PCP-treated embryos was higher than the controls. These characteristics are similar to the Warburg effect, which occurs in human tumors. The microinjection of exogenous ATP confirmed that an additional energy supply could rescue PCP-treated embryos from the developmental delay due to the energy deficit. Taken together, our results demonstrated that PCP causes a Warburg-like effect on zebrafish embryos during gastrulation, and the affected embryos had the phenotype of developmental delay. - Highlights: • We treat zebrafish embryos with PCP at gastrula stage. • PCP acts as an oxidative phosphorylation inhibitor, not an uncoupler, in gastrulation. • Exogenous ATP injection will rescue the development of effected embryos. • The transcriptome of PCP

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

Science.gov (United States)

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

Does dietary fat intake influence oocyte competence and embryo quality by inducing oxidative stress in follicular fluid?

Science.gov (United States)

Kazemi, Ashraf; Ramezanzadeh, Fatemeh; Nasr-Esfahani, Mohammad Hosein; Saboor Yaraghi, Ali Akbar; Ahmadi, Mehdi

2013-12-01

Fat-rich diet may alter oocyte development and maturation and embryonic development by inducing oxidative stress (OS) in follicular environment. To investigate the relationship between fat intake and oxidative stress with oocyte competence and embryo quality. In observational study follicular fluid was collected from 236 women undergoing assisted reproduction program. Malon-di-aldehyde (MDA) levels and total antioxidant capacity (TAC) levels of follicular fluid were assessed as oxidative stress biomarkers. In assisted reproduction treatment cycle fat consumption and its component were assessed. A percentage of metaphase ΙΙ stage oocytes, fertilization rate were considered as markers of oocyte competence and non-fragmented embryo rate, mean of blastomer and good cleavage (embryos with more than 5 cells on 3 days post insemination) rate were considered as markers of embryo quality. The MDA level in follicular fluid was positively related to polyunsaturated fatty acids intake level (p=0.02) and negatively associated with good cleavage rate (p=0.045). Also good cleavage rate (p=0.005) and mean of blastomer (p=0.006) was negatively associated with polyunsaturated fatty acids intake levels. The percentage of metaphase ΙΙ stage oocyte was positively related to the TAC levels in follicular fluid (p=0.046). The relationship between the OS biomarkers in FF and the fertilization rate was not significant. These findings revealed that fat rich diet may induce the OS in oocyte environment and negatively influence embryonic development. This effect can partially be accounted by polyunsaturated fatty acids uptake while oocyte maturation is related to TAC and oocytes with low total antioxidant capacity have lower chance for fertilization and further development.

Does dietary fat intake influence oocyte competence and embryo quality by inducing oxidative stress in follicular fluid?

Science.gov (United States)

Kazemi, Ashraf; Ramezanzadeh, Fatemeh; Nasr-Esfahani, Mohammad Hosein; Saboor Yaraghi, Ali Akbar; Ahmadi, Mehdi

2013-01-01

Background: Fat-rich diet may alter oocyte development and maturation and embryonic development by inducing oxidative stress (OS) in follicular environment. Objective: To investigate the relationship between fat intake and oxidative stress with oocyte competence and embryo quality. Materials and Methods: In observational study follicular fluid was collected from 236 women undergoing assisted reproduction program. Malon-di-aldehyde (MDA) levels and total antioxidant capacity (TAC) levels of follicular fluid were assessed as oxidative stress biomarkers. In assisted reproduction treatment cycle fat consumption and its component were assessed. A percentage of metaphase ΙΙ stage oocytes, fertilization rate were considered as markers of oocyte competence and non-fragmented embryo rate, mean of blastomer and good cleavage (embryos with more than 5 cells on 3 days post insemination) rate were considered as markers of embryo quality. Results: The MDA level in follicular fluid was positively related to polyunsaturated fatty acids intake level (p=0.02) and negatively associated with good cleavage rate (p=0.045). Also good cleavage rate (p=0.005) and mean of blastomer (p=0.006) was negatively associated with polyunsaturated fatty acids intake levels. The percentage of metaphase ΙΙ stage oocyte was positively related to the TAC levels in follicular fluid (p=0.046). The relationship between the OS biomarkers in FF and the fertilization rate was not significant. Conclusion: These findings revealed that fat rich diet may induce the OS in oocyte environment and negatively influence embryonic development. This effect can partially be accounted by polyunsaturated fatty acids uptake while oocyte maturation is related to TAC and oocytes with low total antioxidant capacity have lower chance for fertilization and further development. PMID:24639727

Pro-apoptotic Effect of Pifithrin-α on Preimplantation Porcine Fertilized Embryo Development

Directory of Open Access Journals (Sweden)

Brendan Mulligan

2012-12-01

Full Text Available The aim of this study was to investigate the impact of a reported p53 inhibitor, pifithrin-α (PFT-α, on preimplantation porcine in vitro fertilized (IVF embryo development in culture. Treatment of PFT-α was administered at both early (0 to 48 hpi, and later stages (48 to 168 hpi of preimplantation development, and its impact upon the expression of five genes related to apoptosis (p53, bak, bcl-xL, p66Shc and caspase3, was assessed in resulting d 7 blastocysts, using real-time quantitative PCR. Total cell numbers, along with the number of apoptotic nuclei, as detected by the in situ cell death detection assay, were also calculated on d 7 in treated and non-treated control embryos. The results indicate that PFT-α, when administered at both early and later stages of porcine IVF embryo development, increases the incidence of apoptosis in resulting blastocysts. When administered at early cleavage stages, PFT-α treatment was shown to reduce the developmental competence of porcine IVF embryos, as well as reducing the quality of resulting blastocysts in terms of overall cell numbers. In contrast, at later stages, PFT-α administration resulted in marginally increased blastocyst development rates amongst treated embryos, but did not affect cell numbers. However, PFT-α treatment induced apoptosis and apoptotic related gene expression, in all treated embryos, irrespective of the timing of treatment. Our results indicate that PFT-α may severely compromise the developmental potential of porcine IVF embryos, and is a potent apoptotic agent when placed into porcine embryo culture media. Thus, caution should be exercised when using PFT-α as a specific inhibitor of p53 mediated apoptosis, in the context of porcine IVF embryo culture systems.

Fossil embryos from the Middle and Late Cambrian period of Hunan, south China.

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Dong, Xi-Ping; Donoghue, Philip C J; Cheng, Hong; Liu, Jian-Bo

2004-01-15

Comparative embryology is integral to uncovering the pattern and process of metazoan phylogeny, but it relies on the assumption that life histories of living taxa are representative of their antecedents. Fossil embryos provide a crucial test of this assumption and, potentially, insight into the evolution of development, but because discoveries so far lack phylogenetic constraint, their significance is moot. Here we describe a collection of embryos from the Middle and Late Cambrian period (500 million years ago) of Hunan, south China, that preserves stages of development from cleavage to the pre-hatching embryo of a direct-developing animal comparable to living Scalidophora (phyla Priapulida, Kinorhyncha, Loricifera). The latest-stage embryos show affinity to the Lower Cambrian embryo Markuelia, whose life-history strategy contrasts both with the primitive condition inferred for metazoan phyla and with many proposed hypotheses of affinity, all of which prescribe indirect development. Phylogenetic tests based on these embryological data suggest a stem Scalidophora affinity. These discoveries corroborate, rather than contradict, the predictions of comparative embryology, providing direct historical support for the view that the life-history strategies of living taxa are representative of their stem lineages.

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

Science.gov (United States)

Eto, Tomoo; Takahashi, Riichi; Kamisako, Tsutomu

2015-04-01

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

Time-lapse cinematography of dynamic changes occurring during in vitro development of human embryos.

Science.gov (United States)

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.

Change in radiosensitivity on the development of sea urchin eggs during the early cleavage stage, 2

International Nuclear Information System (INIS)

Nakamura, Izumi

1975-01-01

The effect of cysteamine on the fluctuation of X-ray sensitivity during early cleavage stage of sea urchin eggs expressed by pluteus formation rate was examined. Sea urchin eggs were very resistant to radiation immediately after insemination or after S phase of the cleavage. When irradiation was given just prior to S phase or in the phase of cytokinesis, the eggs were very sensitive. In these sensitive stages, existence of cysteamine during X-irradiation apparently protected eggs against radiation effect. Dose modifying factor increased linearly from 1.8 to 3.4 with increasing dose of cysteamine (25mM-75mM) added. (auth.)

Biopsy of human morula-stage embryos: outcome of 215 IVF/ICSI cycles with PGS.

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Elena E Zakharova

Full Text Available Preimplantation genetic diagnosis (PGD is commonly performed on biopsies from 6-8-cell-stage embryos or blastocyst trophectoderm obtained on day 3 or 5, respectively. Day 4 human embryos at the morula stage were successfully biopsied. Biopsy was performed on 709 morulae from 215 ICSI cycles with preimplantation genetic screening (PGS, and 3-7 cells were obtained from each embryo. The most common vital aneuploidies (chromosomes X/Y, 21 were screened by fluorescence in situ hybridization (FISH. No aneuploidy was observed in 72.7% of embryos, 91% of those developed to blastocysts. Embryos were transferred on days 5-6. Clinical pregnancy was obtained in 32.8% of cases, and 60 babies were born. Patients who underwent ICSI/PGS treatment were compared with those who underwent standard ICSI treatment by examining the percentage of blastocysts, pregnancy rate, gestational length, birth height and weight. No significant differences in these parameters were observed between the groups. Day 4 biopsy procedure does not adversely affect embryo development in vitro or in vivo. The increased number of cells obtained by biopsy of morulae might facilitate diagnostic screening. There is enough time after biopsy to obtain PGD results for embryo transfer on day 5-6 in the current IVF cycle.

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

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Xinyu Liu

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

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

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

2012-06-01

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

Developmental kinetics of the first cell cycles of bovine in vitro produced embryos in relation to their in vitro viability and sex

DEFF Research Database (Denmark)

Holm, Peter; Shukri, Naseer Mahmoud; Vajta, Gabor

1998-01-01

The development of bovine IVP-embryos was observed in a time-lapse culture system to determine cell cycle lengths of 1) embryos that developed into compact morulae (CM) or blastocysts (BL) within 174 h after insemination (viable), 2) embryos that arrested during earlier stages (nonviable) and 3......) male and female embryos. In 4 replicates, inseminated oocytes were cultured on a microscope stage in 3 to 4 groups on a granulosa cell monolayer in supplemented TCM 199. Images were sequentially recorded and stored at 30-min intervals. All embryos that could be identified throughout the culture period...... were included (n=392), and the times of cleavage events noted. After culture, 100 CM or BL were randomly selected for sexing by PCR. BL developed equally well in the time-lapse and control culture systems (36 vs 38. The respective lengths of the first 4 cell cycles of viable embryos were 32.0 + 3.9, g...

Sea urchin akt activity is Runx-dependent and required for post-cleavage stage cell division

KAUST Repository

Robertson, Anthony J.

2013-03-25

In animal development following the initial cleavage stage of embryogenesis, the cell cycle becomes dependent on intercellular signaling and controlled by the genomically encoded ontogenetic program. Runx transcription factors are critical regulators of metazoan developmental signaling, and we have shown that the sea urchin Runx gene runt-1, which is globally expressed during early embryogenesis, functions in support of blastula stage cell proliferation and expression of the mitogenic genes pkc1, cyclinD, and several wnts. To obtain a more comprehensive list of early runt-1 regulatory targets, we screened a Strongylocentrotus purpuratus microarray to identify genes mis-expressed in mid-blastula stage runt-1 morphants. This analysis showed that loss of Runx function perturbs the expression of multiple genes involved in cell division, including the pro-growth and survival kinase Akt (PKB), which is significantly underexpressed in runt-1 morphants. Further genomic analysis revealed that Akt is encoded by two genes in the S. purpuratus genome, akt-1 and akt-2, both of which contain numerous canonical Runx target sequences. The transcripts of both genes accumulate several fold during blastula stage, contingent on runt-1 expression. Inhibiting Akt expression or activity causes blastula stage cell cycle arrest, whereas overexpression of akt-1 mRNA rescues cell proliferation in runt-1 morphants. These results indicate that post-cleavage stage cell division requires Runx-dependent expression of akt.

Sea urchin akt activity is Runx-dependent and required for post-cleavage stage cell division

KAUST Repository

Robertson, Anthony J.; Coluccio, Alison; Jensen, Sarah; Rydlizky, Katarina; Coffman, James A.

2013-01-01

In animal development following the initial cleavage stage of embryogenesis, the cell cycle becomes dependent on intercellular signaling and controlled by the genomically encoded ontogenetic program. Runx transcription factors are critical regulators of metazoan developmental signaling, and we have shown that the sea urchin Runx gene runt-1, which is globally expressed during early embryogenesis, functions in support of blastula stage cell proliferation and expression of the mitogenic genes pkc1, cyclinD, and several wnts. To obtain a more comprehensive list of early runt-1 regulatory targets, we screened a Strongylocentrotus purpuratus microarray to identify genes mis-expressed in mid-blastula stage runt-1 morphants. This analysis showed that loss of Runx function perturbs the expression of multiple genes involved in cell division, including the pro-growth and survival kinase Akt (PKB), which is significantly underexpressed in runt-1 morphants. Further genomic analysis revealed that Akt is encoded by two genes in the S. purpuratus genome, akt-1 and akt-2, both of which contain numerous canonical Runx target sequences. The transcripts of both genes accumulate several fold during blastula stage, contingent on runt-1 expression. Inhibiting Akt expression or activity causes blastula stage cell cycle arrest, whereas overexpression of akt-1 mRNA rescues cell proliferation in runt-1 morphants. These results indicate that post-cleavage stage cell division requires Runx-dependent expression of akt.

Regulation of somatic embryo development in Norway spruce (Picea abies). A molecular approach to the characterization of specific developmental stages

Energy Technology Data Exchange (ETDEWEB)

Sabala, I. [Swedish Univ. of Agricultural Sciences, Uppsala (Sweden). Dept. of Forest Genetics

1998-12-31

Embryo development is a complex process involving a set of strictly regulated events. The regulation of these events is poorly understood especially during the early stages of embryo development. Somatic embryos go through the same developmental stages as zygotic embryos making them an ideal model system for studying the regulation of embryo development. We have used embryogenic cultures of Picea abies to study some aspects of the regulation of embryo development in gymnosperms. The bottle neck during somatic embryogenesis is the switch from the proliferation stage to the maturation stage. This switch is initiated by giving somatic embryos a maturation treatment i.e. the embryos are treated with abscisic acid (ABA). Somatic embryos which respond to ABA by forming mature somatic embryos were stimulated to secret a 70 kDa protein, AF70. The af70 gene was isolated and characterised. The expression of the af70 gene was constitutive in embryos but was highly ABA-induced in seedlings. Moreover, expression of this gene was stimulated during cold acclimation of Picea abies seedlings. A full length Picea abies cDNA clone Pa18, encoding a protein with the characteristics of plant lipid transfer proteins (LTPs), was isolated and characterised. The Pa18 gene is constitutively expressed in embryogenic cultures of Picea abies representing different stages of development as well as in nonembryogenic callus and seedlings. In situ hybridization showed that Pa18 gene is expressed in all embryonic cells of proliferating somatic embryos but the expression of the gene in mature somatic and zygotic embryos is restricted to the outer cell layer. Southern blot analysis at different stringencies was consistent with a single gene. An alteration in expression of Pa18 causes disturbance in the formation of the proper outer cell layer in the maturing somatic embryos. In addition to its influence on embryo development the Pa18 gene product also inhibits growth of Agrobacterium tumefaciens 195

Changes in radiosensitivity of the in vitro fertilized mouse ova during zygotic stage from fertilization to first cleavage

International Nuclear Information System (INIS)

Yamada, Takeshi; Yukawa, Osami; Matsuda, Yoichi; Ohkawa, Akiko

1982-01-01

The radiosensitivity of mouse zygotes fertilized in vitro (BC3F 1 ovum x ICR sperm) to X-rays has been measured as a function of time from fertilization to first cleavage. The dose of X-rays (LD 50 ) required to prevent development of 50% of the zygotes to the blastocyst stage in vitro varied markedly depending on the time of irradiation from about 40 to 400 R. Sensitivity is relatively low shortly after sperm entry and becomes extremely high (LD 50 , 40 R) at the stage just before pronuclear formation (4 to 6 hr after insemination). In the later pronuclear stage (12 hr of fertilization) the sensitivity becomes low (LD 50 , about 400 R), and it increases again just before first cleavage. (author)

Day 4 good morula embryo transfer provided compatible live birth rate with day 5 blastocyst embryo in fresh IVF/ET cycles.

Science.gov (United States)

Li, Ryh-Sheng; Hwu, Yuh-Ming; Lee, Robert Kuo-Kuang; Li, Sheng-Hsiang; Lin, Ming-Huei

2018-02-01

Embryo transfers during cleavage stage (day 2 or day 3) and blastocyst stages (day 5 or day 6) are common in current daily practice in fresh IVF/ET cycles. Data regarding transferring day 4 embryos, morula/compact stage, is still restricted and the grading system is also inconsistent, as between IVF clinics. This study provided a new detailed classification system for morula/compact stage embryos and compared successes rates between day 4 and day 5 ET. This was a retrospective study. A review of medical records from January 1st, 2013, to December 31st 2015, performed for all conventional insemination and ICSI cycles with a GnRH-antagonist protocol at the Infertility Division of MacKay Memorial Hospital in Taipei City, Taiwan. There were 427 cycles included in our study, 107 in study group (day 4 MET) and 320 in control group (day 5 BET). Pregnancy rates and live birth rate were compatible, as between morula embryo transfer (MET) and blastocyst embryo transfer (BET). The implantation rate (36.3% vs. 39.6%, respectively, p = 0.500), clinical pregnancy rate (49.5% vs. 51.9%, respectively, p = 0.737), and live birth rate (42.1% vs. 45.6%, respectively, p = 0.574) were statistically insignificant between groups. The term birth rate was statistically higher in the MET group than in the BET group (95.7% vs. 79.5%, respectively, p = 0.006). When the clinical outcomes between day 4 good MET and day 5 good BET were compared, the results were compatible. The implantation rate (48.8% vs. 41.1%, respectively, p = 0.335), clinical pregnancy rate (55.0% vs. 53.2%, respectively, p = 0.867), and live birth rate (47.5% vs. 47.1%, respectively, p = 1.000) showed no significant difference. The term birth rate was also higher in day 4 good MET group than in day 5 good BET group (100% vs. 78.3%, respectively, p = 0.025). In this study, we performed day 4 MET avoid BET on Sunday. The grading system we provided was more detailed for embryo selection and it was easier to

No Relationship between Embryo Morphology and Successful Derivation of Human Embryonic Stem Cell Lines

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Ström, Susanne; Rodriguez-Wallberg, Kenny; Holm, Frida; Bergström, Rosita; Eklund, Linda; Strömberg, Anne-Marie; Hovatta, Outi

2010-01-01

Background The large number (30) of permanent human embryonic stem cell (hESC) lines and additional 29 which did not continue growing, in our laboratory at Karolinska Institutet have given us a possibility to analyse the relationship between embryo morphology and the success of derivation of hESC lines. The derivation method has been improved during the period 2002–2009, towards fewer xeno-components. Embryo quality is important as regards the likelihood of pregnancy, but there is little information regarding likelihood of stem cell derivation. Methods We evaluated the relationship of pronuclear zygote stage, the score based on embryo morphology and developmental rate at cleavage state, and the morphology of the blastocyst at the time of donation to stem cell research, to see how they correlated to successful establishment of new hESC lines. Results Derivation of hESC lines succeeded from poor quality and good quality embryos in the same extent. In several blastocysts, no real inner cell mass (ICM) was seen, but permanent well growing hESC lines could be established. One tripronuclear (3PN) zygote, which developed to blastocyst stage, gave origin to a karyotypically normal hESC line. Conclusion Even very poor quality embryos with few cells in the ICM can give origin to hESC lines. PMID:21217828

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

Science.gov (United States)

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

2012-12-01

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

Embryo splitting

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Karl Illmensee

2010-04-01

Full Text Available Mammalian embryo splitting has successfully been established in farm animals. Embryo splitting is safely and efficiently used for assisted reproduction in several livestock species. In the mouse, efficient embryo splitting as well as single blastomere cloning have been developed in this animal system. In nonhuman primates embryo splitting has resulted in several pregnancies. Human embryo splitting has been reported recently. Microsurgical embryo splitting under Institutional Review Board approval has been carried out to determine its efficiency for blastocyst development. Embryo splitting at the 6–8 cell stage provided a much higher developmental efficiency compared to splitting at the 2–5 cell stage. Embryo splitting may be advantageous for providing additional embryos to be cryopreserved and for patients with low response to hormonal stimulation in assisted reproduction programs. Social and ethical issues concerning embryo splitting are included regarding ethics committee guidelines. Prognostic perspectives are presented for human embryo splitting in reproductive medicine.

Study of embryonic ploidy: a probable embryo model

Energy Technology Data Exchange (ETDEWEB)

Kundt, Miriam S; Cabrini, Romulo L [Comision Nacional de Energia Atomica, Buenos Aires (Argentina). Dept. de Radiobiologia

2001-07-01

The second polar body (PB) studies in preimplantation mouse embryos were carried out to evaluate the possibility as reference cell to analyze ploidy. For that purpose embryos in a one cell stage [obtained by crossing hybrid females (CBAxC57BL) to NIH males] were cultured in vitro during 72 hs, individually fixed at morula stage and stained with Feulgen. The DNA content of 263 individual nucleus was evaluated cytophotometrically corresponding to 22 compact morulas of normal development. As haploid PB is present in all pre implanted stage, only embryos with one haploid nuclei were considered as normal. In 95.5% (n = 21) of the embryos the PB was present. DNA measurement of 21 PB was 1n {+-} 0.1. By the height sensibility of PB ploidy, the abnormalities were detected by the criterion of >4.1 n and <1.9 n. The results showed that one embryo was completely haploid (1n). The rest of the embryos (n = 20) 222 blastomeres and 20 PB were analyzed. The DNA measurement showed that 92,7% of the blastomeres (n = 206) are between 2 n and 4 n and 7.3% showed ploidy anomalies, regarding the value n of their PB. The period of the cellular cycle was studied in the normal cell ploidy. This study showed that 16.5% of the blastomeres (n = 34) were in the period G1, 70.39% (n =34) in the period S and 13.2% in the period G2 (n = 27). It is concluded that the PB study showed that it has properties as an excellent indicator of internal ploidia: it is present from the moment of the conception, easily recognizable in the perivitelin space in the embryo of one-two cells, remains in interface during the preimplantation development, it is haploid and digitalized pixel by pixel PB study showed the homogeneity of this type of cell, giving a reliable value of ploidy. The properties of the PB and the results showed that the PB could be an excellent indicator for embryonic ploidy studies on genotoxicity, maintaining its original ploidia during the preimplantation development while the blastomeres are

Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6 causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto.

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Takao Sasado

Full Text Available Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68. CPSF6 is a component of the Cleavage Factor Im complex (CFIm which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3'UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3'UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3'UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo.

Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6) causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto.

Science.gov (United States)

Sasado, Takao; Kondoh, Hisato; Furutani-Seiki, Makoto; Naruse, Kiyoshi

2017-01-01

Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC) migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar) is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68). CPSF6 is a component of the Cleavage Factor Im complex (CFIm) which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3'UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3'UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3'UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo.

Extracellular Vesicles from BOEC in In Vitro Embryo Development and Quality.

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Ricaurte Lopera-Vásquez

Full Text Available To evaluate the effect of conditioned media (CM and Extracellular Vesicles (EVs derived from bovine oviduct epithelial cell (BOEC lines on the developmental capacity of bovine zygotes and the quality of embryos produced in vitro, presumptive zygotes were cultured under specific conditions. In experiment 1, zygotes were cultured either on monolayers from BOEC extended culture (E, together with fresh BOEC suspension cells, or with BOEC-CM from fresh or E-monolayers. In experiment 2, EVs were isolated from BOEC-CM and characterized (150-200 nm by Nanosight® and electron microscopy. Zygotes were cultured in the presence of 3x10(5 EVs/mL, 1.5x10(5 EVs/mL or 7.5x10(4 EVs/mL of fresh or frozen BOEC-EVs. In experiment 3, zygotes were cultured in absence of FCS but with EVs from BOEC-E that had been cultured in different culture media. In experiment 4, zygotes were cultured in SOF+5% normal-FCS, or EV-depleted-FCS. In all cases, cleavage rate (Day 2 and blastocyst development (Day 7-9 was assessed. Blastocysts on Days 7/8 were used for quality evaluation through differential cell count, cryotolerance and gene expression patterns. No differences were found among all FCS-containing groups in cleavage rate or blastocyst yield. However, embryos derived from BOEC-CM had more trophectoderm cells, while embryos derived from BOEC-EVs, both fresh and frozen, has more trophectoderm and total cells. More embryos survived vitrification in the BOEC-CM and BOEC-EV groups. In contrast, more embryos survived in the EV-depleted-FCS than in normal-FCS group. Gene expression patterns were modified for PAG1 for embryos cultured with EVs in the presence of FCS and for IFN-T, PLAC8, PAG1, CX43, and GAPDH in the absence of FCS. In conclusion, EVs from FCS have a deleterious effect on embryo quality. BOEC-CM and EVs during in vitro culture had a positive effect on the quality of in vitro produced bovine embryos, suggesting that EVs have functional communication between the

Absence of nucleolus formation in raccoon dog-porcine interspecies somatic cell nuclear transfer embryos results in embryonic developmental failure.

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Jeon, Yubyeol; Nam, Yeong-Hee; Cheong, Seung-A; Kwak, Seong-Sung; Lee, Eunsong; Hyun, Sang-Hwan

2016-08-25

Interspecies somatic cell nuclear transfer (iSCNT) can be a solution for preservation of endangered species that have limited oocytes. It has been reported that blastocyst production by iSCNT is successful even if the genetic distances between donors and recipients are large. In particular, domestic pig oocytes can support the development of canine to porcine iSCNT embryos. Therefore, we examined whether porcine oocytes may be suitable recipient oocytes for Korean raccoon dog iSCNT. We investigated the effects of trichostatin A (TSA) treatment on iSCNT embryo developmental patterns and nucleolus formation. Enucleated porcine oocytes were fused with raccoon dog fibroblasts by electrofusion and cleavage, and blastocyst development and nucleolus formation were evaluated. To our knowledge, this study is the first in which raccoon dog iSCNT was performed using porcine oocytes; we found that 68.5% of 158 iSCNT embryos had the ability to cleave. However, these iSCNT embryos did not develop past the 4-cell stage. Treatment with TSA did not affect iSCNT embryonic development; moreover, the nuclei failed to form nucleoli at 48 and 72 h post-activation (hpa). In contrast, pig SCNT embryos of the control group showed 18.8% and 87.9% nucleolus formation at 48 and 72 hpa, respectively. Our results demonstrated that porcine cytoplasts efficiently supported the development of raccoon dog iSCNT embryos to the 4-cell stage, the stage of porcine embryonic genome activation (EGA); however, these embryos failed to reach the blastocyst stage and showed defects in nucleolus formation.

The effects of melatonin on bovine uniparental embryos development in vitro and the hormone secretion of COCs

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Shujuan Wang

2017-07-01

Full Text Available Melatonin is a unique multifunctional molecule that mediates reproductive functions in animals. In this study, we investigated the effects of melatonin on bovine parthenogenetic and androgenetic embryonic development, oocyte maturation, the reactive oxygen species (ROS levels in parthenogenetic and androgenetic embryos and cumulus—oocyte complexes (COCs hormone secretion with melatonin supplementation at four concentrations (0, 10, 20, and 30 pmol/mL, respectively. The results showed that melatonin significantly promoted the rates of bovine parthenogenetic and androgenetic embryonic cleavage and morula and blastocysts development (P < 0.05. The rate of cleavage was higher in the androgenetic embryo than that in the parthenogenetic embryo. Compared with the parthenogenetic embryos, the androgenetic embryos had a poor developmental competence from morula to blastocyst stage. Moreover, the levels of ROS were significantly lower in the parthenogenetic and androgenetic embryoes with melatonin-treated group than that of the control group (P < 0.05. Melatonin supplemented significantly increased the maturation rate of oocyte in vitro (P < 0.05. More importantly, melatonin significantly promoted the secretion of progesterone and estradiol by COCs (P < 0.05. To reveal the regulatory mechanism of melatonin on steroids synthesis, we found that steroidogenic genes (CYP11A1, CYP19A1 and StAR were upregulated, suggesting that melatonin regulated estradiol and progesterone secretion through mediating the expression of steroidogenic genes (CYP11A1, CYP19A1 and StAR. In addition, MT1 and MT2 were identified in bovine early parthenogenetic and androgenetic embryos using western blot. It could be concluded that melatonin had beneficial effects on bovine oocyte in vitro maturation, COC hormone secretion, early development of subsequent parthenogenetic and androgenetic embryos. It is inferred that melatonin could be used to enhance the efficiency of in

Nucleologenesis and embryonic genome activation are defective in interspecies cloned embryos between bovine ooplasm and rhesus monkey somatic cells

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Han Yong-Mahn

2009-07-01

Full Text Available Abstract Background Interspecies somatic cell nuclear transfer (iSCNT has been proposed as a tool to address basic developmental questions and to improve the feasibility of cell therapy. However, the low efficiency of iSCNT embryonic development is a crucial problem when compared to in vitro fertilization (IVF and intraspecies SCNT. Thus, we examined the effect of donor cell species on the early development of SCNT embryos after reconstruction with bovine ooplasm. Results No apparent difference in cleavage rate was found among IVF, monkey-bovine (MB-iSCNT, and bovine-bovine (BB-SCNT embryos. However, MB-iSCNT embryos failed to develop beyond the 8- or 16-cell stages and lacked expression of the genes involved in embryonic genome activation (EGA at the 8-cell stage. From ultrastructural observations made during the peri-EGA period using transmission electron microscopy (TEM, we found that the nucleoli of MB-iSCNT embryos were morphologically abnormal or arrested at the primary stage of nucleologenesis. Consistent with the TEM analysis, nucleolar component proteins, such as upstream binding transcription factor, fibrillarin, nucleolin, and nucleophosmin, showed decreased expression and were structurally disorganized in MB-iSCNT embryos compared to IVF and BB-SCNT embryos, as revealed by real-time PCR and immunofluorescence confocal laser scanning microscopy, respectively. Conclusion The down-regulation of housekeeping and imprinting genes, abnormal nucleolar morphology, and aberrant patterns of nucleolar proteins during EGA resulted in developmental failure in MB-iSCNT embryos. These results provide insight into the unresolved problems of early embryonic development in iSCNT embryos.

Four stages of hepatic hematopoiesis in human embryos and fetuses.

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Fanni, D; Angotzi, F; Lai, F; Gerosa, C; Senes, G; Fanos, V; Faa, G

2018-03-01

The liver is a major hematopoietic organ during embryonic and fetal development in humans. Its hematopoietic activity starts during the first weeks of gestation and continues until birth. During this period the liver is colonized by undifferentiated hematopoietic stem cells (HSCs) that gradually differentiate and once mature, enter the circulatory system through the hepatic sinusoids, this process is called hepatic hematopoiesis. The morphology of hepatic hematopoiesis, has been studied in humans through the years, and led to a characterization of all the cell types that make up these phenomena. Studies on murine models also helped to describe the extent of hepatic hematopoiesis at different gestational ages. Using this knowledge, we attempted to describe how hepatic hematopoiesis morphologically evolves as gestation progresses, in human embryos and fetuses. Thus, we observed a total of 32 tissue specimens obtained from the livers of embryos and fetuses at different gestational ages. Basing our observations on the four stages of liver hematopoiesis identified by Sasaki and Sonoda in mice, we also described four consecutive stages of liver hematopoiesis in humans, which resulted to be highly similar to those described in murine models.

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

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

2013-10-01

Is the amount of cell-free DNA released by human embryos into culture medium correlated with embryo morphological features? The mitochondrial DNA (mtDNA) content of culture medium is significantly associated with the fragmentation rate on Days 2 and 3 of embryo development, whether the oocyte came from women ≤ 35 or >35 years old. Cellular fragmentation is often utilized as one of the morphological parameters for embryo quality assessment. The amount of cellular fragments is considered to be an important morphological parameter for embryo implantation potential. It has been hypothesized that fragments are apoptotic bodies or anuclear cytoplasmatic pieces of blastomeres, although no definitive conclusion has been drawn about their pathogenesis. Human fertilized oocytes were individually cultured from Day 1 to Days 2 and 3. A total of 800 samples (166 spent media from Day 2 and 634 from Day 3) were enrolled into the present study. Double-stranded DNA (dsDNA) was quantified in 800 spent embryo culture media by Pico Green dye fluorescence assay. After DNA purification, genomic DNA (gDNA) and mtDNA were profiled by specific quantitative PCR. Statistical analyses defined correlations among DNA contents, embryo morphology and maternal age. Different independent tests confirmed the presence of DNA into embryo culture medium and, for the first time, we demonstrate that both gDNA and mtDNA are detectable in the secretome. The amount of DNA is larger in embryos with bad quality cleavage compared with high-grade embryos, suggesting that the DNA profile of culture medium is an objective marker for embryo quality assessment. In particular, DNA profiles are significantly associated with fragmentation feature (total dsDNA: P = 0.0010; mtDNA; P = 0.0247) and advanced maternal age. It is necessary to establish whether DNA profiling of spent embryo culture medium is a robust onsite test that can improve the prediction of blastulation, implantation and/or pregnancy rate. The

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

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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 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.

Nuclear and cellular expression data from the whole 16-cell stage Arabidopsis thaliana embryo and a cell type-specific expression atlas of the early Arabidopsis embryo

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Palovaara, J.P.J.

2017-01-01

SuperSeries contain expression data from the nuclei of cell types involved in patterning events, with focus on root apical stem cell formation, at 16-cell stage, early globular stage and late globular stage in the early Arabidopsis embryo (atlas). Expression data comparing nuclear and cellular RNA

Lipofection of siRNA into bovine 8-16-cell stage embryos using zona removal and the well-of-the-well culture system.

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Ikeda, Shuntaro; Sugimoto, Miki; Kume, Shinichi

2018-04-13

Bovine preimplantation embryos exhibit dramatic biological changes between before and after the 8-16-cell stage. Here we report a simple lipofection method to transfect siRNA into bovine 8-16-cell stage embryos using zona removal and the well-of-the-well (WOW) culture system. Bovine one-cell embryos produced in vitro were freed from the zona pellucida and cultured up to the 8-16-cell stage in WOW dishes. The 8-16-cell embryos were lipofected with siRNA and the transfection efficiency was assessed at 48 h of transfection. Lipofection with a red fluorescent non-targeting siRNA revealed the importance of zona removal for transfection of siRNA into embryos. Using this method, we knocked down the methionine adenosyltransferase 2A (MAT2A) gene, achieving a significant reduction in MAT2A expression (P lipofection', may be useful to analyze gene functions in bovine preimplantation embryos without expensive equipment and skill-intensive techniques.

Dysfunction in gap junction intercellular communication induces aberrant behavior of the inner cell mass and frequent collapses of expanded blastocysts in mouse embryos.

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Togashi, Kazue; Kumagai, Jin; Sato, Emiko; Shirasawa, Hiromitsu; Shimoda, Yuki; Makino, Kenichi; Sato, Wataru; Kumazawa, Yukiyo; Omori, Yasufumi; Terada, Yukihiro

2015-06-01

We investigated the role of gap junctions (GJs) in embryological differentiation, and observed the morphological behavior of the inner cell mass (ICM) by time-lapse movie observation (TLM) with gap junction inhibitors (GJis). ICR mouse embryos were exposed to two types of GJis in CZB medium: oleamide (0 to 50 μM) and 1-heptanol (0 to 10 mM). We compared the rate of blastocyst formation at embryonic day 4.5 (E4.5) with E5.5. We also observed and evaluated the times from the second cleavage to each embryonic developing stage by TLM. We investigated embryonic distribution of DNA, Nanog protein, and Connexin 43 protein with immunofluorescent staining. In the comparison of E4.5 with E5.5, inhibition of gap junction intercellular communication (GJIC) delayed embryonic blastocyst formation. The times from the second cleavage to blastocyst formation were significantly extended in the GJi-treated embryos (control vs with oleamide, 2224 ± 179 min vs 2354 ± 278 min, p = 0.013). Morphological differences were traced in control versus GJi-treated embryos until the hatching stage. Oleamide induced frequent severe collapses of expanded blastocysts (77.4 % versus 26.3 %, p = 0.0001) and aberrant ICM divisions connected to sticky strands (74.3 % versus 5.3 %, p = 0.0001). Immunofluorescent staining indicated Nanog-positive cells were distributed in each divided ICM. GJIC plays an important role in blastocyst formation, collapses of expanded blastocysts, and the ICM construction in mouse embryos.

Effect of vitamin E on preovulatory stage irradiated female mouse expressed as chromosomal abnormalities in generated embryos

International Nuclear Information System (INIS)

Salimi, M.; Mozdarani, H.

2006-01-01

The present study has been carried out to investigate the effects of preovulatory stage gamma-irradiation of female mice in the absence or presence of vitamin E on numerical chromosome abnormalities in 8-cell embryos after mating with non- irradiated males. Materials and Methods: The 8-11 weeks adult female NMRl mice were whole body irradiated at preovulatory stage (post PMSG injection and about 12-18 hours before Injecting HCG) with 4 Gy gamma-rays generated from a cobalt-60 source alone or in combination with 200 IU/kg vitamin E, intraperitoneally administered one hour prior to irradiation. Soon after HCG injection super ovulated irradiated females were mated with non-irradiated males. About 68-h post coitus (p.c), 8-cell embryos were flushed from the oviducts of pregnant mice and were fixed on slides using standard methods in order to screen for metaphase spreads and numerical chromosome abnormalities. Results: In control embryos, 8% of metaphase plates were aneuploidy whereas in preovulatory stage irradiated female mice, about 50% of metaphase plates of embryos showed numerical chromosome aberrations (P nd meiotic division. Reduction of the frequency of chromosome aberrations in the presence of vitamin E is probably due to antioxidant effects of this vitamin, and scavenging free radicals induced by gamma-rays in mice oocytes' environment

Promising System for Selecting Healthy In Vitro–Fertilized Embryos in Cattle

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Sugimura, Satoshi; Akai, Tomonori; Hashiyada, Yutaka; Somfai, Tamás; Inaba, Yasushi; Hirayama, Muneyuki; Yamanouchi, Tadayuki; Matsuda, Hideo; Kobayashi, Shuji; Aikawa, Yoshio; Ohtake, Masaki; Kobayashi, Eiji; Konishi, Kazuyuki; Imai, Kei

2012-01-01

Conventionally, in vitro–fertilized (IVF) bovine embryos are morphologically evaluated at the time of embryo transfer to select those that are likely to establish a pregnancy. This method is, however, subjective and results in unreliable selection. Here we describe a novel selection system for IVF bovine blastocysts for transfer that traces the development of individual embryos with time-lapse cinematography in our developed microwell culture dish and analyzes embryonic metabolism. The system can noninvasively identify prognostic factors that reflect not only blastocyst qualities detected with histological, cytogenetic, and molecular analysis but also viability after transfer. By assessing a combination of identified prognostic factors—(i) timing of the first cleavage; (ii) number of blastomeres at the end of the first cleavage; (iii) presence or absence of multiple fragments at the end of the first cleavage; (iv) number of blastomeres at the onset of lag-phase, which results in temporary developmental arrest during the fourth or fifth cell cycle; and (v) oxygen consumption at the blastocyst stage—pregnancy success could be accurately predicted (78.9%). The conventional method or individual prognostic factors could not accurately predict pregnancy. No newborn calves showed neonatal overgrowth or death. Our results demonstrate that these five predictors and our system could provide objective and reliable selection of healthy IVF bovine embryos. PMID:22590579

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

International Nuclear Information System (INIS)

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

1988-01-01

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 into 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

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

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

2016-04-01

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

Characterizing nuclear and mitochondrial DNA in spent embryo culture media: genetic contamination identified.

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Hammond, Elizabeth R; McGillivray, Brent C; Wicker, Sophie M; Peek, John C; Shelling, Andrew N; Stone, Peter; Chamley, Larry W; Cree, Lynsey M

2017-01-01

To characterize nuclear and mitochondrial DNA (mtDNA) in spent culture media from normally developing blastocysts to determine whether it could be used for noninvasive genetic assessment. Prospective embryo cohort study. Academic center and private in vitro fertilization (IVF) clinic. Seventy patients undergoing intracytoplasmic sperm injection (ICSI) and 227 blastocysts. Culture media assessment, artificial blastocoele fluid collapse and DNA analysis using digital polymerase chain reaction (dPCR), long-range PCR, quantitative PCR (qPCR), and DNA fingerprinting. Presence of nuclear and mtDNA in three different commercial culture media from Vitrolife and Irvine Scientific, spent embryo media assessment at the cleavage and blastocyst stages of development, and analysis of the internal media controls for each patient that had been exposed to identical conditions as embryo media but did not come into contact with embryos. Higher levels of nuclear and mtDNA were observed in the culture media that had been exposed to embryos compared with the internal media controls. Nuclear DNA (∼4 copies) and mtDNA (∼600 copies) could be detected in spent media, and the levels increased at the blastocyst stage. No increase in DNA was detected after artificial blastocoele fluid collapse. Mixed sex chromosome DNA was detected. This originated from contamination in the culture media and from maternal (cumulus) cells. Due to the limited amount of template, the presence of embryonic nuclear DNA could not be confirmed by DNA fingerprinting analysis. Currently DNA from culture media cannot be used for genetic assessment because embryo-associated structures release DNA into the culture medium and the DNA is of mixed origin. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Using SNP array to identify aneuploidy and segmental imbalance in translocation carriers

Directory of Open Access Journals (Sweden)

B. Xiong

2014-12-01

In addition to genetic testing techniques, the embryo biopsy stage (polar body, cleavage embryo or blastocyst and the mode of embryo transfer (fresh or frozen embryos can affect the outcome of PGD. It is now generally recommended that blastomere biopsy should be replaced by blastocyst biopsy to avoid a high mosaic rate and biopsy-related damage to cleavage-stage embryos, which might affect embryo development. However, more clinical data are required to confirm that the technique of SNP array-based PGD (SNP-PGD combined with trophectoderm (TE biopsy and frozen embryo transfer (FET is superior to traditional FISH-PGD combined with Day 3 (D3 blastomere biopsy and fresh embryo transfer.

Peroxidized mineral oil increases the oxidant status of culture media and inhibits in vitro porcine embryo development.

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Martinez, C A; Nohalez, A; Ceron, J J; Rubio, C P; Roca, J; Cuello, C; Rodriguez-Martinez, H; Martinez, E A; Gil, M A

2017-11-01

The use of oils with undetected alterations is a long-recognized problem for in vitro embryo production systems. Since peroxides in oils have been associated with reduced embryo production outcomes, our goals were (1) to evaluate the effects of a batch of mineral oil (MO) that was suspected to be altered on the in vitro production of pig embryos and (2) to determine oil peroxide values throughout culture and the transfer of oxidant agents from oil to culture media. Sunflower oil, which has a completely different chemical composition than MO but a higher oxidative status, and unaltered MO were used as controls. Oocyte maturation, fertilization and embryo development were affected differently depending on the oil overlay used. While the suspected MO was not able to sustain in vitro maturation and fertilization, the oocytes incubated in the presence of sunflower oil were matured and fertilized similarly to those of the unaltered MO group. Moreover, the cleavage rate of presumed zygotes cultured under the suspected MO was severely reduced compared with those cultured under the other oils, and none of the cleaved embryos developed to the blastocyst stage. Although the cleavage rates in the sunflower oil and unaltered MO groups were similar, embryos cultured under sunflower oil also failed to develop to the blastocyst stage. Our results revealed that the suspected MO and sunflower oil had similar levels of peroxides and that these levels were much higher than those of the unaltered MO. The total oxidant status was higher in media incubated under peroxidized oils than in fresh media or media incubated without an oil overlay or under unaltered MO, indicating that oxidant agents were transferred to the incubation media. However, unlike the sunflower oil group, the culture media incubated under the suspected MO had high levels of total oxidant status and low levels of hydrogen peroxide and reactive oxygen species, suggesting the presence of other unknown oxidant agents in

The effects of embryo culture media on the birthweight of singletons via fresh or frozen-thawed embryo transfer: a large-scale retrospective study.

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Gu, Fang; Deng, Mingfen; Gao, Jun; Wang, Zilian; Ding, Chenhui; Xu, Yanwen; Zhou, Canquan

2016-09-19

Embryo culture media used for IVF treatment might affect fetal growth and thus birthweight of the newborns. A retrospective study was conducted in South China using data from 2370 singleton neonates born after IVF/ICSI between 2009 and 2012. Two culture media, i.e., either Vitrolife or SAGE were used as embryo culture media during the study period. Neonates' birthweights were compared between the two embryo culture media groups. Among the 2370 singletons, 1755 cases came from fresh cleavage embryo transfer while 615 were from frozen-thawed cleavage embryo transfer. Within the fresh embryo transfer newborns, no statistical difference was observed in either birthweight (mean ± SD: 3196.0 ± 468.9 versus 3168.4 ± 462.0g, p > 0.05) or adjusted birthweight controlled for gestational age and gender (z-score mean ± SD: 0.11 ± 1.02 versus 0.11 ± 0.99 g, P > 0.05) between the Vitrolife (n = 419) and the SAGE group (n = 1336). Likewise within frozen embryo transfer neotates, no statistical difference of the birthweight (3300.6 ± 441.3 vs.3256.0 ± 466.7 g, P > 0.05) and adjusted birthweight (0.30 ± 0.99 g versus 0.29 ± 0.97 g, P > 0.05) was found between the Vitrolife (n = 202) and the SAGE group (n = 413). The sex ratio [OR1.17, 95 % CI (0.94-1.46)/OR1.1, 95 % CI (0.78-1.54)], rate of small for gestational age [OR1.14, 95 % CI (0.82-1.59)/OR1.06, 95 % CI (0.56-2.02)] and large for gestational age [OR1.07, 95 % CI (0.64-1.76)/OR0.98, 95 % CI (0.47-2.02)] in fresh and frozen-thawed subgourps are all comparable respectively between the two culture media. No group differences were found in the rate of low birthweight and macosomia. Multiple linear regression analysis demonstrated that maternal weight, gestational age, frozen-thawed embryo transfer and infant gender were significantly related to neonatal birthweight (P cultured in SAGE or Vitrolife media after fresh or frozen-thawed cleavage

Preimplantation genetic haplotyping a new application for diagnosis of translocation carrier's embryos- preliminary observations of two robertsonian translocation carrier families.

Science.gov (United States)

Shamash, Jana; Rienstein, Shlomit; Wolf-Reznik, Haike; Pras, Elon; Dekel, Michal; Litmanovitch, Talia; Brengauz, Masha; Goldman, Boleslav; Yonath, Hagith; Dor, Jehoshua; Levron, Jacob; Aviram-Goldring, Ayala

2011-01-01

Preimplantation genetic diagnosis using fluorescence in-situ hybridization (PGD-FISH) is currently the most common reproductive solution for translocation carriers. However, this technique usually does not differentiate between embryos carrying the balanced form of the translocation and those carrying the homologous normal chromosomes. We developed a new application of preimplantation genetic haplotyping (PGH) that can identify and distinguish between all forms of the translocation status in cleavage stage embryos prior to implantation. Polymorphic markers were used to identify and differentiate between the alleles that carry the translocation and those that are the normal homologous chromosomes. Embryos from two families of robertsonian translocation carriers were successfully analyzed using polymorphic markers haplotyping. Our preliminary results indicate that the PGH is capable of distinguishing between normal, balanced and unbalanced translocation carrier embryos. This method will improve PGD and will enable translocation carriers to avoid transmission of the translocation and the associated medical complications to offspring.

Male gender explains increased birthweight in children born after transfer of blastocysts.

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Kaartinen, N M; Kananen, K M; Rodriguez-Wallberg, K A; Tomás, C M; Huhtala, H Sa; Tinkanen, H I

2015-10-01

Does extended embryo culture have a different effect on the birthweight of girls and boys? The mean birthweight of boys born after fresh and frozen-thawed blastocyst transfer was increased compared with those born after cleavage stage embryo transfer. This effect was not detected among girls. Previous studies indicate that newborns from frozen-thawed cleavage stage embryos may present with a higher weight than newborns from fresh embryo transfers. With regard to fresh embryos, newborns after a blastocyst transfer have been reported as having higher birthweights than newborns from cleavage stage embryos. Retrospective multicentre case-control cohort study. All IVF/ICSI treatments were performed in the time-period from January 2008 to March 2014. Birthweight of singletons born at full-term (≥37 weeks), after fresh or frozen blastocyst embryo transfers (n = 277), were compared with weights of children born after fresh or frozen cleavage stage embryo transfers (Day 2-3) (n = 277). The cases and controls were matched by delivery week, and by gender. Data of IVF/ICSI treatments, and the treatments' outcomes were collected and analysed. The birthweight after a fresh blastocyst transfer was significantly higher (mean 3530.6 g) than that after a transfer of cleavage stage embryos (mean 3418.8 g; weight difference 111.8 g, P = 0.047). The weights of newborns after frozen-thawed blastocyst transfers (mean 3647.5 g) and the frozen-thawed cleavage stage embryo transfers (mean 3650.9 g), were similar (weight difference 3.4 g, P = 0.95). The boys born after transfer of frozen-thawed blastocysts had a significantly higher birthweight (mean 3767.9 g) than girls (3525.2 g; weight difference 242.7 g, P = 0.002), whereas the difference of birthweights between genders was only 13.5 g in cleavage stage (P = 0.863). The same effect was seen after fresh blastocyst transfers (weight difference 211.5 g, P = 0.011), but not after fresh Day 2-3 embryo transfers (weight difference 53.6 g, P

Effects of reactive oxygen species levels in prepared culture media on embryo development: a comparison of two media.

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

2014-12-01

This study determined the correlation between the levels of reactive oxygen species (ROS) in prepared culture media and the early development of human embryos. This was an autocontrolled comparison study. A total of 159 patients undergoing in vitro fertilization/intracytoplasmic sperm injection treatment were recruited in this study. The pH values, osmolarity pressures, and ROS levels of 15 batches of two culture media were measured. Sibling oocytes or embryos from individual patients were randomly assigned to two culture groups with Quinn's Advantage Cleavage and Blastocyst media (QAC/QAB) or GIII series cleavage and blastocyst media (G1.3/G2.3). The difference between the two culture groups was analyzed using one-sample t test. The QAC/QAB and G1.3/G2.3 media exhibited similar pH values and osmolarity pressures. However, the prepared QAC/QAB media were characterized to contain lower amounts of ROS than the G1.3/G2.3 media. Furthermore, the blastocysts that developed under the QAC/QAB media were morphologically superior to those that developed under the G1.3/G2.3 media. The elevated ROS levels in culture media were associated with poor development of blastocyst-stage embryos. Measurement of ROS levels may be a valuable process for medium selection or modification. Copyright © 2014. Published by Elsevier B.V.

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

Science.gov (United States)

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.

A role for Aurora C in the chromosomal passenger complex during human preimplantation embryo development

NARCIS (Netherlands)

Santos, Margarida Avo; van de Werken, Christine; de Vries, Marieke; Jahr, Holger; Vromans, Martijn J. M.; Laven, Joop S. E.; Fauser, Bart C.; Kops, Geert J.; Lens, Susanne M.; Baart, Esther B.

BACKGROUND: Human embryos generated by IVF demonstrate a high incidence of chromosomal segregation errors during the cleavage divisions. To analyse underlying molecular mechanisms, we investigated the behaviour of the chromosomal passenger complex (CPC) in human oocytes and embryos. This important

Survival of embryo irradiated with gamma rays by embryo culture in Brassica pekinensis Rupr

International Nuclear Information System (INIS)

Moue, T.

1984-01-01

The effect of irradiation on the survival rates and embryonic development of Brassica pekinensis RUPR. (Varieties; Kashin, Kohai 65 nichi and kairyochitose) was investigated. The purpose of this study was to seek ways of increasing the survival rates of embryos such as B.oleracea obtained through embryo culture techniques after irradiation doses affecting seed fertility and germination, for the purpose of increasing mutation rates. Embryos at different developmental stages ranging from the globular to the early heart stages were irradiated with 20 KR of gamma rays at the daily rate 0L 20 KR or 10 KR (Fig.1 and Table 1). The embryos were excised from ovules 4 to 10 days after irradiation and cultured on White's medium. The shooting and rooting rates on the 34th day of culture were higher at the dose of 10 KR/day than 20 KR/day and were lower when the materials were irradiated at the young embryonic stage (Table 3). Varietal differences in the shooting and rooting rates were also observed. The irradiated embryos survived mainly in the state of callus. It was concluded that the embryo culture technique was successful when applied to irradiated embryos excised at the young embryonic stage and that the technique affected B.pekinensis less than B.oleracea

In vitro culture of individual mouse preimplantation embryos: the role of embryo density, microwells, oxygen, timing and conditioned media.

Science.gov (United States)

Kelley, Rebecca L; Gardner, David K

2017-05-01

Single embryo culture is suboptimal compared with group culture, but necessary for embryo monitoring, and culture systems should be improved for single embryos. Pronucleate mouse embryos were used to assess the effect of culture conditions on single embryo development. Single culture either before or after compaction reduced cell numbers (112.2 ± 3.1; 110.2 ± 3.5) compared with group culture throughout (127.0 ± 3.4; P media volume from 20 µl to 2 µl increased blastocyst cell numbers in single embryos cultured in 5% oxygen (84.4 ± 3.2 versus 97.8 ± 2.8; P Culture in microwell plates for the EmbryoScope and Primo Vision time-lapse systems changed cleavage timings and increased inner cell mass cell number (24.1 ± 1.0; 23.4 ± 1.2) compared with a 2 µl microdrop (18.4 ± 1.0; P media to single embryos increased hatching rate and blastocyst cell number (91.5 ± 4.7 versus 113.1 ± 4.4; P culture before or after compaction is therefore detrimental; oxygen, media volume and microwells influence single embryo development; and embryo-conditioned media may substitute for group culture. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Non-invasive metabolomic profiling of embryo culture media and morphology grading to predict implantation outcome in frozen-thawed embryo transfer cycles.

Science.gov (United States)

Li, Xiong; Xu, Yan; Fu, Jing; Zhang, Wen-Bi; Liu, Su-Ying; Sun, Xiao-Xi

2015-11-01

Assessment of embryo viability is a crucial component of in vitro fertilization and currently relies largely on embryo morphology and cleavage rate. Because morphological assessment remains highly subjective, it can be unreliable in predicting embryo viability. This study investigated the metabolomic profiling of embryo culture media using near-infrared (NIR) spectroscopy for predicting the implantation potential of human embryos in frozen-thawed embryo transfer (FET) cycles. Spent embryo culture media was collected on day 4 after thawed embryo transfer (n = 621) and analysed using NIR spectroscopy. Viability scores were calculated using a predictive multivariate algorithm of fresh embryos with known pregnancy outcomes. The mean viability indices of embryos resulting in clinical pregnancy following FET were significantly higher than those of non-implanted embryos and differed between the 0, 50, and 100 % implantation groups. Notably, the 0 % group index was significantly lower than the 100 % implantation group index (-0.787 ± 0.382 vs. 1.064 ± 0.331, P  0.05). NIR metabolomic profiling of thawed embryo culture media is independent of morphology and correlates with embryo implantation potential in FET cycles. The viability score alone or in conjunction with morphologic grading is a more objective marker for implantation outcome in FET cycles than morphology alone.

Molecular Basis of Meiotic Maturation and Apoptosis of Oocytes, Sperm-Oocyte Interactions and Early Cleavage of Embryos in Mice, Role of Phosphatidylinositol 3-Kinase, Mos, Fas-Fas Ligand, Integrinα6 and MAP Kinase

OpenAIRE

Yumi Hoshino; Ken-ichi Yamanaka; Ikuo Tomioka; Noritaka Fukunaga; Mehdi Abbasi; Eimei Sato

2005-01-01

The interaction between molecular biology and embryology made an extensive progress in the research on gametogenesis, fertilization and early embryogenesis in mice. In this article, molecules involving in meiotic maturation and apoptosis of oocytes, sperm-oocyte interactions and early cleavage of fertilized embryos in mice are described including our recent following experiments. 1) Phosphatidylinositol 3-kinase and Akt participate in the follicle stimulating hormone-induced meiotic maturatio...

Telomere lengthening early in development.

Science.gov (United States)

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.

Flat mount preparation for observation and analysis of zebrafish embryo specimens stained by whole mount in situ hybridization.

Science.gov (United States)

Cheng, Christina N; Li, Yue; Marra, Amanda N; Verdun, Valerie; Wingert, Rebecca A

2014-07-17

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Effect of Albendazole at different concentrations on fertilization and early cleavage in Tetrapygus niger “sea urchin”

Directory of Open Access Journals (Sweden)

Gina Zavaleta Espejo

2013-09-01

Full Text Available In this study we evaluated the effect of Albendazole at different concentrations and exposure times on the process of fertilization and early cleavage in Tetrapygus niger "sea urchin". Each experimental group consisted of 200 mL, of previously filtered seawater at pH 7.3 and temperature of 20 ± 2 °C, plus five drops of eggs and two drops of spermatozoa exposed to different concentrations of Albendazole 400 ppm, 800 ppm and 1200 ppm. Determining the effect of Albendazole was conducted by counting the number of cones of fertilization, as well as the number of cleaving embryos with normal and abnormal. The ANOVA and multiple comparison test Tukey averages showed significant differences between the treatments, namely that increasing the concentration of Albendazole fertilization rate decreases and increases the percentage of embryos with abnormal cleavage, so therefore concluded that the Albendazole at different concentrations and exposure times affects the process of fertilization and early cleavage in T, niger "sea urchin"

Optimizing the number of cleavage stage embryos to transfer on day 3 in women 38 years of age and older: a Society for Assisted Reproductive Technology database study.

Science.gov (United States)

Stern, Judy E; Goldman, Marlene B; Hatasaka, Harry; MacKenzie, Todd A; Surrey, Eric S; Racowsky, Catherine

2009-03-01

To determine the optimal number of day 3 embryos to transfer in women >or=38 years by conducting an evidence-based evaluation. Retrospective analysis of 2000-2004 national SART data. National writing group. A total of 36,103 day 3 embryo transfers in women >or=38 years undergoing their first assisted reproductive technology cycle. None. Logistic regression was used to model the probability of pregnancy, delivery, and multiple births (twin or high order) based on age- and cycle-specific parameters. Pregnancy rates, delivery rates, and multiple rates increased up to transfer of three embryos in 38-year-olds and four in 39-year-olds; beyond this number, only multiple rates increased. In women >or=40 years, delivery rates and multiple rates climbed steadily with increasing numbers transferred. Multivariate analysis confirmed the statistically significant effect of age, number of oocytes retrieved, and embryo cryopreservation on delivery and multiple rates. Maximum FSH level was not an independent predictor by multivariate analysis. Use of intracytoplasmic sperm injection was associated with lowered delivery rate. No more than three or four embryos should be transferred in 38- and 39-year-olds, respectively, whereas up to five embryos could be transferred in >or=40-year-olds. Numbers of embryos to transfer should be adjusted according to number of oocytes retrieved and availability of excess embryos for cryopreservation.

Confinement and clearance of OCT4 in the porcine embryo at stereomicroscopically defined stages around gastrulation

DEFF Research Database (Denmark)

Vejlsted, Morten; Offenberg, Hanne Kjær; Thorup, Flemming

2006-01-01

was selectively observed in the epiblast. A prominent crescent-shaped thickening at the posterior region of the embryonic disk marked the first polarization within this structure reflecting incipient cell ingression. Following differentiation of the epiblast, clearance of OCT4 from the three germ layers......In the areas of developmental biology and embryonic stem cell research, reliable molecular markers of pluripotency and early lineage commitment are sparse in large animal species. In this study, we present morphological and immunohistochemical findings on the porcine embryo in the period around...... gastrulation, days 8-17 postinsemination, introducing a steromicroscopical staging system in this species. In embryos at the expanding hatched blastocyst stage, OCT4 is confined to the inner cell mass. Following detachment of the hypoblast, and formation of the embryonic disk, this marker of pluripotency...

Theory about the Embryo Cryo-Treatment.

Science.gov (United States)

Vladimirov, Iavor K; Tacheva, Desislava; Diez, Antonio

2017-04-01

To create hypothesis, which can give a logical explanation related to the benefits of freezing/thawing embryos. Cryopreservation is not only a technology used for storing embryos, but also a method of embryo treatment that can potentially improve the success rate in infertile couples. From the analysis of multiple results in assisted reproductive technology, which have no satisfactory explanation to date, we found evidence to support a 'therapeutic' effect of the freezing/thawing of embryos on the process of recovery of the embryo and its subsequent implantation. Freezing/thawing is a way to activate the endogenous survival and repair responses in preimplantation embryos. Several molecular mechanisms can explain the higher success rate of ET using thawed embryos compared to fresh ET in women of advanced reproductive age, the higher miscarriage rate in cases of thawed blastocyst ET compared to thawed ET at early cleavage embryo, and the higher perinatal parameters of born children after thawed ET. Embryo thawing induces a stress. Controlled stress is not necessarily detrimental, because it generates a phenomenon that is counteracted by several known biological responses aimed to repair mitochondrial damage of membrane and protein misfolding. The term for favorable biological responses to low exposures to stress is called hormesis. This thesis will summarize the role of cryopreservation in the activation of a hormetic response, preserving the mitochondrial function, improving survival, and having an impact on the process of implantation, miscarriage, and the development of pregnancy.

Vitamin C supplementation enhances compact morulae formation but reduces the hatching blastocyst rate of bovine somatic cell nuclear transfer embryos.

Science.gov (United States)

Li, Qian; Wang, Yong-Sheng; Wang, Li-Jun; Zhang, Hui; Li, Rui-Zhe; Cui, Chen-Chen; Li, Wen-Zhe; Zhang, Yong; Jin, Ya-Ping

2014-08-01

Vitamin C, an antioxidant that reduces reactive oxygen species (ROS) in cells, is capable of significantly improving the developmental competence of porcine and mouse somatic cell nuclear transfer (SCNT) embryos, both in vitro and in vivo. In the present study, the effects of vitamin C on the developmental competence of bovine SCNT embryos were investigated. The results indicated that vitamin C (40 μg/mL) positively affected the scavenging of intracellular ROS, cleavage rate at 24 h (76.67 vs. 68.26%, pvitamin C supplementation did not significantly affect the blastocyst formation rate and proportion of inner cell mass over total cells per blastocyst on day 7. Moreover, vitamin C supplementation obviously impaired the total cell numbers per blastocyst (97.20 ± 11.35 vs. 88.57 ± 10.43, pVitamin C supplementation preferentially improved the viability of bovine SCNT embryos prior to the blastocyst stage, but did not enhance the formation and quality of blastocysts in vitro. In conclusion, the effect of vitamin C on the development of bovine SCNT embryos is complex, and vitamin C is not a suitable antioxidant chemical for the in vitro culture of bovine SCNT embryos.

Phytohemagglutinin facilitates the aggregation of blastomere pairs from Day 5 donor embryos with Day 4 host embryos for chimeric bovine embryo multiplication.

Science.gov (United States)

Simmet, Kilian; Reichenbach, Myriam; Reichenbach, Horst-Dieter; Wolf, Eckhard

2015-12-01

Multiplication of bovine embryos by the production of aggregation chimeras is based on the concept that few blastomeres of a donor embryo form the inner cell mass (ICM) and thus the embryo proper, whereas cells of a host embryo preferentially contribute to the trophectoderm (TE), the progenitor cells of the embryonic part of the placenta. We aggregated two fluorescent blastomeres from enhanced green fluorescent protein (eGFP) transgenic Day 5 morulae with two Day 4 embryos that did not complete their first cleavage until 27 hours after IVF and tested the effect of phytohemagglutinin-L (PHA) on chimeric embryo formation. The resulting blastocysts were characterized by differential staining of cell lineages using the TE-specific factor CDX2 and confocal laser scanning microscopy to facilitate the precise localization of eGFP-positive cells. The proportions of blastocyst development of sandwich aggregates with (n = 99) and without PHA (n = 46) were 85.9% and 54.3% (P chimeric blastocysts analyzed by confocal laser scanning microscopy, nine had eGFP-positive cells (three of them in the ICM, three in the TE, and three in both lineages). When integration in the ICM occurred, the number of eGFP-positive cells in this compartment was 8.3 ± 2.3 (mean ± standard error of the mean). We conclude that PHA is advantageous for the formation of aggregation chimeras, but the approach tested in the present study with only two donor blastomeres and two host embryos did not result in multiplication of genetically valuable donor embryos. Copyright © 2015 Elsevier Inc. All rights reserved.

Arabidopsis mitochondrial protein slow embryo development1 is essential for embryo development

International Nuclear Information System (INIS)

Ju, Yan; Liu, Chunying; Lu, Wenwen; Zhang, Quan; Sodmergen

2016-01-01

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 analysis 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.

Arabidopsis mitochondrial protein slow embryo development1 is essential for embryo development

Energy Technology Data Exchange (ETDEWEB)

Ju, Yan; Liu, Chunying; Lu, Wenwen; Zhang, Quan; Sodmergen, E-mail: sodmergn@pku.edu.cn

2016-05-27

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 analysis 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.

Effects of UV-C irradiation on development of goldfish embryos

International Nuclear Information System (INIS)

Wu Jian; Dai Guifu; Zhang Fengqiu; Lu Lei

2005-01-01

Goldfish embryos at five different developmental stages, from fertilized eggs to heat beating stage, were irradiated by UV rays, and hatching rate, darkly pigmented eye rate and abnormal embryo rate of the irradiated embryos were investigated. Being subjected to very low amount (≤3 min.) of the UV irradiation, the embryos earlier than gastrula stage showed hormesis. However, the embryos at gastrula or heart beating stage were very sensitive to UV irradiation, showing just damage effect, which was very strong even at very low amount of the UV irradiation. The results also showed that development of the gastrula embryos irradiated by the UV rays stopped before darkly pigmented eye state, whereas embryos irradiated at heart beating stage by the UV rays could develop to the darkly pigmented eye stage, though they could not hatch out. (authors)

AFSC/RACE/SAP/Foy: Effects of ocean acidification on embryo stages of Tanner crab: Kodiak Island, Alaska.

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — To study the effects of ocean acidification we examined the effects of ocean acidification on the embryo stages of the economically important southern Tanner crab,...

Manipulating early pig embryos.

Science.gov (United States)

Niemann, H; Reichelt, B

1993-01-01

On the basis of established surgical procedures for embryo recovery and transfer, the early pig embryo can be subjected to various manipulations aimed at a long-term preservation of genetic material, the generation of identical multiplets, the early determination of sex or the alteration of the genetic make-up. Most of these procedures are still at an experimental stage and despite recent considerable progress are far from practical application. Normal piglets have been obtained after cryopreservation of pig blastocysts hatched in vitro, whereas all attempts to freeze embryos with intact zona pellucida have been unsuccessful. Pig embryos at the morula and blastocyst stage can be bisected microsurgically and the resulting demi-embryos possess a high developmental potential in vitro, whereas their development in vivo is impaired. Pregnancy rates are similar (80%) but litter size is reduced compared with intact embryos and twinning rate is approximately 2%. Pig blastomeres isolated from embryos up to the 16-cell stage can be grown in culture and result in normal blastocysts. Normal piglets have been born upon transfer of blastocysts derived from isolated eight-cell blastomeres, clearly underlining the totipotency of this developmental stage. Upon nuclear transfer the developmental capacity of reconstituted pig embryos is low and culture. Sex determination can be achieved either by separation of X and Y chromosome bearing spermatozoa by flow cytometry or by analysing the expression of the HY antigen in pig embryos from the eight-cell to morula stage. Microinjection of foreign DNA has been successfully used to alter growth and development of transgenic pigs, and to produce foreign proteins in the mammary gland or in the bloodstream, indicating that pigs can be used as donors for valuable human pharmaceutical proteins. Another promising area of gene transfer is the increase of disease resistance in transgenic lines of pigs. Approximately 30% of pig spermatozoa bind

Introduction of exogenous DNA into gonads of chick embryos by lipofection and electroporation of stage X blastoderms in vivo.

Science.gov (United States)

Sano, A; Tagami, T; Harumi, T; Matsubara, Y; Naito, M

2003-03-01

1. In order to introduce exogenous DNA into gonads of chick embryos, stage X blastoderms of freshly laid and unincubated eggs were transfected by lipofection and electroporation in vivo. 2. The introduced DNA, green fluorescence protein (GFP) gene, was efficiently expressed in the blastoderms incubated for 24 h (78.8%, 78/99). 3. The GFP gene was present in most of the embryonic bodies and extra-embryonic membranes died by d 10 of incubation, when analysed by polymerase chain reaction. On d 16 to 20 of incubation, the GFP gene was detected in 7.0 to 20.9% of embryos in the heart, liver, stomach and brain, but not in the sartorius muscle. For the gonads, the GFP gene was detected in 22.2% (6/27) of the testes and 6.3% (1/18) of the ovaries examined. 4. These results suggest that it is possible to introduce exogenous DNA into gonads of chick embryos by lipofection and electroporation of stage X blastoderms in vivo.

Annotating and quantifying pri-miRNA transcripts using RNA-Seq data of wild type and serrate-1 globular stage embryos of Arabidopsis thaliana

Directory of Open Access Journals (Sweden)

Daniel Lepe-Soltero

2017-12-01

Full Text Available The genome annotation for the model plant Arabidopsis thaliana does not include the primary transcripts from which MIRNAs are processed. Here we present and analyze the raw mRNA sequencing data from wild type and serrate-1 globular stage embryos of A. thaliana, ecotype Columbia. Because SERRATE is required for pri-miRNA processing, these precursors accumulate in serrate-1 mutants, facilitating their detection using standard RNA-Seq protocols. We first use the mapping of the RNA-Seq reads to the reference genome to annotate the potential primary transcripts of MIRNAs expressed in the embryo. We then quantify these pri-miRNAs in wild type and serrate-1 mutants. Finally, we use differential expression analysis to determine which are up-regulated in serrate-1 compared to wild type, to select the best candidates for bona fide pri-miRNAs expressed in the globular stage embryos. In addition, we analyze a previously published RNA-Seq dataset of wild type and dicer-like 1 mutant embryos at the globular stage [1]. Our data are interpreted and discussed in a separate article [2].

Annotating and quantifying pri-miRNA transcripts using RNA-Seq data of wild type and serrate-1 globular stage embryos of Arabidopsis thaliana.

Science.gov (United States)

Lepe-Soltero, Daniel; Armenta-Medina, Alma; Xiang, Daoquan; Datla, Raju; Gillmor, C Stewart; Abreu-Goodger, Cei

2017-12-01

The genome annotation for the model plant Arabidopsis thaliana does not include the primary transcripts from which MIRNAs are processed. Here we present and analyze the raw mRNA sequencing data from wild type and serrate-1 globular stage embryos of A. thaliana , ecotype Columbia. Because SERRATE is required for pri-miRNA processing, these precursors accumulate in serrate-1 mutants, facilitating their detection using standard RNA-Seq protocols. We first use the mapping of the RNA-Seq reads to the reference genome to annotate the potential primary transcripts of MIRNAs expressed in the embryo. We then quantify these pri-miRNAs in wild type and serrate-1 mutants. Finally, we use differential expression analysis to determine which are up-regulated in serrate-1 compared to wild type, to select the best candidates for bona fide pri-miRNAs expressed in the globular stage embryos. In addition, we analyze a previously published RNA-Seq dataset of wild type and dicer-like 1 mutant embryos at the globular stage [1]. Our data are interpreted and discussed in a separate article [2].

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

Science.gov (United States)

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

2010-12-01

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

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

Science.gov (United States)

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

2016-08-01

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

Neonatal health including congenital malformation risk of 1072 children born after vitrified embryo transfer.

Science.gov (United States)

Belva, F; Bonduelle, M; Roelants, M; Verheyen, G; Van Landuyt, L

2016-07-01

transfer. Data were analysed by multiple linear and logistic regression, adjusted for treatment variables and maternal characteristics. Mothers to infants in the vitrified group were on average slightly older and more often suffering from pregnancy-related hypertensive disorders than mothers to infants in the fresh transfer group. Singletons born after vitrification showed a higher birthweight standard deviation score (SDS) (-0.4 versus -0.7; 95% confidence interval (CI): 0.0-0.3, P = 0.001) and a lower small-for-gestational age rate (AOR: 0.55; 95% CI: 0.34-0.90) in comparison with peers born after fresh embryo transfer. Preterm birth rate and perinatal death rate were comparable between the two groups (AOR: 0.91; 95% CI: 0.57-1.43 and AOR: 0.97; 95% CI: 0.40-2.36). In twins, neonatal outcomes including birthweight SDS, small-for-gestational age and prematurity rates were comparable in the vitrified and the fresh groups, when adjusted for confounders. Furthermore, the rate of major congenital malformations in live borns was comparable between the vitrified group and the fresh group, both in singletons (2.6 versus 2.8%; AOR: 0.91; 95% CI: 0.47-1.78) and in twins (2.4 versus 2.7%; AOR: 0.51; 95% CI: 0.05-5.72). Also, the total malformation rate in the vitrified group (3.4%; 95% CI: 2.4-4.8) did not differ from the rate in the fresh embryo group (3.9%; 95% CI: 3.1-5.0). The embryonic stage at vitrification or fresh transfer (cleavage-stage embryo or blastocyst) did not influence the birth characteristics or malformation rate. The main limitation of this study is the rather small twin group. Therefore, the outcome results for twins should be interpreted cautiously. This study provides evidence that transfer of vitrified Day 3 and Day 5 embryos does not adversely affect the neonatal health of the offspring in comparison with transfer of fresh embryos. Furthermore, neonatal outcomes were not different after transfer of vitrified blastocysts compared with transfer of vitrified

Miniaturized embryo array for automated trapping, immobilization and microperfusion of zebrafish embryos.

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Jin Akagi

Full Text Available 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.

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

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

2015-06-01

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

Pre-incubation of porcine semen reduces the incidence of polyspermy on embryos derived from low quality oocytes

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Cláudio Francisco Brogni

2016-06-01

Full Text Available ABSTRACT: The main cause of low efficiency of in vitro produced porcine embryos is the high polyspermic penetration rates at fertilization, which is aggravated in low quality oocytes. Experiment 1 evaluated the embryo development in high and low quality oocytes. Experiment 2 evaluated the embryo development and quality of low quality oocytes fertilized with sperm pre-incubated during 0h (control, 0.5h, 1h and 1.5h. Experiment 3 investigated fertilization and monospermic rates of the same groups of Experiment 2. Experiment 4 evaluated embryo development, cell density, fertilization and monospermic rates of high quality oocytes using semen pre incubated during the best time observed in the previous experiments. Cleavage and blastocyst rates were analyzed by chi-square test, and remaining data by ANOVA and Tukey test (P≤0.05. The cleavage (74.8 vs 51.7% and blastocyst (33.7 vs 9.8% rates were greater in oocytes of high versus low quality, with no differences in cell density. Fertilization rates (65.6 to 79.5% were not influenced by pre-incubation time. However, semen pre-incubation during 1.5h increased monospermic penetration (53.3% and cleavage rates (92.5% in low quality oocytes. Blastocyst rate was improved with 1.5h of semen pre incubation; however they were still lower than that observed with high quality control oocytes. Ultimately, pre-incubation did not influence fertilization, monospermic penetration, embryo development rates, nor cell density in oocytes of high quality. Low-quality porcine oocytes resulted in better rates of embryo development if in vitro fertilized with sperm pre-incubated for 1.5 hour.

Expression of Aquaporins in Human Embryos and Potential Role of AQP3 and AQP7 in Preimplantation Mouse Embryo Development

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Yun Xiong

2013-05-01

Full Text Available Background/Aims: Water channels, also named aquaporins (AQPs, play crucial roles in cellular water homeostasis. Methods: RT-PCR indicated the mRNA expression of AQPs 1-5, 7, 9, and 11-12, but not AQPs 0, 6, 8, and 10 in the 2∼8-cell stage human embryos. AQP3 and AQP7 were further analyzed for their mRNA expression and protein expression in the oocyte, zygote, 2-cell embryo, 4-cell embryo, 8-cell embryo, morula, and blastocyst from both human and mouse using RT-PCR and immunofluorescence, respectively. Results: AQP3 and AQP7 were detected in all these stages. Knockdown of either AQP3 or AQP7 by targeted siRNA injection into 2-cell mouse embryos significantly inhibited preimplantation embryo development. However, knockdown of AQP3 in JAr spheroid did not affect its attachment to Ishikawa cells. Conclusion: These data demonstrate that multiple aquaporins are expressed in the early stage human embryos and that AQP3 and AQP7 may play a role in preimplantation mouse embryo development.

Preimplantation genetic diagnosis for chromosomal rearrangements with the use of array comparative genomic hybridization at the blastocyst stage.

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Christodoulou, Christodoulos; Dheedene, Annelies; Heindryckx, Björn; van Nieuwerburgh, Filip; Deforce, Dieter; De Sutter, Petra; Menten, Björn; Van den Abbeel, Etienne

2017-01-01

To establish the value of array comparative genomic hybridization (CGH) for preimplantation genetic diagnosis (PGD) in embryos of translocation carriers in combination with vitrification and frozen embryo transfer in nonstimulated cycles. Retrospective data analysis study. Academic centers for reproductive medicine and genetics. Thirty-four couples undergoing PGD for chromosomal rearrangements from October 2013 to December 2015. Trophectoderm biopsy at day 5 or day 6 of embryo development and subsequently whole genome amplification and array CGH were performed. This approach revealed a high occurrence of aneuploidies and structural rearrangements unrelated to the parental rearrangement. Nevertheless, we observed a benefit in pregnancy rates of these couples. We detected chromosomal abnormalities in 133/207 embryos (64.2% of successfully amplified), and 74 showed a normal microarray profile (35.7%). In 48 of the 133 abnormal embryos (36.1%), an unbalanced rearrangement originating from the parental translocation was identified. Interestingly, 34.6% of the abnormal embryos (46/133) harbored chromosome rearrangements that were not directly linked to the parental translocation in question. We also detected a combination of unbalanced parental-derived rearrangements and aneuploidies in 27 of the 133 abnormal embryos (20.3%). The use of trophectoderm biopsy at the blastocyst stage is less detrimental to the survival of the embryo and leads to a more reliable estimate of the genomic content of the embryo than cleavage-stage biopsy. In this small cohort PGD study, we describe the successful implementation of array CGH analysis of blastocysts in patients with a chromosomal rearrangement to identify euploid embryos for transfer. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

The First Human Cloned Embryo.

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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)

Supplementation with sunflower seeds in beef cattle did not impact on oocyte and in vitro embryo production.

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Baltazar, A L; de Mattos, G M; Ropelli, B M; Firetti, Smg; Castilho, C; Pugliesi, G; Maldonado, Mbc; Binelli, M; Silva, Jof; Lupatini, G C; Lafuente, B S; Membrive, Cmb

2018-06-01

Supplementation with compounds rich in linoleic acid, including sunflower seed supplementation, promotes increase in conception rates in cows. We aimed to evaluate whether the sunflower seed (linoleic acid source) supplementation in beef donor females alters the plasma concentrations of cholesterol, triglycerides, HDL and LDL, increases the number and quality of oocytes, increases the cleavage rates and determines an improvement in number and quality of in vitro produced blastocysts. Thus, Nelore females were divided into two groups of 15 animals to receive supplementation with or without sunflower seed for 57 days. Females underwent follicular aspiration and the oocytes were subjected to in vitro embryo production. There was no difference (p > .1) between control group and group supplemented with sunflower seed on the number of displayed follicles; number of aspired oocytes; recovery rate; cleavage rate; number of embryos; number of blastocysts; embryos number of grades I and II; plasma concentrations of total cholesterol, triglycerides; HDL and LDL. Therefore, sunflower seed supplementation in oocyte donors did not increase the number and quality of oocytes, cleavage rates and the number and quality of blastocysts produced in vitro. © 2018 Blackwell Verlag GmbH.

Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi

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Bell Graham

2005-12-01

Full Text Available Abstract Background Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages. Results We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages. Conclusion Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.

The establishment of polarized membrane traffic in Xenopus laevis embryos.

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Roberts, S J; Leaf, D S; Moore, H P; Gerhart, J C

1992-09-01

Delineation of apical and basolateral membrane domains is a critical step in the epithelialization of the outer layer of cells in the embryo. We have examined the initiation of polarized membrane traffic in Xenopus and show that membrane traffic is not polarized in oocytes but polarized membrane domains appear at first cleavage. The following proteins encoded by injected RNA transcripts were used as markers to monitor membrane traffic: (a) VSV G, a transmembrane glycoprotein preferentially inserted into the basolateral surface of polarized epithelial cells; (b) GThy-1, a fusion protein of VSV G and Thy-1 that is localized to the apical domains of polarized epithelial cells; and (c) prolactin, a peptide hormone that is not polarly secreted. In immature oocytes, there is no polarity in the expression of VSV G or GThy-1, as shown by the constitutive expression of both proteins at the surface in the animal and vegetal hemispheres. At meiotic maturation, membrane traffic to the surface is blocked; the plasma membrane no longer accepts the vesicles synthesized by the oocyte (Leaf, D. L., S. J. Roberts, J. C. Gerhart, and H.-P. Moore. 1990. Dev. Biol. 141:1-12). When RNA transcripts are injected after fertilization, VSV G is expressed only in the internal cleavage membranes (basolateral orientation) and is excluded from the outer surface (apical orientation, original oocyte membrane). In contrast, GThy-1 and prolactin, when expressed in embryos, are inserted or released at both the outer membrane derived from the oocyte and the inner cleavage membranes. Furthermore, not all of the cleavage membrane comes from an embryonic pool of vesicles--some of the cleavage membrane comes from vesicles synthesized during oogenesis. Using prolactin as a marker, we found that a subset of vesicles synthesized during oogenesis was only released after fertilization. However, while embryonic prolactin was secreted from both apical and basolateral surfaces, the secretion of oogenic prolactin

Poor prognosis with in vitro fertilization in Indian women compared to Caucasian women despite similar embryo quality.

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Lora K Shahine

Full Text Available BACKGROUND: Disease prevalence and response to medical therapy may differ among patients of diverse ethnicities. Poor outcomes with in vitro fertilization (IVF treatment have been previously shown in Indian women compared to Caucasian women, and some evidence suggests that poor embryo quality may be a cause for the discrepancy. In our center, only patients with the highest quality cleavage stage embryos are considered eligible for extending embryo culture to the blastocyst stage. We compared live birth rates (LBR between Indian and Caucasian women after blastocyst transfer to investigate whether differences in IVF outcomes between these ethnicities would persist in patients who transferred similar quality embryos. METHODOLOGY/PRINCIPAL FINDINGS: In this retrospective cohort analysis, we compared IVF outcome between 145 Caucasians and 80 Indians who had a blastocyst transfer between January 1, 2005 and June 31, 2007 in our university center. Indians were younger than Caucasians by 2.7 years (34.03 vs. 36.71, P = 0.03, were more likely to have an agonist down regulation protocol (68% vs. 43%, P<0.01, and were more likely to have polycystic ovarian syndrome (PCOS, although not significant, (24% vs. 14%, P = 0.06. Sixty eight percent of Indian patients had the highest quality embryos (4AB blastocyst or better transferred compared to 71% of the Caucasians (P = 0.2. LBR was significantly lower in the Indians compared to the Caucasians (24% vs. 41%, P<0.01 with an odds ratio of 0.63, (95%CI 0.46-0.86. Controlling for age, stimulation protocol and PCOS showed persistently lower LBR with an adjusted odds ratio of 0.56, (95%CI 0.40-0.79 in the multivariate analysis. CONCLUSIONS/SIGNIFICANCE: Despite younger age and similar embryo quality, Indians had a significantly lower LBR than Caucasians. In this preliminary study, poor prognosis after IVF for Indian ethnicity persisted despite limiting analysis to patients with high quality embryos transferred

Recruitment and SNARE-mediated fusion of vesicles in furrow membrane remodeling during cytokinesis in zebrafish embryos

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Ming Liwai; Webb, Sarah E.; Lee, Karen W.; Miller, Andrew L.

2006-01-01

Cytokinesis is the final stage in cell division that serves to partition cytoplasm and daughter nuclei into separate cells. Membrane remodeling at the cleavage plane is a required feature of cytokinesis in many species. In animal cells, however, the precise mechanisms and molecular interactions that mediate this process are not yet fully understood. Using real-time imaging in live, early stage zebrafish embryos, we demonstrate that vesicles labeled with the v-SNARE, VAMP-2, are recruited to the cleavage furrow during deepening in a microtubule-dependent manner. These vesicles then fuse with, and transfer their VAMP-2 fluorescent label to, the plasma membrane during both furrow deepening and subsequent apposition. This observation indicates that new membrane is being inserted during these stages of cytokinesis. Inhibition of SNAP-25 (a cognate t-SNARE of VAMP-2), using a monoclonal antibody, blocked VAMP-2 vesicle fusion and furrow apposition. Transient expression of mutant forms of SNAP-25 also produced defects in furrow apposition. SNAP-25 inhibition by either method, however, did not have any significant effect on furrow deepening. Thus, our data clearly indicate that VAMP-2 and SNAP-25 play an essential role in daughter blastomere apposition, possibly via the delivery of components that promote the cell-to-cell adhesion required for the successful completion of cytokinesis. Our results also support the idea that new membrane addition, which occurs during late stage cytokinesis, is not required for furrow deepening that results from contractile band constriction

The effects of MRI on mouse embryos during fetal stage

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Nakamura, Takashi; Sakazaki, Takahiko; Itokawa, Yuka [Suzuka University of Medical Science, Koriyama (Japan)] (and others)

2006-06-15

The effects of Magnetic Resonance Imaging (MRI) on mouse embryos at the early stage of organogenesis were investigated. Pregnant ICR mice were exposed on day 8 of gestation to MRI at 0.5 T for 0.5 hour to 3 hours. The mortality rates of embryos or fetuses, the incidence of external malformations, fetal body weight and sex ratio were observed at day 18 of gestation. A significant increase in embryonic mortality was observed after exposure to either 0.5 T MRI for 0.5 hour or 2 hours. However, the exposure to MRI for 1 hour or 3 hours did not induce any significant increase in embryonic mortality when compared with control. External malformations such as exencephaly, cleft palate and anomalies of tail were observed in all experimental groups exposed to each MRI. A statistically significant increase of external malformations was observed in all groups treated with 0.5 T MRI for 0.5 hour and 3 hours. The incidence of external malformations in the mice group exposed to 0.5 T MRI for 0.5-hour was found to be higher than those of mice group exposed to 0.5 T MRI for 2 hours. The effects of MRI on the external malformations might not to be dose-dependent. There was no statistically significant difference in fetal body weight and sex ratio among each MRI exposure groups.

Laboratory techniques for human embryos.

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

2002-01-01

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

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

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

2010-01-01

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

Comparing 36.5°C with 37°C for human embryo culture: a prospective randomized controlled trial.

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Fawzy, Mohamed; Emad, Mai; Gad, Mostafa A; Sabry, Mohamed; Kasem, Hesham; Mahmoud, Manar; Bedaiwy, Mohamed A

2018-03-27

This prospective, double-blind, randomized controlled trial was designed to evaluate the efficacy of a culture temperature of 36.5°C versus 37°C on human embryo development in vitro. A total of 412 women undergoing IVF were randomized to two groups: the oocytes and embryos of the intervention group were cultured at 36.5°C; those of the control group were cultured at 37°C. Although no significant effect of culture temperature was observed on pregnancy or implantation rates, differences were found in embryo development. Embryo culture at 36.5°C was associated with a significantly higher cleavage rate (OR 1.6, 95% CI 1.03 to 2.51), but a lower fertilization rate, fewer high-quality embryos on day 3, a lower blastocyst formation rate on day 5, and fewer high-quality and cryopreserved blastocysts (OR 0.87, 95% CI 0.78 to 0.98), (OR 0.60, 95% CI 0.53 to 0.69), (OR 0.85, 95% CI 0.75 to 0.97), (OR 0.5, 95% CI 0.44 to 0.56) and (OR 0.77, 95% CI 0.68 to 0.88), respectively, compared with 37°C. On the basis of these results, and in the absence of data on the optimal temperature for each stage of embryo development in vitro, we recommend continuation of the use of 37°C for human embryo culture. Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Assay using embryo aggregation chimeras for the detection of nonlethal changes in X-irradiated mouse preimplantation embryos

International Nuclear Information System (INIS)

Obasaju, M.F.; Wiley, L.M.; Oudiz, D.J.; Miller, L.; Samuels, S.J.; Chang, R.J.; Overstreet, J.W.

1988-01-01

We have developed a short-term in vitro assay for the detection of sublethal effects produced by very low levels of ionizing radiation. The assay utilizes mouse embryo aggregation chimeras consisting of one irradiated embryo paired with an unirradiated embryo whose blastomeres have been labeled with fluorescein isothiocyanate (FITC). X irradiation (from 0.05 to 2 Gy) and chimera construction were performed with four-cell stage embryos, and the chimeras were cultured for 40 h to the morula stage. The morulae were partially dissociated with calcium-free culture medium and viewed under phase contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution of irradiated (unlabeled) and control (FITC labeled) embryos per chimera. In chimeras where neither embryo was irradiated, the ratio of the unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.50 (17.8 +/- 5.6 cells per unlabeled embryo and 17.4 +/- 5.5 cells per FITC-labeled partner embryo). However, in chimeras formed after the unlabeled embryos were irradiated with as little as 0.05 Gy, the ratio of unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.43 (P less than 0.01). The apparent decreases in cell proliferation were not observed in irradiated embryos that were merely cocultured with control embryos, regardless of whether the embryos were zona enclosed or zona free. We conclude that very low levels of radiation induce sublethal changes in cleaving embryos that are expressed as a proliferative disadvantage within two cell cycles when irradiated embryos are in direct cell-to-cell contact with unirradiated embryos

The Effects of ISM1 Medium on Embryo Quality and Outcomes of IVF/ICSI Cycles.

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Hassani, Fatemeh; Eftekhari-Yazdi, Poopak; Karimian, Leila; Rezazadeh Valojerdi, Mojtaba; Movaghar, Bahar; Fazel, Mohammad; Fouladi, Hamid Reza; Shabani, Fatemeh; Johansson, Lars

2013-07-01

The aim of this study is to investigate the effect of ISM1 culture medium on embryo development, quality and outcomes of in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles. This study compares culture medium commonly used in the laboratory setting for oocyte recovery and embryo development with a medium from MediCult. We have assessed the effects of these media on embryo development and newborn characteristics. In this prospective randomized study, fertilized oocytes from patients were randomly assigned to culture in ISM1 (MediCult, cycles: n=293) or routine lab culture medium (G-1TM v5; Vitrolife, cycles: n=290) according to the daily media schedule for oocyte retrieval. IVF or ICSI and embryo transfer were performed with either MediCult media or routine lab media. Embryo quality on days 2/3, cleavage, pregnancy and implantation rates, baby take home rate (BTHR), in addition to the weight and length of newborns were compared between groups. There were similar cleavage rates for ISM1 (86%) vs. G-1TM v5 (88%). We observed a significantly higher percentage of excellent embryos in ISM1 (42.7%) compared to G-1TM v5 (39%, pISM1 had both higher birth weight (3.03 kg) and length (48.8 cm) compared to G-1TM v5 babies that had a birth weight of 2.66 kg and a length of 46.0 cm (pISM1 is a more effective culture medium in generating higher quality embryos, which may be reflected in the characteristics of babies at birth.

Change in radiosensitivity of sea-urchin eggs during early cleavage stages

International Nuclear Information System (INIS)

Nakamura, I.

1977-01-01

When sea-urchin eggs were irradiated with 137 Cs γ-rays, their radiosensitivity, expressed by the percentage which formed pluteus larvae, fluctuated during the early cleavage cycle. Split-dose irradiations were made both in the sensitive and resistant phases. For eggs in the sensitive phase, the effect of the first exposure of 500 rad was not diminished during the interval before the second exposure. Eggs irradiated in the resistant phase were only slightly damaged. Results implied that fluctuations in radiosensitivity of sea-urchin eggs were caused mainly by different degrees of non-repairable damage in each phase of cleavage rather than by different recovery abilities. (author)

Effect of culture medium volume and embryo density on early mouse embryonic development: tracking the development of the individual embryo.

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Dai, Shan-Jun; Xu, Chang-Long; Wang, Jeffrey; Sun, Ying-Pu; Chian, Ri-Cheng

2012-07-01

To determine the optimal volume or density of embryos for the well-of-the-well (WOW) system in order to track the development of individual embryos and to determine whether the WOW system can reverse the negative impact of culturing embryos singly. (1) Mouse embryos (groups of nine at the 2-cell stage) were cultured in 6.25 μl, 12.50 μl, 25.00 μl and 50.00 μl of droplets of culture medium under paraffin oil; (2) Groups of three, six, nine and twelve embryos at the 2-cell stage were cultured in 50 μl of droplet of culture medium under paraffin oil; (3) Groups of nine embryos at the 2-cell stage were cultured in 50 μl of droplet under paraffin oil with or without nine micro-wells made on the bottom of the Petri dish into each of which were placed one of the nine embryos (WOW system). Also single 2-cell stage embryos was cultured individually in 5.5 μl of droplet of culture medium under paraffin oil with or without a single micro-well made on the bottom of the Petri dish (WOW system for single culture). At the end of culture, the percentages of blastocyst development, hatching and hatched blastocysts were compared in each group. The blastocysts were fixed for differential staining. The blastocyst development was significantly higher (P WOW system. The blastocyst development was not improved when single embryo cultured individually in a micro-well was compared to single embryo cultured individually without micro-well. The total cell numbers of blastocysts were significantly higher in group embryo culture than single embryo culture regardless of whether the WOW system was used. In addition, the total cell numbers of blastocysts were significantly higher (P WOW system than without. Group embryo culture is superior to single embryo culture for blastocyst development. The WOW system with 50 μl of droplet of culture medium can be used to track the individual development of embryo cultured in groups while preserving good embryonic development. The reduced

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

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

2013-01-01

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

Genome-Wide DNA Methylation Patterns of Bovine Blastocysts Developed In Vivo from Embryos Completed Different Stages of Development In Vitro.

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Dessie Salilew-Wondim

Full Text Available Early embryonic loss and altered gene expression in in vitro produced blastocysts are believed to be partly caused by aberrant DNA methylation. However, specific embryonic stage which is sensitive to in vitro culture conditions to alter the DNA methylation profile of the resulting blastocysts remained unclear. Therefore, the aim of this study was to investigate the stage specific effect of in vitro culture environment on the DNA methylation response of the resulting blastocysts. For this, embryos cultured in vitro until zygote (ZY, 4-cell (4C or 16-cell (16C were transferred to recipients and the blastocysts were recovery at day 7 of the estrous cycle. Another embryo group was cultured in vitro until blastocyst stage (IVP. Genome-wide DNA methylation profiles of ZY, 4C, 16C and IVP blastocyst groups were then determined with reference to blastocysts developed completely under in vivo condition (VO using EmbryoGENE DNA Methylation Array. To assess the contribution of methylation changes on gene expression patterns, the DNA methylation data was superimposed to the transcriptome profile data. The degree of DNA methylation dysregulation in the promoter and/or gene body regions of the resulting blastocysts was correlated with successive stages of development the embryos advanced under in vitro culture before transfer to the in vivo condition. Genomic enrichment analysis revealed that in 4C and 16C blastocyst groups, hypermethylated loci were outpacing the hypomethylated ones in intronic, exonic, promoter and proximal promoter regions, whereas the reverse was observed in ZY blastocyst group. However, in the IVP group, as much hypermethylated as hypomethylated probes were detected in gene body and promoter regions. In addition, gene ontology analysis indicated that differentially methylated regions were found to affected several biological functions including ATP binding in the ZY group, programmed cell death in the 4C, glycolysis in 16C and genetic

Nucleolar ultrastructure in bovine nuclear transfer embryos

DEFF Research Database (Denmark)

Kanka, J; Smith, S D; Soloy, E

1999-01-01

in nuclear morphology as a transformation of the nucleolus precursor body into a functional rRNA synthesising nucleolus with a characteristic ultrastructure. We examined nucleolar ultrastructure in bovine in vitro produced (control) embryos and in nuclear transfer embryos reconstructed from a MII phase...... at 1 hr after fusion and, by 3 hr after fusion, it was restored again. At this time, the reticulated fibrillo-granular nucleolus had an almost round shape. The nucleolar precursor body seen in the two-cell stage nuclear transfer embryos consisted of intermingled filamentous components and secondary...... time intervals after fusion. In the two-cell stage nuclear transfer embryo, the originally reticulated nucleolus of the donor blastomere had changed into a typical nucleolar precursor body consisting of a homogeneous fibrillar structure. A primary vacuole appeared in the four-cell stage nuclear...

Glassfrog embryos hatch early after parental desertion.

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Delia, Jesse R J; Ramírez-Bautista, Aurelio; Summers, Kyle

2014-06-22

Both parental care and hatching plasticity can improve embryo survival. Research has found that parents can alter hatching time owing to a direct effect of care on embryogenesis or via forms of care that cue the hatching process. Because parental care alters conditions critical for offspring development, hatching plasticity could allow embryos to exploit variation in parental behaviour. However, this interaction of parental care and hatching plasticity remains largely unexplored. We tested the hypothesis that embryos hatch early to cope with paternal abandonment in the glassfrog Hyalinobatrachium fleischmanni (Centrolenidae). We conducted male-removal experiments in a wild population, and examined embryos' response to conditions with and without fathers. Embryos hatched early when abandoned, but extended development in the egg stage when fathers continued care. Paternal care had no effect on developmental rate. Rather, hatching plasticity was due to embryos actively hatching at different developmental stages, probably in response to deteriorating conditions without fathers. Our experimental results are supported by a significant correlation between the natural timing of abandonment and hatching in an unmanipulated population. This study demonstrates that embryos can respond to conditions resulting from parental abandonment, and provides insights into how variation in care can affect selection on egg-stage adaptations.

Glassfrog embryos hatch early after parental desertion

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Delia, Jesse R. J.; Ramírez-Bautista, Aurelio; Summers, Kyle

2014-01-01

Both parental care and hatching plasticity can improve embryo survival. Research has found that parents can alter hatching time owing to a direct effect of care on embryogenesis or via forms of care that cue the hatching process. Because parental care alters conditions critical for offspring development, hatching plasticity could allow embryos to exploit variation in parental behaviour. However, this interaction of parental care and hatching plasticity remains largely unexplored. We tested the hypothesis that embryos hatch early to cope with paternal abandonment in the glassfrog Hyalinobatrachium fleischmanni (Centrolenidae). We conducted male-removal experiments in a wild population, and examined embryos' response to conditions with and without fathers. Embryos hatched early when abandoned, but extended development in the egg stage when fathers continued care. Paternal care had no effect on developmental rate. Rather, hatching plasticity was due to embryos actively hatching at different developmental stages, probably in response to deteriorating conditions without fathers. Our experimental results are supported by a significant correlation between the natural timing of abandonment and hatching in an unmanipulated population. This study demonstrates that embryos can respond to conditions resulting from parental abandonment, and provides insights into how variation in care can affect selection on egg-stage adaptations. PMID:24789892

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

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

1984-11-01

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

Effect of cryopreservation and in vitro culture of bovine fibroblasts on histone acetylation levels and in vitro development of hand-made cloned embryos

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Chacon, L.; Gomez, M.C.; Jenkins, J.A.; Leibo, S.P.; Wirtu, G.; Dresser, B.L.; Pope, C.E.

2011-01-01

In this study, the relative acetylation levels of histone 3 in lysine 9 (H3K9ac) in cultured and cryopreserved bovine fibroblasts was measured and we determined the influence of the epigenetic status of three cultured (C1, C2 and C3) donor cell lines on the in vitro development of reconstructed bovine embryos. Results showed that cryopreservation did not alter the overall acetylation levels of H3K9 in bovine fibroblasts analysed immediately after thawing (frozen/thawed) compared with fibroblasts cultured for a period of time after thawing. However, reduced cleavage rates were noted in embryos reconstructed with fibroblasts used immediately after thawing. Cell passage affects the levels of H3K9ac in bovine fibroblasts, decreasing after P1 and donor cells with lower H3K9ac produced a greater frequency of embryo development to the blastocyst stage. Cryopreservation did not influence the total cell and ICM numbers, or the ICM/TPD ratios of reconstructed embryos. However, the genetic source of donor cells did influence the total number of cells and the trophectoderm cell numbers, and the cell passage influenced the total ICM cell numbers. ?? Copyright Cambridge University Press 2010.

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

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

2014-04-01

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

A cutin fluorescence pattern in developing embryos of some angiosperms

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Ewa Szczuka

2014-01-01

Full Text Available A cuticle visualized by auramine O fluorescence appears on the developing embryos of 9 species belonging to Cruciferae, Caryophyllaceae, Plantaginaceae, Linaceae and Papilionaceae. In the investigated species the formation and extent of fluorescing and non-fluorescing embryonic areas follow a similar pattern. At first the cutin fluorescing layer is formed on the apical part of the proembryo without delimited protoderm. This layer extends and at the late globular stage envelops the embryo proper, except for a cell adjoining the suspensor. Fluorescing cutin persists during the heart stage but disappears from the torpedo embryo. During these stages there is no cutine fluorescence on suspensorial cells. Continuous cutin fluorescence appears again on the surface of the whole embryo by the late torpedo stage. Then fluorescence disappears from the radicular part of U-shaped embryos, but persists on the shoot apex, cotyledons and at least on the upper part of hypocotyl. It is assumed that polarization and nutrition of the embryo may be influenced by cuticular changes.

The Early Stages of Heart Development: Insights from Chicken Embryos

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Johannes G. Wittig

2016-04-01

Full Text Available The heart is the first functioning organ in the developing embryo and a detailed understanding of the molecular and cellular mechanisms involved in its formation provides insights into congenital malformations affecting its function and therefore the survival of the organism. Because many developmental mechanisms are highly conserved, it is possible to extrapolate from observations made in invertebrate and vertebrate model organisms to humans. This review will highlight the contributions made through studying heart development in avian embryos, particularly the chicken. The major advantage of chick embryos is their accessibility for surgical manipulation and functional interference approaches, both gain- and loss-of-function. In addition to experiments performed in ovo, the dissection of tissues for ex vivo culture, genomic, or biochemical approaches is straightforward. Furthermore, embryos can be cultured for time-lapse imaging, which enables tracking of fluorescently labeled cells and detailed analysis of tissue morphogenesis. Owing to these features, investigations in chick embryos have led to important discoveries, often complementing genetic studies in mice and zebrafish. As well as including some historical aspects, we cover here some of the crucial advances made in understanding early heart development using the chicken model.

Peptone and tomato extract induced early stage of embryo development of Dendrobium phalaenopsis Orchid

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Nintya Setiari

2017-04-01

Full Text Available Germination and growth of orchid seeds can be accelerated by the addition of organic supplement and plant extract in culture medium. The objective of this study was to determine the effect of peptone and tomato extract on early stage of embryo development of Dendrobium phalaenopsis orchids. Orchid seeds were sown on NP and VW medium with addition of 10% of CW (NPCW and VWCW.  Five weeks after seed germination, about 58.03% seed germination was observed on VWCW medium, and only 37.45% seed germination on NPCW. Tomato extract and peptone were added in VWCW, resulting VWCWTP medium. After 4-8 weeks on VWCWTP, 94.42% seeds was germinated into plantlet, but only 67.30% germinated seeds on VWCW. To get optimal growth and development of  D.  phalaenopsis orchids embryos in the in vitro condition, supplement of 100 ml.L-1 coconut water, 100 mg.L-1 tomato extract and 2 mg.L-1 peptone into VW basic medium is required.

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

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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.

Embryo selection: the role of time-lapse monitoring.

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Kovacs, Peter

2014-12-15

In vitro fertilization has been available for over 3 decades. Its use is becoming more widespread worldwide, and in the developed world, up to 5% of children have been born following IVF. It is estimated that over 5 million children have been conceived in vitro. In addition to giving hope to infertile couples to have their own family, in vitro fertilization has also introduced risks as well. The risk of multiple gestation and the associated maternal and neonatal morbidity/mortality has increased significantly over the past few decades. While stricter transfer policies have eliminated the majority of the high-order multiples, these changes have not yet had much of an impact on the incidence of twins. A twin pregnancy can be avoided by the transfer of a single embryo only. However, the traditionally used method of morphologic embryo selection is not predictive enough to allow routine single embryo transfer; therefore, new screening tools are needed. Time-lapse embryo monitoring allows continuous, non-invasive embryo observation without the need to remove the embryo from optimal culturing conditions. The extra information on the cleavage pattern, morphologic changes and embryo development dynamics could help us identify embryos with a higher implantation potential. These technologic improvements enable us to objectively select the embryo(s) for transfer based on certain algorithms. In the past 5-6 years, numerous studies have been published that confirmed the safety of time-lapse technology. In addition, various markers have already been identified that are associated with the minimal likelihood of implantation and others that are predictive of blastocyst development, implantation potential, genetic health and pregnancy. Various groups have proposed different algorithms for embryo selection based on mostly retrospective data analysis. However, large prospective trials are needed to study the full benefit of these (and potentially new) algorithms before their

Regional differences in the lateral mobility of plasma membrane lipids in a molluscan embryo

NARCIS (Netherlands)

Speksnijder, J.E.; Dohmen, M.R.; Tertoolen, L.G.J.; Laat, S.W. de

1985-01-01

Regional and temporal differences in plasma membrane lipid mobility have been analyzed during the first three cleavage cycles of the embryo of the polar-lobe-forming mollusc Nassarius reticulatus by the fluorescence photobleaching recovery (FPR) method, using 1,1′-ditetradecyl

Culture medium composition affects the gene expression pattern and in vitro development potential of bovine somatic cell nuclear transfer (SCNT) embryos.

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Arias, María E; Ross, Pablo J; Felmer, Ricardo N

2013-01-01

Different culture systems have been studied that support development of somatic cell nuclear transfer (SCNT) embryos up to the blastocyst stage. However, the use of sequential and two-step culture systems has been less studied. The objective of the present study was to examine the developmental potential and quality of bovine SCNT embryos cultured in different two-step culture media based on KSOM, SOF and the macromolecules FBS and BSA (K-K/FBS, K-S/BSA and K-K/BSA, respectively). No differences were observed in the cleavage rate for any of the culture systems. However, there was a significant difference (Pculture system yielding a higher rate of blastocysts (28%) compared to other treatments (18 and 15%, for K-S/BSA and K-K/BSA, respectively). Although quality of embryos, as assessed by the total number of cells, was not different, the apoptosis index was significantly affected in the sequential culture system (K-S/BSA). Gene expression analysis showed alterations of DNMT1, IGF2, LIF, and PRDX6 genes in embryos cultured in K-S/FBS and of SOD2 in embryos cultured in K-K/BSA. In conclusion, we demonstrated that culture medium may affect not only the developmental potential of SCNT embryos but also, more importantly, the gene expression pattern and apoptotic index, presenting the possibility to manipulate the culture medium composition to modulate global gene expression and improve the overall efficiency of this technique.

Somatic Embryos in Catharanthus roseus: A Scanning Electron Microscopic Study

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Junaid ASLAM

2014-06-01

Full Text Available Catharanthus roseus (L. G. Don is an important medicinal plant as it contains several anti-cancerous compounds, like vinblastine and vincristine. Plant tissue culture technology (organogenesis and embryogenesis has currently been used in fast mass propagating raw materials for secondary metabolite synthesis. In this present communication, scanning electron microscopic (SEM study of somatic embryos was conducted and discussed. The embryogenic callus was first induced from hypocotyls of in vitro germinated seeds on which somatic embryos, differentiated in numbers, particularly on 2,4-D (1.0 mg/L Murashige and Skoog (MS was medium. To understand more about the regeneration method and in vitro formed embryos SEM was performed. The SEM study revealed normal somatic embryo origin and development from globular to heart-, torpedo- and then into cotyledonary-stage of embryos. At early stage, the embryos were clustered together in a callus mass and could not easily be detached from the parental tissue. The embryos were often long cylindrical structure with or without typical notch at the tip. Secondary embryos were also formed on primary embryo structure. The advanced cotyledonary embryos showed prominent roots and shoot axis, which germinated into plantlets. The morphology, structure and other details of somatic embryos at various stages were presented.

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

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

2011-10-01

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

Action of uranium on pre implanted mouse embryos

International Nuclear Information System (INIS)

Kundt, Miriam S.

2001-01-01

The cultured preimplantation embryos are normally employed to evaluate the effects of environmental pollutants specially metals. Embryos were obtained from hybrid females CBA x C57 Bl following induction of super ovulation. They were incubated from 1 cell stage during 120 hs. in M16 cultured medium. Three different experiments were carried out: A, B and C using uranyl nitrate UO 2 (NO 3 ) 2 6H 2 O as source of uranium. In experiment 'A' the embryos were cultivated in the same culture dish containing final U concentrations of 13, 26, 52, 104 and 208 μgU/ml. In experiment 'B' embryos in a one cell stage were placed in culture medium with uranyl nitrate with final U concentrations of 26, 52, 104 μgU/ml. After 24 hours those embryos which had reached the two-cell stage were transferred to another culture dish to which fresh solutions of uranyl nitrate were added, maintaining the same concentrations of the previous one. In experiment 'C' the embryos were cultivated containing final U concentrations of 26, 52 and 104 μgU/ml and they were transferred to another culture dish every day to which fresh solutions of uranyl nitrate were added. Different embryos parameters were analyzed: 1) Development grade; 2) Number of cell per embryo and metaphases index; and 3) Embryo ploidy. 1) Embryos were observed each 24 hs. to evaluate development grade: 2, 4 and 8 cell stage, morula, early -expanded- hatched blastocysts and atresic embryos. No significant differences were observed in the proportion of embryos arrested either in the one-cell or in the two cell stages in control culture medium regarding different concentrations of U, in a total of 4388 embryos analyzed. From 2 cell stage, moment that the embryo begins to synthesize its own ARNm, the delay in embryonic development increased dose dependent. On the other hand, the toxicological effects in the same concentration are increase from 'A' treatment to 'C' treatment. Embriotoxicology effects are evidenced by an increment in

Nuclear reprogramming: kinetics of cell cycle and metabolic progression as determinants of success.

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Sebastian Thomas Balbach

Full Text Available Establishment of totipotency after somatic cell nuclear transfer (NT requires not only reprogramming of gene expression, but also conversion of the cell cycle from quiescence to the precisely timed sequence of embryonic cleavage. Inadequate adaptation of the somatic nucleus to the embryonic cell cycle regime may lay the foundation for NT embryo failure and their reported lower cell counts. We combined bright field and fluorescence imaging of histone H(2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This allowed us to quantitatively analyze cleavage kinetics of cloned embryos and revealed an extended and inconstant duration of the second and third cell cycles compared to fertilized controls generated by intracytoplasmic sperm injection (ICSI. Compared to fertilized embryos, slow and fast cleaving NT embryos presented similar rates of errors in M phase, but were considerably less tolerant to mitotic errors and underwent cleavage arrest. Although NT embryos vary substantially in their speed of cell cycle progression, transcriptome analysis did not detect systematic differences between fast and slow NT embryos. Profiling of amino acid turnover during pre-implantation development revealed that NT embryos consume lower amounts of amino acids, in particular arginine, than fertilized embryos until morula stage. An increased arginine supplementation enhanced development to blastocyst and increased embryo cell numbers. We conclude that a cell cycle delay, which is independent of pluripotency marker reactivation, and metabolic restraints reduce cell counts of NT embryos and impede their development.

An Elk transcription factor is required for Runx-dependent survival signaling in the sea urchin embryo.

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Rizzo, Francesca; Coffman, James A; Arnone, Maria Ina

2016-08-01

Elk proteins are Ets family transcription factors that regulate cell proliferation, survival, and differentiation in response to ERK (extracellular-signal regulated kinase)-mediated phosphorylation. Here we report the embryonic expression and function of Sp-Elk, the single Elk gene of the sea urchin Strongylocentrotus purpuratus. Sp-Elk is zygotically expressed throughout the embryo beginning at late cleavage stage, with peak expression occurring at blastula stage. Morpholino antisense-mediated knockdown of Sp-Elk causes blastula-stage developmental arrest and embryo disintegration due to apoptosis, a phenotype that is rescued by wild-type Elk mRNA. Development is also rescued by Elk mRNA encoding a serine to aspartic acid substitution (S402D) that mimics ERK-mediated phosphorylation of a conserved site that enhances DNA binding, but not by Elk mRNA encoding an alanine substitution at the same site (S402A). This demonstrates both that the apoptotic phenotype of the morphants is specifically caused by Elk depletion, and that phosphorylation of serine 402 of Sp-Elk is critical for its anti-apoptotic function. Knockdown of Sp-Elk results in under-expression of several regulatory genes involved in cell fate specification, cell cycle control, and survival signaling, including the transcriptional regulator Sp-Runt-1 and its target Sp-PKC1, both of which were shown previously to be required for cell survival during embryogenesis. Both Sp-Runt-1 and Sp-PKC1 have sequences upstream of their transcription start sites that specifically bind Sp-Elk. These results indicate that Sp-Elk is the signal-dependent activator of a feed-forward gene regulatory circuit, consisting also of Sp-Runt-1 and Sp-PKC1, which actively suppresses apoptosis in the early embryo. Copyright © 2016 Elsevier Inc. All rights reserved.

Human developmental anatomy: microscopic magnetic resonance imaging (μMRI) of four human embryos (from Carnegie Stage 10 to 20).

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Lhuaire, Martin; Martinez, Agathe; Kaplan, Hervé; Nuzillard, Jean-Marc; Renard, Yohann; Tonnelet, Romain; Braun, Marc; Avisse, Claude; Labrousse, Marc

2014-12-01

Technological advances in the field of biological imaging now allow multi-modal studies of human embryo anatomy. The aim of this study was to assess the high magnetic field μMRI feasibility in the study of small human embryos (less than 21mm crown-rump) as a new tool for the study of human descriptive embryology and to determine better sequence characteristics to obtain higher spatial resolution and higher signal/noise ratio. Morphological study of four human embryos belonging to the historical collection of the Department of Anatomy in the Faculty of Medicine of Reims was undertaken by μMRI. These embryos had, successively, crown-rump lengths of 3mm (Carnegie Stage, CS 10), 12mm (CS 16), 17mm (CS 18) and 21mm (CS 20). Acquisition of images was performed using a vertical nuclear magnetic resonance spectrometer, a Bruker Avance III, 500MHz, 11.7T equipped for imaging. All images were acquired using 2D (transverse, sagittal and coronal) and 3D sequences, either T1-weighted or T2-weighted. Spatial resolution between 24 and 70μm/pixel allowed clear visualization of all anatomical structures of the embryos. The study of human embryos μMRI has already been reported in the literature and a few atlases exist for educational purposes. However, to our knowledge, descriptive or morphological studies of human developmental anatomy based on data collected these few μMRI studies of human embryos are rare. This morphological noninvasive imaging method coupled with other techniques already reported seems to offer new perspectives to descriptive studies of human embryology.

Developmental anatomy of the liver from computerized three-dimensional reconstructions of four human embryos (from Carnegie stage 14 to 23).

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Lhuaire, Martin; Tonnelet, Romain; Renard, Yohann; Piardi, Tullio; Sommacale, Daniele; Duparc, Fabrice; Braun, Marc; Labrousse, Marc

2015-07-01

Some aspects of human embryogenesis and organogenesis remain unclear, especially concerning the development of the liver and its vasculature. The purpose of this study was to investigate, from a descriptive standpoint, the evolutionary morphogenesis of the human liver and its vasculature by computerized three-dimensional reconstructions of human embryos. Serial histological sections of four human embryos at successive stages of development belonging to three prestigious French historical collections were digitized and reconstructed in 3D using software commonly used in medical radiology. Manual segmentation of the hepatic anatomical regions of interest was performed section by section. In this study, human liver organogenesis was examined at Carnegie stages 14, 18, 21 and 23. Using a descriptive and an analytical method, we showed that these stages correspond to the implementation of the large hepatic vascular patterns (the portal system, the hepatic artery and the hepatic venous system) and the biliary system. To our knowledge, our work is the first descriptive morphological study using 3D computerized reconstructions from serial histological sections of the embryonic development of the human liver between Carnegie stages 14 and 23. Copyright © 2015 Elsevier GmbH. All rights reserved.

Melatonin improves the quality of in vitro produced (IVP bovine embryos: implications for blastocyst development, cryotolerance, and modifications of relevant gene expression.

Directory of Open Access Journals (Sweden)

Feng Wang

Full Text Available To evaluate the potential effects of melatonin on the kinetics of embryo development and quality of blastocyst during the process of in vitro bovine embryo culture. Bovine cumulus-oocyte complexes (COCs were fertilized after in vitro maturation. The presumed zygotes were cultured in in vitro culture medium supplemented with or without 10(-7 M melatonin. The cleavage rate, 8-cell rate and blastocyst rate were examined to identify the kinetics of embryo development. The hatched blastocyst rate, mortality rate after thawing and the relevant transcript abundance were measured to evaluate the quality of blastocyst. The results showed that melatonin significantly promoted the cleavage rate and 8-cell embryo yield of in vitro produced bovine embryo. In addition, significantly more blastocysts were observed by Day 7 of embryo culture at the presence of melatonin. These results indicated that melatonin accelerated the development of in vitro produced bovine embryos. Following vitrification at Day 7 of embryo culture, melatonin (10(-7 M significantly increased the hatched blastocyst rate from 24 h to 72 h and decreased the mortality rate from 48 h to 72 h after thawing. The presence of melatonin during the embryo culture resulted in a significant increase in the gene expressions of DNMT3A, OCC, CDH1 and decrease in that of AQP3 after thawing. In conclusion, melatonin not only promoted blastocyst yield and accelerated in vitro bovine embryo development, but also improved the quality of blastocysts which was indexed by an elevated cryotolerance and the up-regulated expressions of developmentally important genes.

The transcription factor FoxB mediates temporal loss of cellular competence for notochord induction in ascidian embryos.

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Hashimoto, Hidehiko; Enomoto, Takashi; Enomoto, Atsushi; Kumano, Gaku; Nishida, Hiroki

2011-06-01

In embryos of the ascidian Halocynthia roretzi, the competence of isolated presumptive notochord blastomeres to respond to fibroblast growth factor (FGF) for induction of the primary notochord decays by 1 hour after cleavage from the 32- to 64-cell stage. This study analyzes the molecular mechanisms responsible for this loss of competence and provides evidence for a novel mechanism. A forkhead family transcription factor, FoxB, plays a role in competence decay by preventing the induction of notochord-specific Brachyury (Bra) gene expression by the FGF/MAPK signaling pathway. Unlike the mechanisms reported previously in other animals, no component in the FGF signal transduction cascade appeared to be lost or inactivated at the time of competence loss. Knockdown of FoxB functions allowed the isolated cells to retain their competence for a longer period, and to respond to FGF with expression of Bra beyond the stage at which competence was normally lost. FoxB acts as a transcription repressor by directly binding to the cis-regulatory element of the Bra gene. Our results suggest that FoxB prevents ectopic induction of the notochord fate within the cells that assume a default nerve cord fate, after the stage when notochord induction has been completed. The merit of this system is that embryos can use the same FGF signaling cascade again for another purpose in the same cell lineage at later stages by keeping the signaling cascade itself available. Temporally and spatially regulated FoxB expression in nerve cord cells was promoted by the ZicN transcription factor and absence of FGF/MAPK signaling.

Changes in protein synthetic activity in early Drosophila embryos mutant for the segmentation gene Krueppel

International Nuclear Information System (INIS)

Bedian, V.; Summers, M.C.; Kauffman, S.A.

1988-01-01

We have identified early embryo proteins related to the segmentation gene Krueppel by [35S]methionine pulse labelling and two-dimensional gel electrophoresis. Protein synthesis differences shared by homozygous embryos of two Krueppel alleles when compared to heterozygous and wild-type embryos are reported. The study was extended to syncytial blastoderm stages by pulse labelling and gel analysis of single embryos, using Krueppel-specific proteins from gastrula stages as molecular markers for identifying homozygous Krueppel embryos. Localized expression of interesting proteins was examined in embryo fragments. The earliest differences detected at nuclear migration stages showed unregulated synthesis in mutant embryos of two proteins that have stage specific synthesis in normal embryos. At the cellular blastoderm stage one protein was not synthesized and two proteins showed apparent shifts in isoelectric point in mutant embryos. Differences observed in older embryos included additional proteins with shifted isoelectric points and a number of qualitative and quantitative changes in protein synthesis. Five of the proteins with altered rates of synthesis in mutant embryos showed localized synthesis in normal embryos. The early effects observed are consistent with the hypothesis that the Krueppel product can be a negative or positive regulator of expression of other loci, while blastoderm and gastrula stage shifts in isoelectric point indicate that a secondary effect of Krueppel function may involve post-translational modification of proteins

Birth of cloned calves from vitrified-warmed zona-free buffalo (Bubalus bubalis) embryos produced by hand-made cloning.

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Saha, Ambikaprasanna; Panda, Sudeepta K; Chauhan, Manmohan S; Manik, Radhey S; Palta, Prabhat; Singla, Suresh K

2013-01-01

The availability of techniques for the vitrification of cloned blastocysts can improve their effective use. The present study compared the developmental competence of buffalo cloned embryos derived from adult (BAF), newborn (BNF) and fetal fibroblast (BFF) before and after vitrification. Despite similar cleavage rates among the three groups, the blastocyst rate was lower for BAF- than BNF- and BFF-derived embryos (30.2±2.2% vs 41.7±1.7% and 39.1±2.1%, respectively; Pcloned buffalo embryos cryopreserved by vitrification can be used to obtain live offspring.

Computer ranking of the sequence of appearance of 73 features of the brain and related structures in staged human embryos during the sixth week of development.

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

1987-09-01

The sequence of events in the development of the brain in human embryos, already published for stages 8-15, is here continued for stages 16 and 17. With the aid of a computerized bubble-sort algorithm, 71 individual embryos were ranked in ascending order of the features present. Whereas these numbered 100 in the previous study, the increasing structural complexity gave 27 new features in the two stages now under investigation. The chief characteristics of stage 16 (approximately 37 postovulatory days) are protruding basal nuclei, the caudal olfactory elevation (olfactory tubercle), the tectobulbar tracts, and ascending fibers to the cerebellum. The main features of stage 17 (approximately 41 postovulatory days) are the cortical nucleus of the amygdaloid body, an intermediate layer in the tectum mesencephali, the posterior commissure, and the habenulo-interpeduncular tract. In addition, a typical feature at stage 17 is the crescentic shape of the lens cavity.

Culture medium composition affects the gene expression pattern and in vitro development potential of bovine somatic cell nuclear transfer (SCNT embryos

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María E Arias

2013-01-01

Full Text Available Different culture systems have been studied that support development of somatic cell nuclear transfer (SCNT embryos up to the blastocyst stage. However, the use of sequential and two-step culture systems has been less studied. The objective of the present study was to examine the developmental potential and quality of bovine SCNT embryos cultured in different two-step culture media based on KSOM, SOF and the macromolecules FBS and BSA (K-K/FBS, K-S/BSA and K-K/BSA, respectively. No differences were observed in the cleavage rate for any of the culture systems. However, there was a significant difference (P<0.01 in the rate of blastocyst development, with the K-K/ FBS culture system yielding a higher rate of blastocysts (28% compared to other treatments (18 and 15%, for K-S/BSA and K-K/BSA, respectively. Although quality of embryos, as assessed by the total number of cells, was not different, the apoptosis index was significantly affected in the sequential culture system (K-S/BSA. Gene expression analysis showed alterations of DNMT1, IGF2, LIF, and PRDX6 genes in embryos cultured in K-S/FBS and of SOD2 in embryos cultured in K-K/BSA. In conclusion, we demonstrated that culture medium may affect not only the developmental potential of SCNT embryos but also, more importantly, the gene expression pattern and apoptotic index, presenting the possibility to manipulate the culture medium composition to modulate global gene expression and improve the overall efficiency of this technique.

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

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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.

Live birth sex ratio after in vitro fertilization and embryo transfer in China--an analysis of 121,247 babies from 18 centers.

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Bu, Zhiqin; Chen, Zi-Jiang; Huang, Guoning; Zhang, Hanwang; Wu, Qiongfang; Ma, Yanping; Shi, Juanzi; Xu, Yanwen; Zhang, Songying; Zhang, Cuilian; Zhao, Xiaoming; Zhang, Bo; Huang, Yuanhua; Sun, Zhengyi; Kang, Yuefan; Wu, Riran; Wu, Xueqing; Sun, Haixiang; Sun, Yingpu

2014-01-01

In order to study the impact of procedures of IVF/ICSI technology on sex ratio in China, we conducted this multi-center retrospective study including 121,247 babies born to 93,895 women in China. There were 62,700 male babies and 58,477 female babies, making the sex ratio being 51.8% (Male: Female  = 107:100). In univariate logistic regression analysis, sex ratio was imbalance toward females of 50.3% when ICSI was preformed compared to 47.7% when IVF was used (Pratio in IVF/ICSI babies was significantly higher toward males in transfers of blastocyst (54.9%) and thawed embryo (52.4%) when compared with transfers of cleavage stage embryo (51.4%) and fresh embryo (51.5%), respectively. Multiple delivery was not associated with sex ratio. However, in multivariable logistic regression analysis after controlling for related factors, only ICSI (adjusted OR =  .90, 95%CI: 0.88-0.93; Pratio in IVF/ICSI babies. In conclusion, the live birth sex ratio in IVF/ICSI babies was influenced by the use of ICSI, which may decrease the percentage of male offspring, or the use of blastocyst transfer, which may increase the percentage of male offspring.

In Vivo Quantitative Study of Sized-Dependent Transport and Toxicity of Single Silver Nanoparticles Using Zebrafish Embryos

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Lee, Kerry J.; Browning, Lauren M.; Nallathamby, Prakash D.; Desai, Tanvi; Cherukui, Pavan K.; Xu, Xiao-Hong Nancy

2012-01-01

Nanomaterials possess distinctive physicochemical properties (e.g., small sizes, high surface area-to-volume ratios) and promise a wide variety of applications, ranging from design of high quality consumer products to effective disease diagnosis and therapy. These properties can lead to toxic effects, potentially hindering advance in nanotechnology. In this study, we have synthesized and characterized purified and stable (non-aggregation) silver nanoparticles (Ag NPs, 41.6±9.1 nm in average diameters), and utilized early-developing (cleavage-stage) zebrafish embryos (critical aquatic and eco- species) as in vivo model organisms to probe diffusion and toxicity of Ag NPs. We found that single Ag NPs (30–72 nm diameters) passively diffused into the embryos through chorionic pores via random Brownian motion and stayed inside the embryos throughout their entire development (120 hours-post-fertilization, hpf). Dose and size dependent toxic effects of the NPs on embryonic development were observed, showing the possibility of tuning biocompatibility and toxicity of the NPs. At lower concentrations of the NPs (≤ 0.02 nM), 75–91% of embryos developed to normal zebrafish. At the higher concentrations of NPs (≥ 0.20 nM), 100% of embryos became dead. At the concentrations in between (0.02–0.2 nM), embryos developed to various deformed zebrafish. Number and sizes of individual Ag NPs embedded in tissues of normal and deformed zebrafish at 120 hpf were quantitatively analyzed, showing deformed zebrafish with higher number of larger NPs than normal zebrafish, and size-dependent nanotoxicity. By comparing with our previous studies of smaller Ag NPs (11.6±3.5 nm), the results further demonstrate striking size-dependent nanotoxicity that, at the same molar concentration, the larger Ag NPs (41.6±9.1 nm) are more toxic than the smaller Ag NPs (11.6±3.5 nm). PMID:22486336

Alternation of apoptotic and implanting genes expression of mouse embryos after re-vitrification

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Majidi Gharenaz, Nasrin; Movahedin, Mansoureh; Mazaheri, Zohreh; Pour beiranvand, Shahram

2016-01-01

Background: Nowadays, oocytes and embryos vitrification has become a routine technique. Based on clinical judgment, re-vitrification maybe required. But little is known about re-vitrification impact on genes expression. Objective: The impact of re-vitrification on apoptotic and implanting genes, Bax, Bcl-2 and ErbB4, at compaction stage embryos were evaluated in this study. Materials and Methods: In this experimental study, 8 cell embryos (n=240) were collected from female mature mice, 60-62 hr post HCG injection. The embryos were divided randomly to 3 groups included: fresh (n=80), vitrified at 8 cell stage (n=80), vitrified at 8 cell stage thawed and re-vitrified at compaction stage (n=80). Embryos were vitrified by using cryolock, (open system) described by Kuwayama. Q-PCR was used to examine the expression of Bax, Bcl2 ErbB4 genes in derived blastocysts. Results: Our result showed that expanded blastocyst rate was similar between vitrified and re-vitrified groups, while re-vitrified embryos showed significant decrease in expanded blastocyst rate comparing with fresh embryos (p=0.03). In addition, significant difference was observed on apoptotic gene expression when comparing re-vitrified and fresh embryos (p=0.004), however expression of Bax and Bcl-2 (apoptotic) genes didn't demonstrate a significant difference between re-vitrified and vitrified groups. The expression rate of ErbB4, an implantation gene was decreased in re-vitrified embryos comparing with fresh embryos (p=0.003), but it was similar between re-vitrified and vitrified embryos. Conclusion: Re-vitrification can alter the expression of Bax, Bcl-2 and ErbB4 genes and developmental rate of mouse embryos in compaction stage. PMID:27679826

Radiosensitivity of Bombyx mori embryos and its modification by thermal shock

International Nuclear Information System (INIS)

Agaev, F.A.; Zakrzhevskaya, D.T.; Yusifov, N.I.; Gaziev, A.I.; AN Azerbajdzhanskoj SSR, Baku

1991-01-01

Radiosensitivity of Bombyx mori embryos on days 3-4 of their development is more than 10 times higher than that of 7-9 day embryos. The rate of DNA synthesis in the embryos correlates with their radiosensitivity. Heat treatment (40 deg C, 60 min) of embryos just before γ-irradiation increases their radioresistance (DMF=+1.6), whereas such a treatment immediately after irradiation reduces the survival rate of embryos as compared to the controls irradiated without heat treatment (DMA=-1.5). The radiomodifying effect of the thermal shock on the Bombyx mori embryos is the same with exposure at both the radioresistant and the radiosensitive stage of their development. However, it is more pronounced at the radiosensitive stage

Developmental toxicity and oxidative stress induced by gamma irradiation in zebrafish embryos.

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Hu, Miao; Hu, Nan; Ding, Dexin; Zhao, Weichao; Feng, Yongfu; Zhang, Hui; Li, Guangyue; Wang, Yongdong

2016-11-01

This study aimed to evaluate the biological effects of gamma irradiation on zebrafish embryos. Different doses of gamma rays (0.01, 0.05, 0.1, 0.5 and 1 Gy) were used to irradiate zebrafish embryos at three developmental stages (stage 1, 6 h post-fertilization (hpf); stage 2, 12 hpf; stage three, 24 hpf), respectively. The survival, malformation and hatching rates of the zebrafish embryos were measured at the morphological endpoint of 96 hpf. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione S-transferase (GST) were assayed. Morphology analysis showed that gamma irradiation inhibited hatching and induced developmental toxicity in a dose-dependent manner. Interestingly, after irradiation the malformation rate changed not only in a dose-dependent manner but also in a developmental stage-dependent manner, indicating that the zebrafish embryos at stage 1 were more sensitive to gamma rays than those at other stages. Biochemical analysis showed that gamma irradiation modulated the activities of antioxidant enzymes in a dose-dependent manner. A linear relationship was found between GPx activity and irradiation dose in 0.1-1 Gy group, and GPx was a suitable biomarker for gamma irradiation in the dose range from 0.1 to 1 Gy. Furthermore, the activities of SOD, CAT, GR and GPx of the zebrafish embryos at stage 3 were found to be much higher than those at other stages, indicating that the zebrafish embryos at stage 3 had a greater ability to protect against gamma rays than those at other stages, and thus the activities of antioxidant enzymes changed in a developmental stage-dependent manner.

Developmental toxicity and oxidative stress induced by gamma irradiation in zebrafish embryos

International Nuclear Information System (INIS)

Hu, Miao; Hu, Nan; Ding, Dexin; Zhao, Weichao; Feng, Yongfu; Zhang, Hui; Li, Guangyue; Wang, Yongdong

2016-01-01

This study aimed to evaluate the biological effects of gamma irradiation on zebrafish embryos. Different doses of gamma rays (0.01, 0.05, 0.1, 0.5 and 1 Gy) were used to irradiate zebrafish embryos at three developmental stages (stage 1, 6 h post-fertilization (hpf); stage 2, 12 hpf; stage three, 24 hpf), respectively. The survival, malformation and hatching rates of the zebrafish embryos were measured at the morphological endpoint of 96 hpf. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione S-transferase (GST) were assayed. Morphology analysis showed that gamma irradiation inhibited hatching and induced developmental toxicity in a dose-dependent manner. Interestingly, after irradiation the malformation rate changed not only in a dose-dependent manner but also in a developmental stage-dependent manner, indicating that the zebrafish embryos at stage 1 were more sensitive to gamma rays than those at other stages. Biochemical analysis showed that gamma irradiation modulated the activities of antioxidant enzymes in a dose-dependent manner. A linear relationship was found between GPx activity and irradiation dose in 0.1-1 Gy group, and GPx was a suitable biomarker for gamma irradiation in the dose range from 0.1 to 1 Gy. Furthermore, the activities of SOD, CAT, GR and GPx of the zebrafish embryos at stage 3 were found to be much higher than those at other stages, indicating that the zebrafish embryos at stage 3 had a greater ability to protect against gamma rays than those at other stages, and thus the activities of antioxidant enzymes changed in a developmental stage-dependent manner. (orig.)

Developmental toxicity and oxidative stress induced by gamma irradiation in zebrafish embryos

Energy Technology Data Exchange (ETDEWEB)

Hu, Miao; Hu, Nan; Ding, Dexin; Zhao, Weichao; Feng, Yongfu; Zhang, Hui; Li, Guangyue; Wang, Yongdong [University of South China, Key Discipline Laboratory for National Defense for Biotechnology in Uranium Mining and Hydrometallurgy, Hengyang, Hunan Province (China)

2016-11-15

This study aimed to evaluate the biological effects of gamma irradiation on zebrafish embryos. Different doses of gamma rays (0.01, 0.05, 0.1, 0.5 and 1 Gy) were used to irradiate zebrafish embryos at three developmental stages (stage 1, 6 h post-fertilization (hpf); stage 2, 12 hpf; stage three, 24 hpf), respectively. The survival, malformation and hatching rates of the zebrafish embryos were measured at the morphological endpoint of 96 hpf. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione S-transferase (GST) were assayed. Morphology analysis showed that gamma irradiation inhibited hatching and induced developmental toxicity in a dose-dependent manner. Interestingly, after irradiation the malformation rate changed not only in a dose-dependent manner but also in a developmental stage-dependent manner, indicating that the zebrafish embryos at stage 1 were more sensitive to gamma rays than those at other stages. Biochemical analysis showed that gamma irradiation modulated the activities of antioxidant enzymes in a dose-dependent manner. A linear relationship was found between GPx activity and irradiation dose in 0.1-1 Gy group, and GPx was a suitable biomarker for gamma irradiation in the dose range from 0.1 to 1 Gy. Furthermore, the activities of SOD, CAT, GR and GPx of the zebrafish embryos at stage 3 were found to be much higher than those at other stages, indicating that the zebrafish embryos at stage 3 had a greater ability to protect against gamma rays than those at other stages, and thus the activities of antioxidant enzymes changed in a developmental stage-dependent manner. (orig.)

Somatic Embryos in Catharanthus roseus: A Scanning Electron Microscopic Study

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Junaid ASLAM

2014-06-01

Full Text Available Catharanthus roseus (L. G. Don is an important medicinal plant as it contains several anti-cancerous compounds, like vinblastine and vincristine. Plant tissue culture technology (organogenesis and embryogenesis has currently been used in fast mass propagating raw materials for secondary metabolite synthesis. In this present communication, scanning electron microscopic (SEM study of somatic embryos was conducted and discussed. The embryogenic callus was first induced from hypocotyls of in vitro germinated seeds on which somatic embryos, differentiated in numbers, particularly on 2,4-D (1.0 mg/L Murashige and Skoog (MS was medium. To understand more about the regeneration method and in vitro formed embryos SEM was performed. The SEM study revealed normal somatic embryo origin and development from globular to heart-, torpedo- and then into cotyledonary-stage of embryos. At early stage, the embryos were clustered together in a callus mass and could not easily be detached from the parental tissue. The embryos were often long cylindrical structure with or without typical notch at the tip. Secondary embryos were also formed on primary embryo structure. The advanced cotyledonary embryos showed prominent roots and shoot axis, which germinated into plantlets. The morphology, structure and other details of somatic embryos at various stages were presented.

Effects of alpha particles on zebrafish embryos

International Nuclear Information System (INIS)

Yum, E.H.W.; Choi, V.W.Y.; Yu, K.N.; Li, V.W.T.; Cheng, S.H.

2008-01-01

Full text: Ionizing radiation such as X-ray and alpha particles can damage cellular macromolecules, which can lead to DNA single- and double-strand breaks. In the present work, we studied the effects of alpha particles on dechorionated zebrafish embryos. Thin polyallyldiglycol carbonate (PADC) films with a thickness of 16 μm were prepared from commercially available PADC films (with thickness of 100 μm) by chemical etching and used as support substrates for holding zebrafish embryos for alpha-particle irradiation. These films recorded alpha-particle hit positions, quantified the number and energy of alpha particles actually incident on the embryo cells, and thus enabled the calculation of the dose absorbed by the embryo cells. Irradiation was made at 1.25 hours post fertilization (hpf) with various absorbed dose. TdT-mediated dUTP Nick-End Labeling (TUNEL) assay was performed on the embryos at different time stages after irradiation. Marked apoptosis was detected only in embryos at earlier time stages. The results showed that DNA double-strand break during zebrafish embryogenesis can be induced by alpha-particle irradiation, which suggests that zebrafish is a potential model for assessing the effects of alpha-particle radiation

Characterization of the onset of embryonic control and early development in the bovine embryo

International Nuclear Information System (INIS)

Barnes, F.L.

1988-01-01

Bovine embryos were used to determine if morphological and molecular features of early development are similar to in vivo recovered bovine embryos and to determine at what level early bovine development is regulated. Radiolabeling of IVP embryos and in vivo recovered embryos with 35 S-methionine for SDS-polyacrylamide gel electrophoresis reveals that these embryos are equivalent. Few differences in protein profiles are observed between 1- and early 4-cell embryos. A change in protein profiles begins at the mid 4-cell stage and continues into the 8-cell stage. Few differences in protein profiles are observed between 1- and early 4-cell embryos. A change in protein profiles begins at the mid 4-cell stage and continues into the 8-cell stage. Few differences in protein profiles are observed between late 8-cells and morulae. This transition is α-amanitin sensitive therefore due to de novo embryonic transcription. Embryonic transcription is partially responsible for terminating the post-transcriptionally regulated period of early bovine development. Argentophillic nucleolar organizing regions (Ag-NORs) indicate onset of nucleolar activation. Ag-NORs were absent in 2- and 4-cell IVP embryos and rarely occurred in 8-cell IVP embryos cultured in vitro. IVP 1- and 2-cell embryos cultured to blastocysts in sheep oviducts demonstrated Ag-NORs. Thus the lack of nucleolar activation of IVP embryos cultured in vitro is culture induced between the 2- and 8-cell stage

Alternation of apoptotic and implanting genes expression of mouse embryos after re-vitrification

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Nasrin Majidi Gharenaz

2016-08-01

Full Text Available Background: Nowadays, oocytes and embryos vitrification has become a routine technique. Based on clinical judgment, re-vitrification maybe required. But little is known about re-vitrification impact on genes expression. Objective: The impact of re-vitrification on apoptotic and implanting genes, Bax, Bcl-2 and ErbB4, at compaction stage embryos were evaluated in this study. Materials and Methods: In this experimental study, 8 cell embryos (n=240 were collected from female mature mice, 60-62 hr post HCG injection. The embryos were divided randomly to 3 groups included: fresh (n=80, vitrified at 8 cell stage (n=80, vitrified at 8 cell stage thawed and re-vitrified at compaction stage (n=80. Embryos were vitrified by using cryolock, (open system described by Kuwayama. Q-PCR was used to examine the expression of Bax, Bcl2 ErbB4 genes in derived blastocysts. Results: Our result showed that expanded blastocyst rate was similar between vitrified and re-vitrified groups, while re-vitrified embryos showed significant decrease in expanded blastocyst rate comparing with fresh embryos (p=0.03. In addition, significant difference was observed on apoptotic gene expression when comparing re-vitrified and fresh embryos (p=0.004, however expression of Bax and Bcl-2 (apoptotic genes didn't demonstrate a significant difference between re-vitrified and vitrified groups. The expression rate of ErbB4, an implantation gene was decreased in re-vitrified embryos comparing with fresh embryos (p=0.003, but it was similar between re-vitrified and vitrified embryos. Conclusion: Re-vitrification can alter the expression of Bax, Bcl-2 and ErbB4 genes and developmental rate of mouse embryos in compaction stage

Preimplantation diagnosis of repeated miscarriage due to chromosomal translocations using metaphase chromosomes of a blastomere biopsied from 4- to 6-cell-stage embryos.

Science.gov (United States)

Tanaka, Atsushi; Nagayoshi, Motoi; Awata, Shoichiro; Mawatari, Yoshifumi; Tanaka, Izumi; Kusunoki, Hiroshi

2004-01-01

To evaluate the safety and accuracy of karyotyping the blastomere chromosomes at metaphase in the natural cell cycle for preimplantation diagnosis. A pilot study. A private infertility clinic and a university laboratory. Eleven patients undergoing IVF and preimplantation diagnosis. Intact human embryos at the 4- to 6-cell stage and human-mouse heterokaryons were cultured and checked hourly for disappearance of the nuclear envelope. After it disappeared, the metaphase chromosomes were analyzed by fluorescence in situ hybridization. Percentage of analyzable metaphase plates and safety and accuracy of the method. The success rate of electrofusion to form human-mouse heterokaryons was 87.1% (27/31), and analyzable chromosomes were obtained from 77.4% (24/31) of the heterokaryons. On the other hand, disappearance of the nuclear envelope occurred in 89.5% (17/19) of the human embryos and it began earlier than that in the heterokaryons. Analyzable chromosomes were obtained and their translocation sites were identified in all blastomeres biopsied from the 17 embryos. After the biopsy, 67.0% of the embryos could develop to the blastocyst stage. The natural cell cycle method reported herein requires frequent observation, but it is safe, with no artificial effects on the chromosomes and without loss of or damage to blastomeres, which occurred with the electrofusion method. Using the natural cell cycle method, we could perform preimplantation diagnosis with nearly 100% accuracy.

The Effects of ISM1 Medium on Embryo Quality and Outcomes of IVF/ICSI Cycles

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Fatemeh Shabani

2013-01-01

Full Text Available Background: The aim of this study is to investigate the effect of ISM1 culture mediumon embryo development, quality and outcomes of in vitro fertilization/intracytoplasmicsperm injection (IVF/ICSI cycles. This study compares culture medium commonly usedin the laboratory setting for oocyte recovery and embryo development with a mediumfrom MediCult. We have assessed the effects of these media on embryo development andnewborn characteristics.Materials and Methods: In this prospective randomized study, fertilized oocytesfrom patients were randomly assigned to culture in ISM1 (MediCult, cycles:n=293 or routine lab culture medium (G-1TM v5; Vitrolife, cycles: n=290 accordingto the daily media schedule for oocyte retrieval. IVF or ICSI and embryotransfer were performed with either MediCult media or routine lab media. Embryoquality on days 2/3, cleavage, pregnancy and implantation rates, baby take homerate (BTHR, in addition to the weight and length of newborns were comparedbetween groups.Results: There were similar cleavage rates for ISM1 (86% vs. G-1TM v5 (88%. Weobserved a significantly higher percentage of excellent embryos in ISM1 (42.7% comparedto G-1TM v5 (39%, p<0.05. Babies born after culture in ISM1 had both higherbirth weight (3.03 kg and length (48.8 cm compared to G-1TM v5 babies that had a birthweight of 2.66 kg and a length of 46.0 cm (p<0.001 for both.Conclusion: This study suggests that ISM1 is a more effective culture medium ingenerating higher quality embryos, which may be reflected in the characteristics ofbabies at birth.

Computer ranking of the sequence of appearance of 40 features of the brain and related structures in staged human embryos during the seventh week of development.

Science.gov (United States)

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

1988-08-01

The sequence of events in the development of the brain in human embryos, already published for stages 8-17, is here continued for stages 18 and 19. With the aid of a computerized bubble-sort algorithm, 58 individual embryos were ranked in ascending order of the features present. The increasing structural complexity provided 40 new features in these two stages. The chief characteristics of stage 18 (approximately 44 postovulatory days) are rapidly growing basal nuclei; appearance of the extraventricular bulge of the cerebellum (flocculus), of the superior cerebellar peduncle, and of follicles in the epiphysis cerebri; and the presence of vomeronasal organ and ganglion, of the bucconasal membrane, and of isolated semicircular ducts. The main features of stage 19 (approximately 48 days) are the cochlear nuclei, the ganglion of the nervus terminalis, nuclei of the prosencephalic septum, the appearance of the subcommissural organ, the presence of villi in the choroid plexuses of the fourth and lateral ventricles, and the stria medullaris thalami.

Impact of GnRH analogues on oocyte/embryo quality and embryo development in in vitro fertilization/intracytoplasmic sperm injection cycles: a case control study

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Rigó János

2009-09-01

Full Text Available Abstract Background Despite the clinical outcomes of ovarian stimulation with either GnRH-agonist or GnRH-antagonist analogues for in vitro fertilization (IVF being well analysed, the effect of analogues on oocyte/embryo quality and embryo development is still not known in detail. The aim of this case-control study was to compare the efficacy of a multiple-dose GnRH antagonist protocol with that of the GnRH agonist long protocol with a view to oocyte and embryo quality, embryo development and IVF treatment outcome. Methods Between October 2001 and December 2008, 100 patients were stimulated with human menopausal gonadotrophin (HMG and GnRH antagonist in their first treatment cycle for IVF or intracytoplasmic sperm injection (ICSI. One hundred combined GnRH agonist + HMG (long protocol cycles were matched to the GnRH antagonist + HMG cycles by age, BMI, baseline FSH levels and by cause of infertility. We determined the number and quality of retrieved oocytes, the rate of early-cleavage embryos, the morphology and development of embryos, as well as clinical pregnancy rates. Statistical analysis was performed using Wilcoxon's matched pairs rank sum test and McNemar's chi-square test. P Results The rate of cytoplasmic abnormalities in retrieved oocytes was significantly higher with the use of GnRH antagonist than in GnRH agonist cycles (62.1% vs. 49.9%; P Conclusion Antagonist seemed to influence favourably some parameters of early embryo development dynamics, while other morphological parameters seemed not to be altered according to GnRH analogue used for ovarian stimulation in IVF cycles.

Closure of the vertebral canal in human embryos and fetuses

NARCIS (Netherlands)

Mekonen, Hayelom K.; Hikspoors, Jill P. J. M.; Mommen, Greet; Kruepunga, Nutmethee; Köhler, S. Eleonore; Lamers, Wouter H.

2017-01-01

The vertebral column is the paradigm of the metameric architecture of the vertebrate body. Because the number of somites is a convenient parameter to stage early human embryos, we explored whether the closure of the vertebral canal could be used similarly for staging embryos between 7 and 10weeks of

Effects of exogenous melatonin on in vivo embryo viability and oocyte competence of undernourished ewes after weaning during the seasonal anestrus.

Science.gov (United States)

Vázquez, M I; Abecia, J A; Forcada, F; Casao, A

2010-09-01

This study investigated the effects of exogenous melatonin on embryo viability and oocyte competence in post-partum undernourished ewes during the seasonal anestrus. At parturition (mid-Feb), 36 adult Rasa Aragonesa ewes were assigned to one of two groups: treated (+MEL) or not treated (-MEL) with a subcutaneous implant of melatonin (Melovine(R), CEVA) on the day of lambing. After 45 d of suckling, lambs were weaned, ewes were synchronized using intravaginal pessaries, and fed to provide 1.5x (Control, C) or 0.5x (Low, L) times daily maintenance requirements. Thus, ewes were divided into four groups: C-MEL, C+MEL, L-MEL, and L+MEL. At estrus (Day=0), ewes were mated. At Day 5 after estrus, embryos were recovered by mid-ventral laparotomy and classified based on their developmental stage and morphology. After embryo collection, ovaries were recovered and oocytes were classified and selected for use in in vitro fertilization (IVF). Neither diet nor melatonin treatment had a significant effect on ovulation rate and on the number of ova recovered per ewe. Melatonin treatment significantly improved the number of fertilized embryos/corpus luteum (CL) (-MEL: 0.35 +/- 0.1, +MEL: 0.62 +/- 0.1; P = 0.08), number of viable embryos/CL (-MEL: 0.23 +/- 0.1, +MEL: 0.62 +/- 0.1; P competence of oocytes during IVF. Treatment groups did not differ significantly in the number of healthy oocytes used for IVF, number of cleaved embryos, or number of blastocysts and, consequently, the groups had similar cleavage and blastocyst rates. In conclusion, melatonin treatments improved ovine embryo viability during anestrus, particularly in undernourished post-partum ewes, although the effects of melatonin did not appear to be mediated at the oocyte competence level. Copyright 2010 Elsevier Inc. All rights reserved.

Regional localization of suspensor mRNAs during early embryo development.

Science.gov (United States)

Weterings, K; Apuya, N R; Bi, Y; Fischer, R L; Harada, J J; Goldberg, R B

2001-11-01

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

Regional Localization of Suspensor mRNAs during Early Embryo Development

Science.gov (United States)

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

2001-01-01

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

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

Science.gov (United States)

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.

Early aberrations in chromatin dynamics in embryos produced under In vitro conditions

DEFF Research Database (Denmark)

Deshmukh, Rahul Shahaji; Østrup, Olga; Strejcek, Frantisek

2012-01-01

standard to that of embryos produced by IVF, parthenogenetic activation (PA), or SCNT. In contrast to IV embryos, chromatin spatial and temporal dynamics in PA, IVF, and SCNT embryos were altered; starting with aberrant chromatin-nuclear envelope interactions at the two-cell stage, delayed chromatin...... decondensation and nucleolar development at the four-cell stage, and ultimately culminating in failure of proper first lineage segregation at the blastocyst stage, demonstrated by poorly defined inner cell mass. Interestingly, in vitro produced (IVP) embryos also lacked a heterochromatin halo around nucleolar...

Effect of arachidonic acid supplementation and cyclooxygenase/lipoxygenase inhibition on the development of early bovine embryos Influência do ácido araquidónico e da inibição da ciclo-oxigenase ou lipo-oxigenase no desenvolvimento inicial de embriões bovinos

Directory of Open Access Journals (Sweden)

Rosa Maria Pereira

2006-04-01

Full Text Available The effect of arachidonic acid (AA cascade on bovine embryo development in a granulosa cell co-culture system was studied. Arachidonic acid (100 µM was supplemented from 1-cell to 8-16 cell block stage (first three days of co-culture and from 1-cell to hatching. Specific cyclooxygenase (indomethacin, 28 µM and lipoxygenase (nordihydroguaiaretic acid - NDGA, 28 µM inhibitors were used from 1-cell to 8-16 cell block stage with AA. Embryo development was assessed by cleavage, day 7-day 8 and hatched embryo rates and by measuring growth rates through development stages found in days 7-10 of culture (day 0 = insemination day. Embryo quality was scored at day 8. A 6.5-10.4% increase on cleavage rate after AA supplementation was found. This AA supplementation from 1-cell to hatching delayed embryo growth rate beyond day 7 and a reduction on hatching rate was detected. When AA supplementation was restricted to the first three days of co-culture those negative effects were overcome. Also, indomethacin and NDGA prevented the positive effect of AA and induced a significant reduction on cleavage, respectively. NDGA further decreased day 7 embryo rate and quality. Results suggest that AA has a two-phase action on bovine embryos, promoting early development and impairing embryo growth from day 7 onwards and hatching rates. Both cyclooxygenase and lipoxygenase were found to be important pathways to promote cleavage.Estudou-se a influência da cascata do ácido araquidónico (AA no desenvolvimento de embriões bovinos produzidos in vitro em co-cultura com células da granulosa. Os embriões foram suplementados com AA (100 µM desde o estádio de 1 célula até 8-16 células (primeiros três dias de co-cultura ou até a eclosão. Introduziram-se inibidores específicos da ciclo-oxigenase (indometacina, 28 µM e da lipo-oxigenase (ácido nordihidroguaiarético - NDGA, 28 µM, juntamente com o ácido araquidónico, desde o estádio de 1 célula até 8-16 c

Gonadotropin Releasing Hormone Agonists or Antagonists for Preimplantation Genetic Diagnosis (PGD)? A Prospective Randomised Trial.

Science.gov (United States)

Verpoest, Willem; De Vos, Anick; De Rycke, Martine; Parikh, Shruti; Staessen, Catherine; Tournaye, Herman; De Vos, Michel; Vloeberghs, Veerle; Blockeel, Christophe

2017-11-10

The use of GnRH analogue medication is essential in reproductive medicine to avoid premature ovulation by pituitary suppression for the duration of ovarian stimulation by gonadotrophins. The type of pituitary suppression by either GnRH agonist analogues versus GnRH antagonist analogues may result in different embryological hence clinical results. Preimplantation genetic diagnosis is a subtype of IVF in which embryos are created for genetic diagnosis of hereditary disorders in order to avoid genetically affected children. Embryological quality hence ovarian stimulation in preimplantation genetic diagnosis is crucial as genetic selection will reduce the number of available embryos to a fraction of the total. The aim of this study was to assess the efficiency of GnRH antagonist versus GnRH agonist treatment for pituitary suppression in ovarian stimulation for PGD, by proxy of number and quality of embryos at cleavage stage available for biopsy. We conducted a prospective randomised controlled trial comparing pituitary suppression by GnRH antagonist versus GnRH agonist in ovarian stimulation for PGD. The primary outcome measure was the number of embryos of sufficient quality for biopsy at cleavage stage. Secondary outcome parameters were the number of blastocysts available of top quality, and clinical pregnancy rate. There was no difference in number of oocytes retrieved, embryos at cleavage stage available for biopsy or embryo quality. The clinical pregnancy rate was higher in the GnRH agonist group; however the sample size was insufficient to allow conclusions. The use of GnRH agonist versus antagonist treatment does not result in differences in a number of oocytes, embryos or embryo quality in ovarian stimulation for preimplantation genetic diagnosis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Estádios de desenvolvimento embrionário e localização do embrião zigótico em sementes de citros Embryo development stage and the location of embryo zygotic in the seed of citrine

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Valtemir Gonçalves Ribeiro

1999-08-01

Full Text Available Objetivou-se estudar o comportamento de embriões zigóticos e nucelares aos 120, 130, 140 e 150 dias após serem efetuadas hibridações controladas entre a laranjeira 'Natal' (Citrus sinensis Osb. e o parental masculino Poncirus trifoliata (L. Raf. Em cada data, as sementes foram removidas, e os embriões excisados foram caracterizados em estádios de desenvolvimento (globular, cordiforme, torpedo e cotiledonar; coloração (clorofilado ou não; e localização na semente (próximo à micrópila ou mais interiormente. A partir dessas características, foram construídas tabelas de contingência para testar hipóteses de independência entre elas, mediante o teste exato de Fisher e chi² (qui-quadrado. Relações de dependência foram verificadas entre as características: estádios de desenvolvimento embrionário com a localização na semente; estádios de desenvolvimento embrionário com a natureza da plântula (zigótica ou nucelar; e entre natureza da plântula com a localização do embrião na semente. Verificou-se que os embriões zigóticos excisados de frutos com 130 a 150 dias da hibridação controlada, localizam-se, em grande maioria, próximos à região micropilar da semente, em estádio globular e cordiforme de desenvolvimento.Controlled hybridizations between 'Natal' orange variety (Citrus sinensis Osb. and male parent Poncirus trifoliata (L. Raf. were performed in order to study zygotic and nucellar embryos behaviour at 120, 130, 140 and 150 days. At very date the seeds were removed and the embryos excised to characterize: development stage (globular, cordiform, torpedo and cotyledonal; colour (with or without chlorophyll; and location in the seed (close to the micropyle or more internal in the seed. From these characteristics contingency tables were evaluated to test independency hypothesis through Fisher test and chi² (chi-square. Dependence relationships were observed between the following characteristics: stages of the

Experimental analysis of control mechanisms in somite segmentation in avian embryos. II. Reduction of material in the gastrula stages of the chick.

Science.gov (United States)

Bellairs, R; Veini, M

1984-02-01

A new theory of control of somite segmentation in chick embryos is proposed. This supposses that tiny clusters of already programmed cells are present throughout the presumptive somite area at stage 4, but that in order to fulfill their destiny they probably depend on the addition of further cells from the primitive streak. Evidence is based on the two groups of experiments: a) Experiments involving transection across the primitive streak at various stages, (which results in a 'tail' which possesses mesodermal derivatives) and across the segmental plate (which results in a 'tail' lacking mesodermal derivatives). b) Experiments in which parts of embryos have been explanted with or without their primitive streak. It is suggested that the initial clusters of pre-programmed cells move further and further posteriorly, developing into somitomeres (the precursors of true somites) only as they receive re-inforcements from the primitive streak or, ultimately, from the tail bud.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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

Dose estimation in embryo or fetus in external fields

International Nuclear Information System (INIS)

Gregori, Beatriz N.

2001-01-01

The embryo or the fetus can be irradiated as result of radiological procedures of diagnosis of therapy in where the beam effects directly on the same one or in tissues or peripherical organs. Some authors have suggested that in the first stages of the pregnancy the dose in ovaries can be the good estimated of the dose in embryo or fetus. In advanced conditions of the development, probably also in the early stage, is more appropriated to specify the dose in the embryo or fetus equal of the uterus. The dose in the uterus is a good estimated so much for external irradiation as for radionuclides incorporation

Is it time for a paradigm shift in understanding embryo selection?

Science.gov (United States)

Gleicher, Norbert; Kushnir, Vitaly A; Barad, David H

2015-01-11

Embryo selection has been an integral feature of in vitro fertilization (IVF) almost since its inception. Since the advent of extended blastocyst stage embryo culture, and especially with increasing popularity of elective single embryo transfer (eSET), the concept of embryo selection has increasingly become a mainstay of routine IVF. We here, however, argue that embryo selection via blastocyst stage embryo transfer (BSET), as currently practiced, at best improves IVF outcomes only for a small minority of patients undergoing IVF cycles. For a large majority BSET is either ineffective or, indeed, may actually be harmful by decreasing IVF pregnancy chances. Overall, only a small minority of patients, thus, benefit from prolonged embryo culture, while BSET, as a tool to enhance IVF outcomes, is increasingly utilized as routine care in IVF for all patients. Since newer methods of embryo selection, like preimplantation genetic screening (PGS) and closed system embryo incubation with time-lapse photography are practically dependent on BSET, these concepts of embryo selection, currently increasingly adopted in mainstream IVF, require reconsideration. They, automatically, transfer the downsides of BSET, including decreases in IVF pregnancy chances in some patients, to these new procedures, and in addition raise serious questions about cost-effectiveness.

Developmental competence of porcine chimeric embryos produced by aggregation

DEFF Research Database (Denmark)

Li, Juan; Jakobsen, Jannik E.; Xiong, Qiang

2015-01-01

The purpose of our study was to compare the developmental competence and blastomere allocation of porcine chimeric embryos formed by micro-well aggregation. Chimeras were created by aggregating either two blastomeres originating from 2-cell embryos or two whole embryos, where embryos were produced...... either by parthenogenetic activation (PA) or handmade cloning (HMC). Results showed that the developmental competence of chimeric embryos, evaluated based on their blastocyst rate and total cell number per blastocyst, was increased when two whole 2-cell stage embryos (PA or HMC) were aggregated....... In comparison, when two blastomeres were aggregated, the developmental competence of the chimeric embryos decreased if the blastomeres were either from PA or from HMC embryos, but not if they were from different sources, i.e. one PA and one HMC blastomere. To evaluate the cell contribution in embryo formation...

Xenopus laevis embryos and tadpoles as models for testing for ...

African Journals Online (AJOL)

The toxicity of bio available Zn, Cu, Pb, and Cd on the life stages of Xenopus laevis embryos and tadpoles was investigated. Cu and Cd were found to affect the hatching success of the embryos, with a strong negative relationship existing between the increase in Cu concentrations and the hatching of the embryos.

In vitro embryo culture of rarely endangered musella lasiocarpa (musaceae) with embryo dormancy

International Nuclear Information System (INIS)

Anjun, T.

2014-01-01

Musella lasiocarpa (Musaceae) is an ornamental annually producing many viable seeds, but seldom recruited by seeds in the wild. One mature Musella seed has a small mushroom-shaped embryo without discernible organ differentiation. Therefore, freshly-harvested mature seeds are dormant. When the seeds gradually finished differentiation during warm stratification at 23 degree C, they germinated to 82%. Besides, extracted embryos from fresh seeds did not germinate on the basal medium of Murshige and Skoog medium (MS) supplemented with 3% sucrose and 0.8% agar, but they were induced to form calli and root by media. The optimum medium for inducing calli was MS + 1.0 mg/L 6-BA + 0.05 mg/L NAA + 100 mg/L Vc with the highest proliferation coefficient (7.3) in 35 days. Moreover, the embryos from the 6-month warm stratified seeds could proliferate on the suitable medium. The optimal medium for rooting was MS + 0.5 mg/L 2, 4-D + Vitamin C 100 mg/L. The results confirmed that both the embryo developmental stage and appropriate combination of chemicals significantly affected seed germination and In vitro embryo culture of this species. (author)

Dynamic Subcellular Localization of Iron during Embryo Development in Brassicaceae Seeds

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Miguel A. Ibeas

2017-12-01

Full Text Available Iron is an essential micronutrient for plants. Little is know about how iron is loaded in embryo during seed development. In this article we used Perls/DAB staining in order to reveal iron localization at the cellular and subcellular levels in different Brassicaceae seed species. In dry seeds of Brassica napus, Nasturtium officinale, Lepidium sativum, Camelina sativa, and Brassica oleracea iron localizes in vacuoles of cells surrounding provasculature in cotyledons and hypocotyl. Using B. napus and N. officinale as model plants we determined where iron localizes during seed development. Our results indicate that iron is not detectable by Perls/DAB staining in heart stage embryo cells. Interestingly, at torpedo development stage iron localizes in nuclei of different cells type, including integument, free cell endosperm and almost all embryo cells. Later, iron is detected in cytoplasmic structures in different embryo cell types. Our results indicate that iron accumulates in nuclei in specific stages of embryo maturation before to be localized in vacuoles of cells surrounding provasculature in mature seeds.

Effect of Fibroblast Growth Factor 2 (FGF2 and Insulin Transferrin Selenium (ITS on In Vitro Maturation, Fertilization and Embryo Development in Sheep

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Sukanta Mondal

2015-08-01

Full Text Available The present study evaluated the effect of fibroblast growth factor 2 (FGF2 and insulin-transferrin-selenium (ITS to the in vitro maturation and embryo culture media on ovine oocyte maturation, cleavage and embryo development. Oocytes having more than five layers of unexpanded cumulus cells and granular homogenous ooplasm were cultured into 50 μL droplets of eight different culture systems: (i TCM-199 (Tissue Culture Medium-199; (ii TCM-199+10 ng/mL FGF2; (iii TCM-199+20 ng/mL FGF2; (iv TCM-199+30 ng/mL FGF2; (v TCM-199+10 ng/mL ITS; (vi TCM-199+20 ng/mL ITS; (vii TCM-199+30 ng/mL ITS and (viii TCM-199+20 ng/mL ITS+20 ng/mL FGF2 in a CO2 incubator at 38.50C for 24 h. All the oocyte culture media were supplemented with 10% FBS, FSH (10 μg/mL and gentamicin (50 µg/mL. The maturation rate was assessed based on the degree of expansion of cumulus cells and identifying first polar body extrusion into perivitelline space. The matured oocytes were inseminated with 1 to 2 million spermatozoa/mL in Brackett and Oliphant medium and the cleavage rate was checked after 42-48 h post insemination and further cultured for 6-7 days. Maturation and cleavage rates were significantly higher (P<0.05 in the oocytes cultured in TCM-199 +10% FBS+FSH (10 μg/mL supplemented with both 20 ng/mL ITS and 20 ng/mL FGF2 as compared to the control. It was concluded that the supplementation of ITS and FGF2 in maturation medium was beneficial for improving maturation and cleavage rates of sheep oocytes. The addition of ITS and FGF2 in embryo culture medium did not improve the development of sheep embryos.

Impact of motorboats on fish embryos depends on engine type.

Science.gov (United States)

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.

High dose progesterone effects the growth of early chick embryo

International Nuclear Information System (INIS)

Iqbal, I.; Qamar, K.

2014-01-01

Objective: To find out the effect of high dose progesterone on the development of early chick embryo. Study Design: Lab based randomized controlled trial. Place and Duration of study: This study was carried out in Army Medical College and Post Graduate Institute of Poultry Sciences, Rawalpindi from June 2010 - December 2010. Material and Methods: Forty five specific pathogen free, fertile, eggs of Fyoumi species of chick were selected at zero hour of incubation. They were incubated at 37.5oC and 75% relative humidity for 26 hrs until the embryos reached stage 8 of the development. Then on stage 8 the eggs were divided into three groups consisting of 15 eggs per group. The first group (GI) was incubated without any operation. The second (G2) and third groups (G3) were injected with two and twenty times more than physiologic does of progesterone respectively. After 48 hours of incvbation, all embryos were examined for their development under light microscopy. Results: All the embryos of G1 and G2 showed normal development according to their stage of development, while 4 out of 11 embryos of G3 were under developed and their survival rate was also less. Conclusion: Exogenous progesterone at levels twenty times above its physiologic range effects the development of chick embryos. Further studies are needed to explain the mechanisms of this effect. (author)

MORPHOLOGICAL CHANGES DURING THE DEVELOPMENT OF SOMATIC EMBRYOS OF SAGO (Metroxylon sagu Rottb.

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Pauline D. Kasi

2016-10-01

Full Text Available Development of somatic embryos of sago (Metroxylon sagu Rottb. on agar-solidified medium are highly varied producing heterogeneous seedlings. Understanding of this phenomenon may help in improving the cultural procedures and conditions of sagosomatic embryogenesis to obtain uniform seedlings in a large scale. This experiment was conducted at the laboratory for plant cell culture and micropropagation, Indonesian Biotechnology Research Institute for Estate Crops from January to March 2006 to examine morphological changes i.e. color and development stages of sago during their somatic embryo development on an agar-solidified medium. Twenty single globular somatic embryos of sago with specific color (yellowish, greenish, and reddish were cultured in a Petri dish supplemented with a solid medium. The medium was a micronutrients-modified MS (MMS with half strength of macronutrients containing 0.01 mg l-1 ABA, 2 mg l-1 kinetin, 20 g l-1 sucrose, 0.5 g l-1 activated charcoal, and 2 g l-1 gelrite. Parameter observed was the percentage of embryo’s number based on color and developmental stage. The result showed that at the end of 6-week culture passage, most originally greenish (80.8% and reddish (95.8% embryos remained unchanged in their colors, whereas almost half of the originally yellowish embryos turned to greenish and only 30%remained yellowish. At the same time, single globular embryos have changed gradually into the next developmental stages, although not all of the embryos were germinated. The initial color of embryo affected the rate of the developmental stage changes. Yellowish and greenish globular embryos developed more rapidly into cotyledon or germinant stages at 58% and 55% respectively, in 6 weeks than the reddish ones (41%. Therefore, the yellowish and greenish embryos are the best sources of material for in vitro mass propagation and synthetic seed production of sago.

Parent-of-origin dependent gene-specific knock down in mouse embryos

International Nuclear Information System (INIS)

Iqbal, Khursheed; Kues, Wilfried A.; Niemann, Heiner

2007-01-01

In mice hemizygous for the Oct4-GFP transgene, the F1 embryos show parent-of-origin dependent expression of the marker gene. F1 embryos with a maternally derived OG2 allele (OG2 mat /-) express GFP in the oocyte and during preimplantation development until the blastocyst stage indicating a maternal and embryonic expression pattern. F1-embryos with a paternally inherited OG2 allele (OG2 pat /-) express GFP from the 4- to 8-cell stage onwards showing only embryonic expression. This allows to study allele specific knock down of GFP expression. RNA interference (RNAi) was highly efficient in embryos with the paternally inherited GFP allele, whereas embryos with the maternally inherited GFP allele showed a delayed and less stringent suppression, indicating that the initial levels of the target transcript and the half life of the protein affect RNAi efficacy. RT-PCR analysis revealed only minimum of GFP mRNA. These results have implications for studies of gene silencing in mammalian embryos

Biotransformation in the zebrafish embryo -temporal gene transcription changes of cytochrome P450 enzymes and internal exposure dynamics of the AhR binding xenobiotic benz[a]anthracene.

Science.gov (United States)

Kühnert, Agnes; Vogs, Carolina; Seiwert, Bettina; Aulhorn, Silke; Altenburger, Rolf; Hollert, Henner; Küster, Eberhard; Busch, Wibke

2017-11-01

Not much is known about the biotransformation capability of zebrafish (Danio rerio) embryos. For understanding possible toxicity differences to adult fish, it might be crucial to understand the biotransformation of chemicals in zebrafish embryos i.e. as part of toxicokinetics. The biotransformation capabilities were analysed for two different stages of zebrafish embryos in conjunction with the internal concentrations of a xenobiotic. Zebrafish embryos of the late cleavage/early blastula period (2-26 hpf) and the early pharyngula period (26-50 hpf) were exposed for 24 h to the AhR binding compound benz[a]anthracene (BaA). Time dependent changes in cyp transcription (cyp1a, cyp1b1, cyp1c1 and cyp1c2) as well as concentration & time-dependent courses of BaA in the fish embryo and the exposure medium were analysed. Additionally, the CYP mediated formation of biotransformation products was investigated. We found correlations between transcriptional responses and the internal concentration for both exposure types. These correlations were depending on the start of the exposure i.e. the age of the exposed embryo. While no significant induction of the examined gene transcripts was observed in the first 12 h of exposure beginning in the blastula period a correlation was apparent when exposure started later i.e. in the pharyngula period. A significant induction of cyp1a was detected already after 1.5 h of BaA exposure. Gene transcripts for cyp1b1, cyp1c1 and cyp1c2 showed expressions distinctly different from cyp1a and were, in general, less inducible by BaA in both exposure windows. The toxicokinetic analysis showed that the biotransformation capability was fivefold higher in the older fish embryos. Biotransformation products of phase I reactions were found between 32 hpf and 50 hpf and were tentatively identified as benz[a]anthracene-phenol and benz[a]anthracene-dihydrodiol-epoxide. In conclusion, not only duration but also onset of exposure in relation to the

Developmental Competence and Epigenetic Profile of Porcine Embryos Produced by Two Different Cloning Methods.

Science.gov (United States)

Liu, Ying; Lucas-Hahn, Andrea; Petersen, Bjoern; Li, Rong; Hermann, Doris; Hassel, Petra; Ziegler, Maren; Larsen, Knud; Niemann, Heiner; Callesen, Henrik

2017-06-01

The "Dolly" based cloning (classical nuclear transfer, [CNT]) and the handmade cloning (HMC) are methods that are nowadays routinely used for somatic cloning of large domestic species. Both cloning protocols share several similarities, but differ with regard to the required in vitro culture, which in turn results in different time intervals until embryo transfer. It is not yet known whether the differences between cloned embryos from the two protocols are due to the cloning methods themselves or the in vitro culture, as some studies have shown detrimental effects of in vitro culture on conventionally produced embryos. The goal of this study was to unravel putative differences between two cloning methods, with regard to developmental competence, expression profile of a panel of developmentally important genes and epigenetic profile of porcine cloned embryos produced by either CNT or HMC, either with (D5 or D6) or without (D0) in vitro culture. Embryos cloned by these two methods had a similar morphological appearance on D0, but displayed different cleavage rates and different quality of blastocysts, with HMC embryos showing higher blastocyst rates (HMC vs. CNT: 35% vs. 10%, p cloned embryos were similar on D0, but differed on D6. In conclusion, both cloning methods and the in vitro culture may affect porcine embryo development and epigenetic profile. The two cloning methods essentially produce embryos of similar quality on D0 and after 5 days in vitro culture, but thereafter both histone acetylation and gene expression differ between the two types of cloned embryos.

Ultrastructural studies of Biomphalaria glabrata (Say, 1818) embryo

International Nuclear Information System (INIS)

Kikuchi, O.K.; Okazaki, K.; Kawano, T.; Ribeiro, A.A.G.F.C.

1988-09-01

Ultrastructural studies of Biomphalaria glabrata embryos (MOllusca: Gastropoda), and important snail vector of schistosomiasis has not been explored. In the present work it was evaluated a suitable electron microscopical technique for embryos processing. Promising results was obtained with double fixation in 1% glutaraldehyde plus 1% osmium tetroxide in 0.05 M cacodylate buffer (pH 7.4), preliminary staining overnight in 1% uranyl acetate and embedding in EPON or Polylite under vacuum. It was used embryos at young trochophore stage wich is characterized by active organogenesis. Some ultrastructural aspects of B. glabrata embryos cells are presented. (author) [pt

A Review of the Teratogenic Factors Effect on Embryo

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Manzarbanoo Shojaei fard

2017-02-01

Full Text Available Background & Objectives: Teratology is a branch of embryology science that studies causes, mechanisms and abnormal pattern development. Embryo growth traumatic factors during pregnancy are called teratogens that some teratogens pass the placental barrier and cause adverse effect during development stages and malformation, however a drug may improve general health of the mother, but it might be poisonous for embryo and cause diverse malformation. Since study of embryo health and risk factor in this stage is important, the aim of this review article was the investigation of some types of teratosgens (such as radiation, infectious agents, heat disorders, maternal conditions and particularly the effect of teratogenic drugs on embryo including some legal drugs (such as acetaminophen, thalidomide, acyclovir, sedatives and anticonvulsants and illegal drugs (such as nicotine, alcohol, cocaine and marijuana. Conclusion: In general, teratogens depending on the type and duration of exposure in pregnancyperiod, adversely affect embryo and cause various disorders. A better understanding of these teratogens can contribute to prevent these defects, since many other drugs with similar effects and lower teratogenicity can be used to improve mothers’ health.

Ultrastructural changes in goat interspecies and intraspecies reconstructed early embryos

DEFF Research Database (Denmark)

Tao, Yong; Gheng, Lizi; Zhang, Meiling

2008-01-01

and dispered gradually from the 4-cell period. The nucleolus of GC and GG embryos changed from electron dense to a fibrillo-granular meshwork at the 16-cell stage, showing that nucleus function in the reconstructed embryos was activated. The broken nuclear envelope and multiple nucleoli in one blastomere......- and intraspecies reconstructed embryos have a similar pattern of developmental change to that of in vivo-produced embryos for ZP, rough ER, Gi and nucleolus, but differ for mitochondria, LD, vesicles, nucleus and gap junction development. In particular, the interspecies cloned embryos showed more severe...

Endometrial thickness significantly affects clinical pregnancy and live birth rates in frozen-thawed embryo transfer cycles.

Science.gov (United States)

Bu, Zhiqin; Wang, Keyan; Dai, Wei; Sun, Yingpu

2016-07-01

In order to explore the relationship between endometrial thickness on the day of embryo transfer and pregnancy outcomes in frozen-thawed embryo transfer (FET) cycles, we retrospectively analyzed data from 2997 patients undergoing their first FET cycles from January 2010 to December 2012. All patients were divided into three groups (Group A, ≤8 mm; Group B, 9-13 mm; Group C, ≥14 mm) according to the endometrial thickness on embryo transfer day. Compared with patients in the other two groups, patients with thin endometrial thickness in Group A had significantly lower clinical pregnancy rate (33.4%, 41.3% and 45.4%, p birth rate (23.8%, 32.2% and 34.0%, p confidence interval (CI): 1.10-1.77, p birth rate (aOR: 1.50; 95% CI: 1.16-1.95, p < 0.01) were significant. We conclude that for patients undergoing FET, endometrial thickness on the embryo transfer day significantly affects IVF outcomes in cleavage embryo transfer cycles independent of other factors.

Blastocyst Morphology Holds Clues Concerning The Chromosomal Status of The Embryo

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Rita de Cassia Savio Figueira

2015-07-01

Full Text Available Background: Embryo morphology has been proposed as an alternative marker of chromosomal status. The objective of this retrospective cohort study was to investigate the association between the chromosomal status on day 3 of embryo development and blastocyst morphology. Materials and Methods: A total of 596 embryos obtained from 106 cycles of intracytoplasmic sperm injection (ICSI followed by preimplantation genetic aneuploidy screening (PGS were included in this retrospective study. We evaluated the relationship between blastocyst morphological features and embryonic chromosomal alteration. Results: Of the 564 embryos with fluorescent in situ hybridization (FISH results, 200 reached the blastocyst stage on day 5 of development. There was a significantly higher proportion of euploid embryos in those that achieved the blastocyst stage (59.0% compared to embryos that did not develop to blastocysts (41.2% on day 5 (P<0.001. Regarding blastocyst morphology, we observed that all embryos that had an abnormal inner cell mass (ICM were aneuploid. Embryos with morphologically normal ICM had a significantly higher euploidy rate (62.1%, P<0.001. As regards to the trophectoderm (TE morphology, an increased rate of euploidy was observed in embryos that had normal TE (65.8% compared to embryos with abnormal TE (37.5%, P<0.001. Finally, we observed a two-fold increase in the euploidy rate in high-quality blastocysts with both high-quality ICM and TE (70.4% compared to that found in low-quality blastocysts (31.0%, P<0.001. Conclusion: Chromosomal abnormalities do not impair embryo development as aneuploidy is frequently observed in embryos that reach the blastocyst stage. A high-quality blastocyst does not represent euploidy of chromosomes 13, 14, 15, 16, 18, 21, 22, X and Y. However, aneuploidy is associated with abnormalities in the ICM morphology. Further studies are necessary to confirm whether or not the transfer of blastocysts with low-quality ICM should be

In vitro development rate of preimplantation rabbit embryos cultured with different levels of melatonin.

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Mehaisen, Gamal Mohamed Kamel; Saeed, Ayman Moustafa

2015-02-01

This study aimed to investigate the effect of melatonin supplementation at different levels in culture medium on embryo development in rabbits. Embryos of 2-4 cells, 8-16 cells and morula stages were recovered from nulliparous Red Baladi rabbit does by laparotomy technique 24, 48 and 72 h post-insemination, respectively. Normal embryos from each stage were cultured to hatched blastocyst stages in either control culture medium (TCM-199 + 20% fetal bovine serum) or control supplemented with melatonin at 10(-3) M, 10(-6) M or 10(-9) M. No effect of melatonin was found on development of embryos recovered at 24 h post-insemination. The high level of melatonin at 10(-3) M adversely affected the in vitro development rates of embryos recovered at 48 h post-insemination (52 versus 86, 87 and 80% blastocyst rate; 28 versus 66, 78 and 59% hatchability rate for 10(-3) M versus 10(-9) M, 10(-6) M and control, respectively, P< 0.05). At the morula stage, melatonin at 10-3 M significantly increased the in vitro development of embryos (92% for 10(-3) M versus 76% for control, P < 0.05), while the hatchability rate of these embryos was not improved by melatonin (16-30% versus 52% for melatonin groups versus control, P < 0.05). Results show that a moderate level of melatonin (10(-6) M) may improve the development and hatchability rates of preimplantation rabbit embryos. The addition of melatonin at a 10-3 M concentration enhances the development of rabbit morulae but may negatively affect the development of earlier embryos. More studies are needed to optimize the use of melatonin in in vitro embryo culture in rabbits.

Assessment of swim-up and discontinuous density gradient in sperm sex preselection for bovine embryo production

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A.C Lucio

2012-06-01

Full Text Available The purpose of this work was to associate the modified swim-up method with centrifugation in density gradient for the separation of X-bearing spermatozoa. Sperm viability and integrity were evaluated through the Trypan Blue/Giemsa staining method. Quality control of centrifuged spermatozoa was performed in in vitro produced embryos. The results were validated by the sex ratio of in vitro produced embryos using PCR by Y- specific sequences present in bovine male genomic DNA. After determining genetic sex of in vitro produced embryos, the results showed difference (P<0.05 in deviation of sex ratio when comparing the control group (45.2% females with the other spermatozoa selection procedures (60.6% females (P<0.05. The sperm selection methods are capable of selecting X-bearing spermatozoa without compromising the spermatozoa fertility (cleavage and blastocyst rates, 70% and 26%, respectively and were considered relevant methods to be introduced in bovine in vitro produced embryo programs.

Development and quality of porcine parthenogenetically activated embryos after removal of zona pellucida

DEFF Research Database (Denmark)

Li, Rong; Liu, Ying; Pedersen, Hanne Skovsgaard

2013-01-01

at all developmental stages, but the difference was only significant at the five-cell stage. When compared with development of zona-intact embryos, ZP removal decreased the overall blastocyst percentage (83.9 ± 2.0 vs. 72.5 ± 2.9, respectively) and especially the percentage of good morphology (grades 1......, the developmental percentages, the frequency of apoptosis, and robustness after removal of the ZP by pronase. Three experiments were made between zona-free PA embryos and zona-intact embryos: (1) determination of the timing of developmental stages using time-lapse observations for 6 days; (2) determination...

Fruit, seed and embryo development of different cassava (Manihot ...

African Journals Online (AJOL)

Fruit, seed and embryo developments of different cassava (Manihot esculenta Crantz) genotypes, as well as embryo rescue, were investigated. The fruits of three genotypes after uncontrolled open pollination presented the same progressive development with similar sizes at different stages. There are large differences in ...

Homologous recombination and non-homologous end-joining repair pathways in bovine embryos with different developmental competence

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Henrique Barreta, Marcos [Universidade Federal de Santa Catarina, Campus Universitario de Curitibanos, Curitibanos, SC (Brazil); Laboratorio de Biotecnologia e Reproducao Animal-BioRep, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Garziera Gasperin, Bernardo; Braga Rissi, Vitor; Cesaro, Matheus Pedrotti de [Laboratorio de Biotecnologia e Reproducao Animal-BioRep, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Ferreira, Rogerio [Centro de Educacao Superior do Oeste-Universidade do Estado de Santa Catarina, Chapeco, SC (Brazil); Oliveira, Joao Francisco de; Goncalves, Paulo Bayard Dias [Laboratorio de Biotecnologia e Reproducao Animal-BioRep, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Bordignon, Vilceu, E-mail: vilceu.bordignon@mcgill.ca [Department of Animal Science, McGill University, Ste-Anne-De-Bellevue, QC (Canada)

2012-10-01

This study investigated the expression of genes controlling homologous recombination (HR), and non-homologous end-joining (NHEJ) DNA-repair pathways in bovine embryos of different developmental potential. It also evaluated whether bovine embryos can respond to DNA double-strand breaks (DSBs) induced with ultraviolet irradiation by regulating expression of genes involved in HR and NHEJ repair pathways. Embryos with high, intermediate or low developmental competence were selected based on the cleavage time after in vitro insemination and were removed from in vitro culture before (36 h), during (72 h) and after (96 h) the expected period of embryonic genome activation. All studied genes were expressed before, during and after the genome activation period regardless the developmental competence of the embryos. Higher mRNA expression of 53BP1 and RAD52 was found before genome activation in embryos with low developmental competence. Expression of 53BP1, RAD51 and KU70 was downregulated at 72 h and upregulated at 168 h post-insemination in response to DSBs induced by ultraviolet irradiation. In conclusion, important genes controlling HR and NHEJ DNA-repair pathways are expressed in bovine embryos, however genes participating in these pathways are only regulated after the period of embryo genome activation in response to ultraviolet-induced DSBs.

Homologous recombination and non-homologous end-joining repair pathways in bovine embryos with different developmental competence

International Nuclear Information System (INIS)

Henrique Barreta, Marcos; Garziera Gasperin, Bernardo; Braga Rissi, Vitor; Cesaro, Matheus Pedrotti de; Ferreira, Rogério; Oliveira, João Francisco de; Gonçalves, Paulo Bayard Dias; Bordignon, Vilceu

2012-01-01

This study investigated the expression of genes controlling homologous recombination (HR), and non-homologous end-joining (NHEJ) DNA-repair pathways in bovine embryos of different developmental potential. It also evaluated whether bovine embryos can respond to DNA double-strand breaks (DSBs) induced with ultraviolet irradiation by regulating expression of genes involved in HR and NHEJ repair pathways. Embryos with high, intermediate or low developmental competence were selected based on the cleavage time after in vitro insemination and were removed from in vitro culture before (36 h), during (72 h) and after (96 h) the expected period of embryonic genome activation. All studied genes were expressed before, during and after the genome activation period regardless the developmental competence of the embryos. Higher mRNA expression of 53BP1 and RAD52 was found before genome activation in embryos with low developmental competence. Expression of 53BP1, RAD51 and KU70 was downregulated at 72 h and upregulated at 168 h post-insemination in response to DSBs induced by ultraviolet irradiation. In conclusion, important genes controlling HR and NHEJ DNA-repair pathways are expressed in bovine embryos, however genes participating in these pathways are only regulated after the period of embryo genome activation in response to ultraviolet-induced DSBs.

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

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Jin, Jun-Xue; Kang, Jin-Dan; Li, Suo; Jin, Long; Zhu, Hai-Ying; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun, E-mail: yinxj33@msn.com

2015-01-02

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

Effects of N, N-dimethylglycine on the development of in vitro produced bovine embryos.

Science.gov (United States)

Takahashi, Toshikiyo; Itoh, Ryu; Nagai, Takashi

2009-06-01

This study investigated the effects of N, N-Dimethylglycine (DMG) on the development of in vitro produced (IVP) bovine embryos. IVP embryos were obtained by in vitro fertilization of in vitro matured oocytes for 6 h. In Experiment 1, IVP embryos were cultured in mSOFaa supplemented with bovine serum albumin but without glucose (SOF1) for 4 days, transferred to mSOFaa (with 5% fetal bovine serum and 1.5 mM glucose; SOF2) supplemented with 0 (control), 0.1,1 or 10 microM DMG and cultured for an additional 7 days (11 days in total) to assess their development in vitro. When cultured in the medium with 0.1 microM DMG, a significantly higher number of IVP embryos developed to the blastocyst and hatched blastocyst stages (40.3 and 40.8%, respectively) compared with the other groups (18.7-31.0% and 15.0-28.7%, respectively; PDMG for 4 days, transferred to SOF2 with or without 0.1 microM DMG and further cultured as in Experiment 1; DMG was added to either SOF1 or SOF2 and to both of them to assess its exposure effects on embryo development. When cultured continuously with DMG for 11 days, significantly higher rates of IVP embryos developed into blastocyst and hatched blastocyst stages (39.0 and 47.7%, respectively) compared with the other groups (31.0-32.2% and 29.5-31.0%, respectively; PDMG to IVC medium after 7 days of IVC. When DMG was added to IVC medium, the ratio of embryos developed to advanced developmental stages (No. of embryos developed to the blastocyst and expanded blastocyst stages/No. of embryos developed to the morula stage) was 28.7% (86/3) and 7 times higher than that of those cultured without DMG, 4.0% (52/13). These results suggest that addition of 0.1 microM DMG to mSOFaa during IVC of IVP bovine embryos has a promoting effect on their development.

Testing the embryo, testing the fetus.

Science.gov (United States)

Ehrich, K; Farsides, B; Williams, C; Scott, Rosamund

2007-12-01

This paper stems from an ethnographic, multidisciplinary study that explored the views and experiences of practitioners and scientists on social, ethical and clinical dilemmas encountered when working in the area of PGD for serious genetic disorders. We focus here on staff perceptions and experiences of working with embryos and helping women/couples to make choices that will result in selecting embryos for transfer and disposal of 'affected' embryos, compared to the termination of affected pregnancies following PND. Analysis and discussion of our data led us to consider the possible advantages of PGD and whether a gradualist account of the embryo's and fetus's moral status can account for all of these, particularly since a gradualist account concentrates on the significance of time (developmental stage) and makes no comment as to the significance of place (in-vitro, in-utero).

In vivo and in vitro development of Tibetan antelope (Pantholops hodgsonii interspecific cloned embryos

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Guanghua SU,Lei CHENG,Yu GAO,Kun LIU,Zhuying WEI,Chunling BAI,Fengxia YIN,Li GAO,Guangpeng LI,Shorgan BOU

2014-02-01

Full Text Available The Tibetan antelope is endemic to the Tibetan Plateau, China, and is now considered an endangered species. As a possible rescue strategy, the development of embryos constructed by interspecies somatic cell nuclear transfer (iSCNT was examined. Tibetan antelope fibroblast cells were transferred into enucleated bovine, ovine and caprine oocytes. These cloned embryos were then cultured in vitro or in the oviducts of intermediate animals. Less than 0.5% of the reconstructed antelope-bovine embryos cultured in vitro developed to the blastocyst stage. However, when the cloned antelope-bovine embryos were transferred to caprine oviducts, about 1.6% of the embryos developed to the blastocyst stage. In contrast, only 0.7% of the antelope-ovine embryos developed to the morula stage and none developed to blastocysts in ovine oviducts. The treatment of donor cells and bovine oocytes with trichostatin A did not improve the embryo development even when cultured in the oviducts of ovine and caprine. When the antelope-bovine embryos, constructed from oocytes treated with roscovitine or trichostatin A, were cultured in rabbit oviducts 2.3% and 14.3% developed to blastocysts, respectively. It is concluded that although some success was achieved with the protocols used, interspecies cloning of Tibetan antelope presents difficulties still to be overcome. The mechanisms resulting in the low embryo development need investigation and progress might require a deeper understanding of cellular reprogramming.

Tripolar mitosis and partitioning of the genome arrests human preimplantation development in vitro.

Science.gov (United States)

Ottolini, Christian S; Kitchen, John; Xanthopoulou, Leoni; Gordon, Tony; Summers, Michael C; Handyside, Alan H

2017-08-29

Following in vitro fertilisation (IVF), only about half of normally fertilised human embryos develop beyond cleavage and morula stages to form a blastocyst in vitro. Although many human embryos are aneuploid and genomically imbalanced, often as a result of meiotic errors inherited in the oocyte, these aneuploidies persist at the blastocyst stage and the reasons for the high incidence of developmental arrest remain unknown. Here we use genome-wide SNP genotyping and meiomapping of both polar bodies to identify maternal meiotic errors and karyomapping to fingerprint the parental chromosomes in single cells from disaggregated arrested embryos and excluded cells from blastocysts. Combined with time lapse imaging of development in culture, we demonstrate that tripolar mitoses in early cleavage cause chromosome dispersal to clones of cells with identical or closely related sub-diploid chromosome profiles resulting in intercellular partitioning of the genome. We hypothesise that following zygotic genome activation (ZGA), the combination of genomic imbalance and partial genome loss disrupts the normal pattern of embryonic gene expression blocking development at the morula-blastocyst transition. Failure to coordinate the cell cycle in early cleavage and regulate centrosome duplication is therefore a major cause of human preimplantation developmental arrest in vitro.

Zona pellucida damage to human embryos after cryopreservation and the consequences for their blastomere survival and in-vitro viability.

Science.gov (United States)

Van Den Abbeel, E; Van Steirteghem, A

2000-02-01

The study objective was to quantify zona pellucida (ZP) damage in cryopreserved human embryos. The influence of two different freezing containers was investigated, and the influence of freezing damage on the survival and viability of the embryos evaluated. ZP damage did not differ according to whether embryos originated from in-vitro fertilization (IVF) cycles or from IVF cycles in association with intracytoplasmic sperm injection (ICSI). The freezing container, however, significantly influenced the occurrence of ZP damage after cryopreservation. More damage was observed when the embryos were frozen-thawed using plastic cryovials than using plastic mini-straws (16.6% versus 2.3%; P plastic mini-straws. The further cleavage of frozen-thawed embryos suitable for transfer was not different whether there was ZP damage or not; however, it was higher when there was 100% blastomere survival as compared with when some blastomeres were damaged (79.0% versus 43.7%; P plastic mini-straws. In conclusion, the aim of a cryopreservation programme should be to have as many fully intact embryos as possible after thawing. Increased ZP damage might indicate a suboptimal cryopreservation procedure.

Nucleic acid metabolism in sea urchin embryos and its alteration after x-irradiation

International Nuclear Information System (INIS)

Kimura, I.

1974-01-01

Nucleic acid metabolism observed during embryogenesis of the sea urchin (Hemicentrotus pulcherrimus) and its alteration after x irradiation were studied on both qualitative and quantitative bases. MAK chromatographic analysis has revealed that the stage-dependent synthesis of RNA occurred during embryogenesis: some RNA families were observed specifically for early cleavage stage, not being observed at stages later than gastrulation. Further, they were modified by irradiation pari passu with delay and inhibition of cleavage. These results were discussed in comparison with our previous results on normal and regenerating rat liver

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

Energy Technology Data Exchange (ETDEWEB)

Osychenko, A A; Zalesskii, A D; Krivokharchenko, A S; Zhakhbazyan, A K; Nadtochenko, V A [N N Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow (Russian Federation); Ryabova, A V [A M Prokhorov General Physics Institute, Russian Academy of Sciences, Moscow (Russian Federation)

2015-05-31

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

EVALUATION OF ETHINYLESTRADIOL (EE2 EFFECT ON EMBRYO DEVELOPMENT IN COMMON CARP (CYPRINUS CARPIO

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GABI DUMITRESCU

2009-10-01

Full Text Available Worldwide, the scientific researches performed during the last years are focused on the determination of the negative effects caused by natural and antropogeneous chemical compounds on aquatic species; these species are more exposed to most pollutants than the land species, for the simple reason that the aquatic environment is the last destination for most residues. Our research team proposed to test the toxic effect caused by ethinylestradiol on embryo development in common carp (Cyprinus carpio. Common carp embryos were purchased from the fish farm S.C. Acva Prod S.R.L. Cefa, Bihor County these were obtained by artificial reproduction. After taking and selection, the fecundated spawns were introduced in 10 Nunk culture plates of 45 ml, where we introduced 40 ml water, too. We created 3 batches, with two replications, namely: batch 1 – control, batch 2 – in water, we added ethinylestradiol (EE2 in concentration of 1.5 ng L-1 and batch 3 – we added in water a concentration of 7 ng L-1 EE2. During the incubation, the Nunk plates were kept in breeding aquariums, at a temperature of 24°C. Successive to the supervision of embryos in batch 3, 48 hours post-fecundation, we could observe evolution stagnations, 70% of them being in the stage of 40 somites of the segmentation period. At the same age, 100% of the control batch- embryos entered the stage of advanced faringula, and in batch 2 all embryos were in the stage of incipient faringula. 60-72 hours post-fecundation, all embryos in the batch 3 died, 90% in the 40 somite stage of the segmentation period and 10% in the stage of incipient faringula. 85 hours post-fecundation, all embryos belonging to the control batch were in the larva stage, while in batch 2, 90% were in the larva stage and 10% died in the stage of advanced faringula.

Consistent left-right asymmetry cannot be established by late organizers in Xenopus unless the late organizer is a conjoined twin.

Science.gov (United States)

Vandenberg, Laura N; Levin, Michael

2010-04-01

How embryos consistently orient asymmetries of the left-right (LR) axis is an intriguing question, as no macroscopic environmental cues reliably distinguish left from right. Especially unclear are the events coordinating LR patterning with the establishment of the dorsoventral (DV) axes and midline determination in early embryos. In frog embryos, consistent physiological and molecular asymmetries manifest by the second cell cleavage; however, models based on extracellular fluid flow at the node predict correct de novo asymmetry orientation during neurulation. We addressed these issues in Xenopus embryos by manipulating the timing and location of dorsal organizer induction: the primary dorsal organizer was ablated by UV irradiation, and a new organizer was induced at various locations, either early, by mechanical rotation, or late, by injection of lithium chloride (at 32 cells) or of the transcription factor XSiamois (which functions after mid-blastula transition). These embryos were then analyzed for the position of three asymmetric organs. Whereas organizers rescued before cleavage properly oriented the LR axis 90% of the time, organizers induced in any position at any time after the 32-cell stage exhibited randomized laterality. Late organizers were unable to correctly orient the LR axis even when placed back in their endogenous location. Strikingly, conjoined twins produced by late induction of ectopic organizers did have normal asymmetry. These data reveal that although correct LR orientation must occur no later than early cleavage stages in singleton embryos, a novel instructive influence from an early organizer can impose normal asymmetry upon late organizers in the same cell field.

Metabolite profiling of somatic embryos of Cyclamen persicum in comparison to zygotic embryos, endosperm and testa

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Traud eWinkelmann

2015-08-01

Full Text Available Somatic embryogenesis has been shown to be an efficient in vitro plant regeneration system for many crops such as the important ornamental plant Cyclamen persicum, for which this regeneration pathway of somatic embryogenesis is of interest for the vegetative propagation of parental lines as well as elite plants. However, somatic embryogenesis is not commercially used in many crops due to several unsolved problems, such as malformations, asynchronous development, deficiencies in maturation and germination of somatic embryos. In contrast, zygotic embryos in seeds develop and germinate without abnormalities in most cases. Instead of time-consuming and labor-intensive experiments involving tests of different in vitro culture conditions and plant growth regulator supplements, we follow a more directed approach. Zygotic embryos served as a reference and were compared to somatic embryos in metabolomic analyses allowing the future optimization of the in vitro system. The aims of this study were to detect differences in the metabolite profiles of torpedo stage somatic and zygotic embryos of C. persicum. Moreover, major metabolites in endosperm and testa were identified and quantified.Two sets of extracts of two to four biological replicates each were analyzed. In total 52 metabolites were identified and quantified in the different tissues. One of the most significant differences between somatic and zygotic embryos was that the proline concentration in the zygotic embryos was about 40 times higher than that found in somatic embryos. Epicatechin, a scavenger for reactive oxygen species, was found in highest abundance in the testa. Sucrose, the most abundant metabolite was detected in significantly higher concentrations in zygotic embryos. Also, a yet unknown trisaccharide, was significantly enriched in zygotic embryos.

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

Science.gov (United States)

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 shell-less chick embryo culturing technique, reproduced successfully under local circumstances

International Nuclear Information System (INIS)

Zareen, N.; Khan, Y.

2008-01-01

The goal of this project was to demonstrate shell-less chick embryo culturing as a potential experimental model in the field of developmental anatomy. Freshly laid, fertilized chicken eggs of Egyptian Fayoumi breed were obtained from Poultry Research Institute Punjab, Rawalpindi. The fertilized chicken eggs were preincubated for 33 hours under standard conditions of 37.5 degree C and 65-75% humidity, to bring them to stage 9 (29-33 hours embryo, 7 somites) of Hamburger and Hamilton staging system. After this period, the eggs were taken out of the incubator, placed horizontally, wiped with 70% ethanol and permitted to air-dry for 10 minutes to reduce contamination from the egg surface and also to ensure that the embryo was properly positioned. The eggs contents were then transferred into the culture containers by cracking the undersides against an edge. The formation and growth of the embryonic membranes, the central nervous system - beginning from the vesicle stage, the circulatory system - including the heart, the eyes, beak, limbs, skin, feathers, wings and folding of the body were directly observed. Repeated successful culturing was attempted, tracing the developmental process of the embryo upto the 15th day of embryonic life at least after which the survivability period varied in different embryo cultures. The most advanced age reached in this project was day 19 of the embryonic life, which in researchers understanding is the latest developmental stage in shellless environment described as yet. The normal hatching time of this breed is 21-22 days. The size of these embryos was smaller as compared to the embryos of the same age that carried out their development inside their shells. (author)

Chronological Reorganization of Microtubules, Actin Microfilaments, and Chromatin during the First Cell Cycle in Swamp Buffalo (Bubalus bubalis Embryos

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Vibuntita Chankitisakul

2010-01-01

Full Text Available This paper aimed to study the dynamics of early embryonic development, in terms of redistribution of cytoskeleton (microtubules, actin microfilaments and chromatin configurations during the first cell cycle in swamp buffalo embryos. Oocytes were matured and fertilized in vitro, and they were fixed at various time points after IVF. At 6 h after IVF, 44.4% matured oocytes were penetrated by spermatozoa. Partial ZP digestion, however, did not improve fertilization rate compared to control (P>.05. At 12 h after IVF, the fertilized oocytes progressed to the second meiotic division and formed the female pronucleus simultaneously with the paternal chromatin continued to decondense. A sperm aster was observed radiating from the base of the decondensing sperm head. At 18 h after IVF, most presumptive zygotes had reached the pronuclear stage. The sperm aster was concurrently enlarged to assist the migration and apposition of pronuclei. Cell cleavage was facilitated by microfilaments and firstly observed by 30 h after IVF. In conclusion, the cytoskeleton actively involves with the process of fertilization and cleavage in swamp buffalo oocytes. The centrosomal material is paternally inherited. Fertilization failure is predominantly caused by poor sperm penetration. However, partial digestion of ZP did not improve fertilization rate.

EGF increases expression and activity of PAs in preimplantation rat embryos and their implantation rate

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Har-Vardi Iris

2007-01-01

Full Text Available Abstract Background Embryo implantation plays a major role in embryogenesis and the outcome of pregnancy. Plasminogen activators (PAs have been implicated in mammalian fertilization, early stages of development and embryo implantation. As in-vitro developing embryos resulted in lower implantation rate than those developed in-vivo we assume that a reduced PAs activity may be involved. In the present work we studied the effect of EGF on PAs activity, quantity and embryo implantation. Methods Zygotes were flushed from rat oviducts on day one of pregnancy and grown in-vitro in R1ECM supplemented with EGF (10 ng/ml and were grown up to the blastocyst stage. The control groups were grown in the same medium without EGF. The distribution and quantity of the PAs were examined using fluorescence immunohistochemistry followed by measurement of PAs activity using the chromogenic assay. Implantation rate was studied using the embryo donation model. Results PAs distribution in the embryos was the same in EGF treated and untreated embryos. Both PAs were localized in the blastocysts' trophectoderm, supporting the assumption that PAs play a role in the implantation process in rats. EGF increased the quantity of uPA at all stages studied but the 8-cell stage as compared with controls. The tissue type PA (tPA content was unaffected except the 8-cell stage, which was increased. The activity of uPA increased gradually towards the blastocyst stage and more so due to the presence of EGF. The activity of tPA did not vary with the advancing developmental stages although it was also increased by EGF. The presence of EGF during the preimplantation development doubled the rate of implantation of the treated group as compared with controls.

Classification of embryo sacs in the Eragrostis curvula Complex

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T. B. Vorster

1984-12-01

Full Text Available At each of 17 collecting points between Johannesburg and Brits in the Transvaal, three plants which belong to the  Eragrostis curvula Complex were collected and studied. A total o f 3 902 embryo sacs was examined in this sample. Of the embryo sacs examined, 3 306 were apomictic by means of diplospory, whereas 99 were sexual monosporic Polygonum-type embryo sacs. One hundred and nineteen embryo sacs were abnormal or divergent, and 378 were degenerated. There are indications that seasonal climatic fluctuations may be responsible for embryo sacs developing abnormally or degenerating. Simple and multiple correlations confirmed that sexual embryo sacs usually do not develop abnormally or degenerate during the later developmental stages. This finding lends credence to both the system of classification of individual embryo sacs and to the validity of the estimate of the proportion of sexuality of the plants sampled at each sampling point.

Human embryo research and the 14-day rule.

Science.gov (United States)

Pera, Martin F

2017-06-01

In many jurisdictions, restrictions prohibit the culture of human embryos beyond 14 days of development. However, recent reports describing the successful maintenance of embryos in vitro to this stage have prompted many in the field to question whether the rule is still appropriate. This Spotlight article looks at the original rationale behind the 14-day rule and its relevance today in light of advances in human embryo culture and in the derivation of embryonic-like structures from human pluripotent stem cells. © 2017. Published by The Company of Biologists Ltd.

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

Science.gov (United States)

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

1997-07-01

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

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

Energy Technology Data Exchange (ETDEWEB)

Park, Chan Jin; Song, Sang Ha; Kim, Dae Han; Gye, Myung Chan, E-mail: mcgye@hanyang.ac.kr

2017-01-15

Highlights: • Nickel inhibited the development of external gill in B. orientalis embryos. • The 168 h LC{sub 50} and EC{sub 50} values of nickel were 33.8 and 5.4 μM, respectively, in embryos. • Nickel induced abnormal tail development of embryos. • NF stage 26–31 was the most sensitive window for embryos to nickel exposure. • Nickel affected the calcium-dependent myogenic gene expression in embryos. - Abstract: The developmental toxicity of nickel was examined in the embryos of Bombina orientalis, a common amphibian in Korea. Based on a standard frog embryo teratogenesis assay, the LC{sub 50} and EC{sub 50} for malformation of nickel after 168 h of treatment were 33.8 μM and 5.4 μM, respectively. At a lethal concentration (100 μM), nickel treatment decreased the space between gill filaments and caused epithelial swelling and abnormal fusion of gill filaments. These findings suggest that nickel affects the functional development of gills, leading to embryonic death. At sublethal concentrations (1–10 μM), nickel produced multiple embryonic abnormalities, including bent tail and tail dysplasia. At 10 μM, nickel significantly decreased tail length and tail muscle fiber density in tadpoles, indicating inhibition of myogenic differentiation. Before hatching, the pre-muscular response to muscular response stages (stages 26–31) were the most sensitive period to nickel with respect to tail muscle development. During these stages, MyoD mRNA was upregulated, whereas myogenic regulatory factor 4 mRNA was downregulated by 0.1 μM nickel. Calcium-dependent kinase activities in muscular response stage embryos were significantly decreased by nickel, whereas these activities were restored by exogenous calcium. In tadpoles, 10 μM nickel significantly decreased the expression of the myosin heavy chain and the 12/101 muscle marker protein in the tail. Expression was restored by exogenous calcium. Our results indicate that nickel affects muscle development by

Developmental effects of mercury on Etheostoma caeruleum and E. spectabile: Predictable biomarkers of stress

Energy Technology Data Exchange (ETDEWEB)

Sharp, J.R. [Southeast Missouri State Univ., Cape Girardeau, MO (United States). Dept. of Biology

1995-12-31

Etheostoma caeruleum and E. spectabile are sympatric teleostean species of the Family Percidae. The ova diameters and incubation times are different: E. caeruleum (1.9mm and 12-d), E. spectabile (1.2mm and 8-d). For both species, cleavage stage (4--8 cell), mid-blastula, mid-neurula, and early-eye stage embryos were exposed to + {minus}1 a 24-h static-renewal test of 0, 10, 20, 40, 60, 80, 100 {micro}gHg {sup ++}L{sup {minus}1} to assess the effects of stage-specific initial mercury exposure on the embryo-larval responses. In addition, cleavage stage embryos were exposed to a 1-d, 2-d, and 4-d static-renewal toxicity test to determine the influence that exposure duration to mercury has on embryolarval responses. Five replicates of 10 embryos each were incubated at 18 C for each concentration and exposure variation. Embryos were allowed to develop until all had hatched or died. Four embryonic responses were assessed for each species and exposure protocol: 96-h LC50, AB50, SH50 and VH50. The typical nonstressor specific terata were noted for each species with an increase in percent of embryos expressing abnormal developmental patterns with increase mercury concentrations and severity of exposure. These included dwarfism, cephalic complications, ophthalmic abnormalities, cardiovascular abnormalities, various edema, and haemorrhagia. Hatching success and viability of hatch were likewise reduced with increasing severity of exposure and mercury concentration. Previously undetected terata that were observed in the first hatch included scoliosis, lordosis, kyphosis, synarthrodic jaws, and grossly enlarged yolk sacs.

Killing of preimplantation mouse embryos by main ingredients of cleansers AS and LAS

International Nuclear Information System (INIS)

Nomura, T.; Hata, S.; Shibata, K.; Kusafuka, T.

1987-01-01

When main ingredients of cleansers, alcohol sulfate (AS) and linear alkylbenzene sulfonate (LAS), were applied to the dorsal skin of pregnant JCL:ICR mice during preimplantation period, significant numbers of embryos collected from the oviducts and uteri on day 3 showed severe deformity or remained at the morula stage. Most of abnormal embryos were fragmented or remained at the 1-8 cell stages, and they were either dead or dying. Similar results were observed with commercially obtained kitchen detergent and hair shampoo. Fertilized eggs may be specifically sensitive to synthetic detergents. Very low doses of X-rays also induced significant yields of abnormal embryos. Major difference between X-rays and detergents was that X-ray-induced abnormality appeared at the morula or blastocyst stage, while detergent-induced one did at the earlier stages. (Auth.)

Rape embryogenesis. III. Embryo development in time

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Teresa Tykarska

2014-01-01

Full Text Available It was found that the growth curve of the rape embryo axis is of triple sigmoid type. Embryo growth occurs in 3 phases corresponding to 3 different periods of development. Phase I includes growth of the apical cell up to it's division into two layers of octants. Phase II comprises the increase of the spherical proembryo to the change of its symmetry from radial to bilateral. Phase III includes, growth of the embryo from the heart stage up to the end of embryogenesis. In each phase the relative growth rate increases drastically and then diminishes. The differences in growth intensity during the same phase are several-fold. The growth intensity maximum of the embryo axis occurs in phase II. The phasic growth intensity maxima occur: in phase I during apical cell elongation, :before its division, and in phases II and III in the periods of cell division ;growth in globular and torpedo-shaped -shaped embryos.

High Aneuploidy Rates Observed in Embryos Derived from Donated Oocytes are Related to Male Aging and High Percentages of Sperm DNA Fragmentation

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Javier García-Ferreyra

2015-01-01

Full Text Available Capsule Male aging effects on aneuploidy rates in embryos. Objective Paternal age is associated with decreasing sperm quality; however, it is unknown if it influences chromosomal abnormalities in embryos. The objective of this study is to evaluate if the aneuploidy rates in embryos are affected by advanced paternal age. Methods A total of 286 embryos, obtained from 32 in vitro fertilization/intracytoplasmic sperm injection cycles with donated oocytes in conjunction with preimplantation genetic diagnosis, were allocated according to paternal age in three groups: Group A: ≤39 years (n = 44 embryos; Group B: 40-49 years (n = 154 embryos; and Group C: ≥50 years (n = 88 embryos. Fertilization rates, embryo quality at day 3, blastocyst development, and aneuploidy embryo rates were then compared. Results There was no difference in the seminal parameters (volume, concentration, and motility in the studied groups. Fertilization rate, percentages of zygotes underwent cleavage, and good quality embryos on day 3 were similar between the three evaluated groups. The group of men ≥50 years had significantly more sperm with damaged DNA, low blastocyst development rate, and higher aneuploidy rates in embryos compared to the other two evaluated groups ( P 50 years old.

Centrioles in the beginning of human development.

OpenAIRE

Sathananthan, A H; Kola, I; Osborne, J; Trounson, A; Ng, S C; Bongso, A; Ratnam, S S

1991-01-01

We demonstrate the presence of centrioles in fertilized human oocytes at syngamy. Single or double centrioles within centrosomes were detected by transmission electron microscopy at one pole of the first cleavage spindle in normal and dispermic embryos (25-26 hr after insemination). Sperm centrioles were also closely associated with the male pronucleus (16-20 hr after insemination) in pronuclear stage embryos. A tripolar spindle derived from a tripronuclear embryo is also demonstrated with tw...

Determination of gene expression patterns using high-throughput RNA in situ hybridizaion to whole-mount Drosophila embryos

Energy Technology Data Exchange (ETDEWEB)

Weiszmann, R.; Hammonds, A.S.; Celniker, S.E.

2009-04-09

We describe a high-throughput protocol for RNA in situ hybridization (ISH) to Drosophila embryos in a 96-well format. cDNA or genomic DNA templates are amplified by PCR and then digoxigenin-labeled ribonucleotides are incorporated into antisense RNA probes by in vitro transcription. The quality of each probe is evaluated before ISH using a RNA probe quantification (dot blot) assay. RNA probes are hybridized to fixed, mixed-staged Drosophila embryos in 96-well plates. The resulting stained embryos can be examined and photographed immediately or stored at 4oC for later analysis. Starting with fixed, staged embryos, the protocol takes 6 d from probe template production through hybridization. Preparation of fixed embryos requires a minimum of 2 weeks to collect embryos representing all stages. The method has been used to determine the expression patterns of over 6,000 genes throughout embryogenesis.

Photoreversible UV-inactivation of messenger RNA in an insect embryo (Smittia spec., chironomidae, diptera)

International Nuclear Information System (INIS)

Jaeckle, H.; Kalthoff, K.

1980-01-01

Smittia embryos were UV-irradiated during intravitelline cleavage while nuclei are heavily shielded by yolk-rich cytoplasm and do not synthesize detectable amounts of RNA. Irradiation at 265, 285 and 295 nm wavelength caused biological inactivation, and pyrimidine dimer formation in maternal RNA. Marked effects on protein synthesis were also observed: (1) the overall rate of 35 S-methionine incorporation in vivo was reduced to less than half of the normal rate, (2) two dimensional gel electrophoresis revealed quantitative variations in the synthetic rate of some polypeptides and the appearance of new ones in UV-irradiated embryos, (3) translation of polyadenylated RNA from Smittia embryos in a cell-free system was inhibited by UV-irradiation in vivo, (4) the apparent degradation during early embryogenesis, of maternal polyadenylated RNA was retarded in UV-irradiated embryos. Exposure to light (400 nm) after UV caused partial photoreversal of all UV effects observed. This is the first data showing that animal mRNA, after UV-irradiation, can be photoreactivated in vivo. The results also strongly suggest that the photorepairable lesions consist of pyrimidine dimers generated in a photosensitized reaction. (author)

Effect of the cryopreservation method used, the embryonic stage and the use of conjugated linoleic acid isomers on the cryotolerance of in vitro-produced bovine embryos

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Luciana Simões Rafagnin Marinho

2015-12-01

Full Text Available Conjugated linoleic acid (CLA might be able to improve the cryotolerance of in vitro-produced (IVP embryos. The effect of two CLA isomers on the cryotolerance of bovine IVP embryos, as well as that of the stage of embryonic development and the method used for cryopreservation was evaluated by three experiments. In Experiment 1, oocytes (n = 3,917 were fertilized in vitro and cultured with 0, 50, 100, or 200 ?M trans-10, cis-12 (t10, c12 CLA. In Experiment 2, fertilized oocytes (n = 2,131 were cultured with 100 ?M t10, c12 or cis-9, trans-11 (c9, t11 CLA, or a combination of both isomers. The embryos were vitrified at the blastocyst (BL or the expanded blastocyst (EB stage. In Experiment 3, oocytes (n = 1,720 were fertilized and cultured with or without 100 ?M t10, c12 CLA, and the blastocysts were vitrified or frozen. Blastocyst development rate as well as the rates of re-expansion and hatching after thawing was recorded. Moreover, the mean cell number and mRNA expression of acetyl-CoA carboxylase (ACC1 and stearoyl-CoA desaturase (SCD1 as well as fatty acid synthase (FASN multienzyme complex were determined. In Experiment 1, the highest concentration of t10, c12 CLA that did not reduce blastocyst development rate was 100 ?M. In Experiment 2, the rates of re-expansion and hatching among the EBs obtained through IVP after supplementation with t10, c12 CLA (73.1% and 57.7%, with c9, t11 CLA (80.0% and 68.6%, with the combination (78.3% and 52.2%, and with the control group (85.4% and 58.3% were similar. At the BL stage, the rates of re-expansion and hatching were lower than those at the EB stage, and CLA combination allowed a hatching rate (8.0% lower than that observed in the control group (40.0%. In Experiment 3, the hatching rates for vitrified EBs (vitrified control; 67.4% and vitrified CLA EBs (65.8% were higher than those obtained for frozen EBs, exposed (13.3% or not exposed (28.6% to CLA. In addition, in Experiment 3, the hatching rate was

HSPC117 deficiency in cloned embryos causes placental abnormality and fetal death

International Nuclear Information System (INIS)

Wang, Yingying; Hai, Tang; Liu, Zichuan; Zhou, Shuya; Lv, Zhuo; Ding, Chenhui; Liu, Lei; Niu, Yuyu; Zhao, Xiaoyang; Tong, Man; Wang, Liu; Jouneau, Alice; Zhang, Xun; Ji, Weizhi; Zhou, Qi

2010-01-01

Somatic cell nuclear transfer (SCNT) has been successfully used in many species to produce live cloned offspring, albeit with low efficiency. The low frequency of successful development has usually been ascribed to incomplete or inappropriate reprogramming of the transferred nuclear genome. Elucidating the genetic differences between normal fertilized and cloned embryos is key to understand the low efficiency of SCNT. Here, we show that expression of HSPC117, which encodes a hypothetical protein of unknown function, was absent or very low in cloned mouse blastocysts. To investigate the role of HSPC117 in embryo development, we knocked-down this gene in normal fertilized embryos using RNA interference. We assessed the post-implantation survival of HSPC117 knock-down embryos at 3 stages: E9 (prior to placenta formation); E12 (after the placenta was fully functional) and E19 (post-natal). Our results show that, although siRNA-treated in vivo fertilized/produced (IVP) embryos could develop to the blastocyst stage and implanted without any difference from control embryos, the knock-down embryos showed substantial fetal death, accompanied by placental blood clotting, at E12. Furthermore, comparison of HSPC117 expression in placentas of nuclear transfer (NT), intracytoplasmic sperm injection (ICSI) and IVP embryos confirmed that HSPC117 deficiency correlates well with failures in embryo development: all NT embryos with a fetus, as well as IVP and ICSI embryos, had normal placental HSPC117 expression while those NT embryos showing reduced or no expression of HSPC117 failed to form a fetus. In conclusion, we show that HSPC117 is an important gene for post-implantation development of embryos, and that HSPC117 deficiency leads to fetal abnormalities after implantation, especially following placental formation. We suggest that defects in HSPC117 expression may be an important contributing factor to loss of cloned NT embryos in vivo.

Expression of nucleolar-related proteins in porcine preimplantation embryos produced in vivo and in vitro

DEFF Research Database (Denmark)

Bjerregaard, Bolette; Wrenzycki, Christine; Strejcek, Frantisek

2004-01-01

The expression of nucleolar-related proteins was studied as an indirect marker of the ribosomal RNA (rRNA) gene activation in porcine embryos up to the blastocyst stage produced in vivo and in vitro. A group of the in vivo-developed embryos were cultured with alpha-amanitin to block the de novo...... proteins pRb and p130, which are involved in cell-cycle regulation, was assessed by semiquantitative RT-PCR up to the blastocyst stage. Toward the end of third cell cycle, the nuclei in non-alpha-amanitin-treated, in vivo-produced embryos displayed different stages of transformation of the nuclear...... was delayed in porcine embryos produced in vitro compared to the in vivo-derived counterparts with respect to mRNAs encoding PAF53 and UBF. Moreover, differences existed in the mRNA expression patterns of pRb between in vivo- and in vitro-developed embryos. These findings show, to our knowledge for the first...

Single-cell analysis of differences in transcriptomic profiles of oocytes and cumulus cells at GV, MI, MII stages from PCOS patients.

Science.gov (United States)

Liu, Qiwei; Li, Yumei; Feng, Yun; Liu, Chaojie; Ma, Jieliang; Li, Yifei; Xiang, Huifen; Ji, Yazhong; Cao, Yunxia; Tong, Xiaowen; Xue, Zhigang

2016-12-22

Polycystic ovary syndrome (PCOS) is a common frequent endocrine disorder among women of reproductive age. Although assisted reproductive techniques (ARTs) are used to address subfertility in PCOS women, their effectiveness is not clear. Our aim was to compare transcriptomic profiles of oocytes and cumulus cells (CCs) between women with and without PCOS, and assess the effectiveness of ARTs in treating PCOS patients. We collected oocytes and CCs from 16 patients with and without PCOS patients to categorize them into 6 groups according to oocyte nuclear maturation. Transcriptional gene expression of oocyte and CCs was determined via single-cell RNA sequencing. The ratio of fertilization and cleavage was higher in PCOS patients than in non-PCOS patients undergoing ARTs, and there was no difference in the number of high-quality embryos between the groups. Differentially expressed genes including PPP2R1A, PDGFRA, EGFR, GJA1, PTGS2, TNFAIP6, TGF-β1, CAV1, INHBB et al. were investigated as potential causes of PCOS oocytes and CCs disorder at early stages, but their expression returned to the normal level at the metaphase II (MII) stage via ARTs. In conclusion, ARTs can improve the quality of cumulus-oocyte complex (COC) and increase the ratio of fertilization and cleavage in PCOS women.

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

Science.gov (United States)

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

The type of GnRH analogue used during controlled ovarian stimulation influences early embryo developmental kinetics

DEFF Research Database (Denmark)

Muñoz, Manuel; Cruz, María; Humaidan, Peter

2013-01-01

in the Instituto Valenciano de Infertilidad (IVI) Alicante and the Instituto Universitario-IVI Valencia, Spain, 2817 embryos deriving from 400 couples undergoing oocyte donation were analysed. After controlled ovarian stimulation and IVF/intracytoplamic sperm injection, the timing of embryonic cleavages......OBJECTIVE: To explore if the GnRH analogue used for controlled ovarian stimulation (COS) and the ovulation triggering factor (GnRH agonist+hCG triggering versus GnRH antagonist+GnRH agonist triggering) affect embryo development and kinetics. STUDY DESIGN: In a retrospective cohort study...... was assessed by a video time-lapse system. The results were analysed using Student's t test for comparison of timings (hours) and Chi-squared test for comparison of proportions. A p-value...

Radiation- and drug-induced DNA repair in mammalian oocytes and embryos

International Nuclear Information System (INIS)

Pedersen, R.A.; Brandriff, B.

1979-01-01

A review of studies showing ultraviolet- or drug-induced unscheduled DNA synthesis in mammalian oocytes and embryos suggests that the female gamete has an excision repair capacity from the earliest stages of oocyte growth. The oocyte's demonstrable excision repair capacity decreases at the time of meiotic maturation for unknown reasons, but the fully mature oocyte maintans a repair capacity, in contrast to the mature sperm, and contributes this to the zygote. Early embryo cells maintain relatively constant levels of excision repair until late fetal stages, when they lose their capacity for excision repair. These apparent changes in excision repair capacity do not have a simple relationship to known differences in radiation sensitivity of germ cells and embryos

Selection of Norway spruce somatic embryos by computer vision

Science.gov (United States)

Hamalainen, Jari J.; Jokinen, Kari J.

1993-05-01

A computer vision system was developed for the classification of plant somatic embryos. The embryos are in a Petri dish that is transferred with constant speed and they are recognized as they pass a line scan camera. A classification algorithm needs to be installed for every plant species. This paper describes an algorithm for the recognition of Norway spruce (Picea abies) embryos. A short review of conifer micropropagation by somatic embryogenesis is also given. The recognition algorithm is based on features calculated from the boundary of the object. Only part of the boundary corresponding to the developing cotyledons (2 - 15) and the straight sides of the embryo are used for recognition. An index of the length of the cotyledons describes the developmental stage of the embryo. The testing set for classifier performance consisted of 118 embryos and 478 nonembryos. With the classification tolerances chosen 69% of the objects classified as embryos by a human classifier were selected and 31$% rejected. Less than 1% of the nonembryos were classified as embryos. The basic features developed can probably be easily adapted for the recognition of other conifer somatic embryos.

Ca²⁺ influx-linked protein kinase C activity regulates the β-catenin localization, micromere induction signalling and the oral-aboral axis formation in early sea urchin embryos.

Science.gov (United States)

Yazaki, Ikuko; Tsurugaya, Toko; Santella, Luigia; Chun, Jong Tai; Amore, Gabriele; Kusunoki, Shinichiro; Asada, Akiko; Togo, Tatsuru; Akasaka, Koji

2015-06-01

Sea urchin embryos initiate cell specifications at the 16-cell stage by forming the mesomeres, macromeres and micromeres according to the relative position of the cells in the animal-vegetal axis. The most vegetal cells, micromeres, autonomously differentiate into skeletons and induce the neighbouring macromere cells to become mesoendoderm in the β-catenin-dependent Wnt8 signalling pathway. Although the underlying molecular mechanism for this progression is largely unknown, we have previously reported that the initial events might be triggered by the Ca2+ influxes through the egg-originated L-type Ca2+ channels distributed asymmetrically along the animal-vegetal axis and through the stretch-dependent Ca2+channels expressed specifically in the micromere at the 4th cleavage. In this communication, we have examined whether one of the earliest Ca2+ targets, protein kinase C (PKC), plays a role in cell specification upstream of β-catenin. To this end, we surveyed the expression pattern of β-catenin in early embryos in the presence or absence of the specific peptide inhibitor of Hemicentrotus pulcherrimus PKC (HpPKC-I). Unlike previous knowledge, we have found that the initial nuclear entrance of β-catenin does not take place in the micromeres, but in the macromeres at the 16-cell stage. Using the HpPKC-I, we have demonstrated further that PKC not only determines cell-specific nucleation of β-catenin, but also regulates a variety of cell specification events in the early sea urchin embryos by modulating the cell adhesion structures, actin dynamics, intracellular Ca2+ signalling, and the expression of key transcription factors.

Functional analysis of lysosomes during mouse preimplantation embryo development.

Science.gov (United States)

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

2013-01-01

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

Polyamine levels during the development of zygotic and somatic embryos of Pinus radiata

Science.gov (United States)

Rakesh Minocha; Dale R. Smith; Cathie Reeves; Kevin D. Steele; Subhash C. Minocha

1999-01-01

Changes in the cellular content of three polyamines (putrescine, spermidine and spermine) were compared at different stages of development in zygotic and somatic embryos of Pinus radiata D. Don. During embryo development, both the zygotic and the somatic embryos showed a steady increase in spermidine content, with either a small decrease or no...

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

Science.gov (United States)

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

2008-01-01

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

Influence of culture medium composition on relative mRNA abundances in domestic cat embryos.

Science.gov (United States)

Hribal, R; Jewgenow, K; Braun, B C; Comizzoli, P

2013-04-01

Different culture conditions have been used to produce domestic cat embryos. As part of the in vitro procedures, the medium composition significantly affects the quality of the embryo development also. Quality assessments based on cleavage kinetics and blastomere symmetry are useful, but embryos also can differ in their relative gene expression patterns despite similar morphological characteristics. The aim of this study was to compare cat embryos produced with two different in vitro culture systems routinely used in two different laboratories [Smithsonian Conservation Biology Institute, Washington D.C., USA (SCBI) and Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany (IZW)]. Specifically, relative mRNA expression patterns of critical genes for pre-implantation embryo development were assessed in both conditions. Embryos were produced in parallel in both culture systems by IVF using frozen-thawed ejaculated semen in the United States and fresh epididymal sperm in Germany. Success of embryo development in vitro was recorded as well as relative mRNA abundances [DNA methyltransferases 1 and 3A (DNMT1, DNMT3A), gap junction protein alpha 1 (GJA1), octamer-binding transcription factor 4 [OCT4], insulin-like growth factors 1 and 2 receptors (IGF1R, IGF2R), beta-actin (ACTB)] in pools of days 4-5 morulae by semi-quantitative RT-PCR assay. Percentages of cleaved embryos were similar (p > 0.05) between both culture systems, regardless of the location. OCT4 mRNA abundance was higher (p culture system compared with those from the IZW system when epididymal sperm was used for IVF. No clear correlation between the expression pattern and the culture system could be found for all other genes. It is suggested that OCT4 expression might be affected by the media composition in some conditions and can be the indicator of a better embryo quality. © 2012 Blackwell Verlag GmbH.

Genetic effects on mammalian development during and after implantation

Energy Technology Data Exchange (ETDEWEB)

Pedersen, R A; Spindle, A I

1976-05-01

The effects of gene expression on early mouse embryo differentiation were investigated by measuring the developmental rate of embryos homozygous for the yellow allele (A/sup y/) of the agouti locus and by determining the growth response of normal embryos treated with 5-bromodeoxyuridine (Brd U). The earliest effect of A/sup y/ homozygosity observed in cultured embryos was a delay in cleavage from the 2-cell to the 4-cell stage. Although A/sup y//A/sup y/ embryos were capable of some post-blastocyst development in vitro if the zona pellucida was removed, their inner cell masses disappeared during attachment and trophoblast outgrowth. The period of gene expression that is responsible for the abnormal development of A/sup y//A/sup y/ embryos may thus be between the 2-cell stage and implantation. It is therefore suggested that gene expression necessary for in vitro hatching, attachment, trophoblast outgrowth and inner cell mass development occurs between the morula and late blastocyst stages.

Depletion of primordial germ cells (PGCs) by X-irradiation to extraembryonic region of chicken embryos and expression of xenotransplanted quail PGCs

International Nuclear Information System (INIS)

Atsumi, Y.; Yazawa, S.; Usui, F.; Nakamura, Y.; Yamamoto, Y.; Tagami, T.; Hiramatsu, K.; Kagami, H.; Ono, T.

2009-01-01

The generation of germline chimeras by the transfer of primordial germ cells (PGCs) requires incorporation of the PGCs of the donor into the gonadal tissue of the recipient embryo. We investigated the utility of soft x-irradiation with application of a lead (12 x 3 x 0.25 mm, - 0.1 g) shield to the embryo proper for the production of chicken-quail germline chimeras. Chicken embryos shielded during irradiation for 120s (- 7.2 Gy) at stages 13 to 17 showed a hatchability of 35% (106/301), whereas the hatchability of unshielded embryos was 26% (27/105). The relative population of gonadal PGCs at stage 30 for embryos irradiated at stage 13 with or without shielding was 13 and 5%, respectively, of the value for nonirradiated controls. Chicken embryos irradiated at stages 13 or 14 with or without shielding and transfused with quail embryonic blood containing PGCs each exhibited - 130 relative population of donor PGCs in the left gonad at stage 30. Xenotransplanted hatchlings exhibited donor-derived PGCs as detected by Southern hybridization and PCR. Exposure of chicken embryos to - 7.2 Gy of x-radiation at stage 13 with the application of a lead shield to the embryo proper is thus a feasible approach to depletion of endogenous germ cells and the production of chicken-quail germline chimeras

Difference in birth weight of consecutive sibling singletons is not found in oocyte donation when comparing fresh versus frozen embryo replacements.

Science.gov (United States)

Galliano, Daniela; Garrido, Nicolás; Serra-Serra, Vicente; Pellicer, Antonio

2015-12-01

, cryopreservation technique, cleavage stage of the embryos, and time that embryos remained frozen did not reveal any significant trends. This study comparing siblings from OD cycles, and eliminating the independent variables that affect early events in pregnancy, revealed no difference in duration of gestation and live birth weights between fetuses obtained after the replacement of fresh or frozen embryos. Moreover, no clinical, phenotypic, or laboratory factors appeared to be relevant, once statistically controlled. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Effect of the microenvironment and embryo density on developmental characteristics and gene expression profile of bovine preimplantative embryos cultured in vitro.

Science.gov (United States)

Hoelker, Michael; Rings, Franka; Lund, Qamaruddin; Ghanem, Nasser; Phatsara, Chirawath; Griese, Josef; Schellander, Karl; Tesfaye, Dawit

2009-03-01

The Well of the Well (WOW) system has been developed to culture embryos in small groups or to track the development of single embryos. In the present study, we aimed to examine the effects of the microenvironment provided by the WOW system and embryo density on developmental rates, embryo quality and preimplantative gene expression profile of the resulting embryos. Embryos cultured in a group of 16 reached the blastocyst stage at a significantly lower level than zygotes cultured in a group of 50 (22.2 vs 30.3%), whereas zygotes cultured in WOW were able to compensate against low embryo densities, reaching a blastocyst rate as high as embryos cultured in a group of 50 (31.3 vs 30.3%). Moreover, embryos derived from WOW culture did not differ in terms of differential cell counts and apoptotic cell index compared with controls. The gene expression analysis revealed 62 transcripts to be upregulated and 33 transcripts to be downregulated by WOW culture. Comparing the in vivo derived blastocysts with the blastocysts derived from WOW culture, and group culture, expression of ATP5A1, PLAC8 and KRT8 was more similar to the embryos derived from WOW culture, whereas expression of S100A10 and ZP3 genes was more similar to blastocysts cultured in a group. In conclusion, microenvironment as well as embryo density significantly affected developmental rates. While subsequent blastocysts did not differ in terms of differential cell counts and apoptotic cell index, significant differences were observed in terms of the relative abundance of transcripts in the resulting embryos.

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

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Prabuddha Gupta

Full Text Available 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.

The effects of platelet lysate on maturation, fertilization and embryo development of NMRI mouse oocytes at germinal vesicle stage.

Science.gov (United States)

Pazoki, Hassan; Eimani, Hussein; Farokhi, Farah; Shahverdi, Abdol-Hossein; Tahaei, Leila Sadat

2016-04-01

Improving in vitro maturation could increase the rate of pregnancy from oocytes matured in vitro. Consequently, patients will be prevented from using gonadotropin with its related side effects. In this study, the maturation medium was enriched by platelet lysate (PL), then maturation and subsequent developments were monitored. Oocytes at germinal vesicle stage with cumulus cells (cumulus-oocyte complex) and without cumulus cells (denuded oocytes) were obtained from mature female mice. The maturation medium was enriched by 5 and 10 % PL and 5 % PL + 5 % fetal bovine serum (FBS) as experimental groups; the control groups' media consisted of 5 and 10 % FBS. After 18 h, the matured oocytes were collected and, after fertilization, subsequent development was monitored. The rates of maturation, fertilization and 2-cell embryo development for the denuded oocyte groups in experimental media 5 % PL and 5 % PL + 5 % FBS were significantly higher than those of the control groups ( P platelet lysate could improve the maturation rate in the absence of granulosa cells compared to media with FBS. This extract also had positive effects on fertilization and embryo development.

Regional differences in the lateral mobility of plasma membrane lipids in a molluscan embryo

OpenAIRE

Speksnijder, J.E.; Dohmen, M.R.; Tertoolen, L.G.J.; Laat, S.W. de

1985-01-01

Regional and temporal differences in plasma membrane lipid mobility have been analyzed during the first three cleavage cycles of the embryo of the polar-lobe-forming mollusc Nassarius reticulatus by the fluorescence photobleaching recovery (FPR) method, using 1,1′-ditetradecyl 3,3,3′,3′-tetramethylindocarbocyanine iodide (C14diI) as a fluorescent lipid probe. During this period of development the lateral diffusion coefficient of membrane lipids is consistently greater in the vegetal polar lob...

In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence.

Science.gov (United States)

Gerger, R P C; Ribeiro, E S; Forell, F; Bertolini, L R; Rodrigues, J L; Ambrósio, C E; Miglino, M A; Mezzalira, A; Bertolini, M

2010-02-18

The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovine oocytes were manually bisected and selected by DNA staining. One or two enucleated hemi-cytoplasts were paired and fused with an adult skin somatic cell. Cultured skin cells from an adult Nellore cow harvested at three distinct culture confluence levels (70-80, 80-90, and >95%) were used for construction of embryos and hemi-embryos. After activation, structures were cultured in vitro as one embryo (1 x 100%) or as aggregates of two hemi-embryos (2 x 50%) per microwell. Fusion, cleavage and blastocyst rates were compared using the chi(2) test. The fusion rate for hemi-embryos (51.4%) was lower than for embryos (67.6%), with no influence of degree of cell confluence. However, blastocyst rates improved linearly (7.0, 17.5, and 29.4%) with increases in cell confluence. We conclude that degree of cell culture confluence significantly influences subsequent embryo development; use of a cell population in high confluence (>90%) for nuclear transfer significantly improved blastocyst yield after cloning.

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

Energy Technology Data Exchange (ETDEWEB)

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

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, at sufficient concentrations and in specific forms, can be toxic to biological systems. The effects of fly ash contamination on exposed fish populations depend on the magnitude and duration of exposure, with the most significant risk considered to be the effects of specific ash constituents, especially selenium, on fish early life stages. Uptake by adult female fish of fly ash constituents through the food chain and subsequent maternal transfer of contaminants to the developing eggs is thought to be the primary route of selenium exposure to larval fish (Woock and others 1987, Coyle and others 1993, Lemly 1999, Moscatello and others 2006), but direct contact of the fertilized eggs and developing embryos to ash constituents in river water and sediments is also a potential risk factor (Woock and others 1987, Coyle and others 1993, Jezierska and others 2009). To address the risk of fly ash from the Kingston spill to the reproductive health of downstream fish populations, ORNL has undertaken a series of studies in collaboration with TVA including: (1) a field study of the bioaccumulation of fly ash constituents in fish ovaries and the reproductive condition of sentinel fish species in reaches of the Emory and Clinch Rivers affected by the fly ash spill; (2) laboratory tests of the potential toxicity of fly ash from the spill area on fish embryonic and larval development (reported in the

The effects of X-rays on chicken embryos

International Nuclear Information System (INIS)

Wendt, E.

1981-01-01

The radiosensitivity of the chickens embryo changes in the course of its 21 days of development. A period of relatively high resistance in the early stages of development (1. to 3. day of incubation), is followed by an increase of sensitivity from the 4. day onwards. In 1- to 3-day-old embryos, X-rays cause nonspecific malformations in those organs which are in a phenocritical period at the moment of irradiation. In mature embryos (4. to 20. day of incubation) characteristic biochemical changes in the metabolism of proteins and amino-acids as well as the nitrogen excretion can be observed as the predominant radiation effects. (orig.)

Patterns of free calcium in zebrafish embryos

NARCIS (Netherlands)

Creton, R; Speksnijder, JE; Jaffe, LF

Direct knowledge of Ca2+ patterns in vertebrate development is largely restricted to early stages, in which they control fertilization, ooplasmic segregation and cleavage. To explore new roles of Ca2+ in vertebrate development, we injected the Ca2+ indicator aequorin into zebrafish eggs and imaged

Protein phosphorylation during coconut zygotic embryo development

International Nuclear Information System (INIS)

Islas-Flores, I.; Oropeza, C.; Hernandez-Sotomayor, S.M.T.

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 [gamma-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

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

Science.gov (United States)

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

2014-02-01

Currently, in vitro-produced embryos derived by ovum pick up (OPU) and in vitro fertilization (IVF) technologies represent approximately one-third of the embryos worldwide in cattle. Nevertheless, the culture of small groups of embryos from an individual egg donor is an issue that OPU-IVF laboratories have to face. In this work, we tested whether the development and quality of the preimplantation embryos in vitro cultured in low numbers (five embryos) could be improved by the addition of epidermal growth factor, insulin, transferrin and selenium (EGF-ITS) or by the WOW system. With this aim, immature oocytes recovered from slaughtered heifers were in vitro matured and in vitro fertilized. Presumptive zygotes were then randomly cultured in four culture conditions: one large group (LG) (50 embryos/500 μl medium) and three smaller groups [five embryos/50 μl medium without (control) or with EGF-ITS (EGF-ITS) and five embryos per microwell in the WOW system (WOW)]. Embryos cultured in LG showed a greater ability to develop to blastocyst stage than embryos cultured in smaller groups, while the blastocyst rate of WOW group was significantly higher than in control. The number of cells/blastocyst in LG was higher than control or WOW, whereas the apoptosis rate per blastocyst was lower. On the other hand, the addition of EGF-ITS significantly improved both parameters compared to the control and resulted in similar embryo quality to LG. In conclusion, the WOW system improved embryo development, while the addition of EGF-ITS improved the embryo quality when smaller groups of embryos were cultured. © 2013 Blackwell Verlag GmbH.

In vitro production of buffalo embryos from stepwise vitrified immature oocytes.

Science.gov (United States)

Abd-Allah, Saber Mohammed

2009-01-01

This study was conducted to produce buffalo embryos in vitro from stepwise vitrified immature oocytes. Cumulus oocyte complexes (COCs) were obtained from the ovaries of slaughtered buffalo and were collected from the local abattoir. Selected COCs were exposed to a vitrification solution consisting of 40% ethylene glycol (EG) plus 0.3 M trehalose and 20% polyvinyl pyrrolidone (PVP) for 1 min and loaded in 0.25 ml plastic mini-straws containing 100 microl of 10% sucrose. The loaded cryostraws were cryopreserved by stepwise vitrification and were stored in liquid nitrogen for 4 to 6 months. Data analysis revealed a high percentage of post-thawing morphologically normal immature oocytes (80.7%) with a low percentage of damaged oocytes. There were no significant differences in the maturation (82.1%), cleavage (47.6%) and buffalo embryo development (15.4%) produced by the stepwise vitrified immature oocytes in comparison to the three observations in fresh oocytes (88.3%, 50.4% and 19.4%, respectively, p<0.05).

In vitro production of buffalo embryos from stepwise vitrified immature oocytes

Directory of Open Access Journals (Sweden)

Saber Mohammed Abd-Allah

2009-09-01

Full Text Available This study was conducted to produce buffalo embryos in vitro from stepwise vitrified immature oocytes. Cumulus oocyte complexes (COCs were obtained from the ovaries of slaughtered buffalo and were collected from the local abattoir. Selected COCs were exposed to a vitrification solution consisting of 40% ethylene glycol (EG plus 0.3 M trehalose and 20% polyvinyl pyrrolidone (PVP for 1 min and loaded in 0.25 ml plastic mini-straws containing 100 µl of 10% sucrose. The loaded cryostraws were cryopreserved by stepwise vitrification and were stored in liquid nitrogen for 4 to 6 months. Data analysis revealed a high percentage of post-thawing morphologically normal immature oocytes (80.7% with a low percentage of damaged oocytes. There were no significant differences in the maturation (82.1%, cleavage (47.6% and buffalo embryo development (15.4% produced by the stepwise vitrified immature oocytes in comparison to the three observations in fresh oocytes (88.3%, 50.4% and 19.4%, respectively, p<0.05.

Evaluation of cell number and DNA content in mouse embryos cultivated with uranium

International Nuclear Information System (INIS)

Kundt, Mirian S.; Cabrini, Romulo L.

2000-01-01

The evaluation of the degree of development, the number of cells and the DNA content, were used to evaluate the embryotoxicity of uranium. Embryos at a one cell stage were cultured with uranyl nitrate hexahydrate (UN) at a final concentration of uranium (U) of 26, 52 and 104 μgU/ml. At 24 hs of culture, the embryos at the 2 cell stage, were put in new wells with the same concentrations of U as the previous day, until the end of the period of incubation at 72 hs. At 72 hs of culture, 87% of the original one cell embryos were at morula stage, and in those cultivated with uranium, the percentage decreased significantly to 77; 63.24 and 40.79% respectively for the different U concentrations. Those embryos that exhibited a normal morphology, were selected and fixed on slides. The number of cells per embryo was evaluated in Giemsa stained preparations. The DNA content was evaluated cytophotometrically in Feulgen stained nuclei. The number of cells decreased significantly from 20,3 ± 5.6 in the control to 19 ± 6; 14 ± 3 and 13.9 ± 5.6 for the different concentrations. All the embryos evaluated showed one easy recognizable polar body, which was used a haploid indicator (n). The content of DNA was measured in a total of 20 control embryos and 16 embryos cultivated with UN. In control embryos, 92,7% of the nuclei presented a normal ploidy from 2n to 4n, 2,9% nuclei were hypoploid and 4,4% were hyperploid. The percentage of hypoploid nuclei rose in a dose-dependent fashion to 3.45; 44.45 and 50.34% respectively for the embryos cultured at the different U concentrations. The results indicate that U is embryotoxic, that its effects are dose dependent at the concentrations used in this study and that even those embryos that show a normal morphology, can be genetically affected. We show that the model employed is extremely sensitive. It is possible to use the preimplantation embryos, as a model to test the effect of possibly mutagenic agents of the nuclear industry. (author)

Cotyledonary somatic embryos of Pinus pinaster Ait. most closely resemble fresh, maturing cotyledonary zygotic embryos: biological, carbohydrate and proteomic analyses.

Science.gov (United States)

Morel, Alexandre; Trontin, Jean-François; Corbineau, Françoise; Lomenech, Anne-Marie; Beaufour, Martine; Reymond, Isabelle; Le Metté, Claire; Ader, Kevin; Harvengt, Luc; Cadene, Martine; Label, Philippe; Teyssier, Caroline; Lelu-Walter, Marie-Anne

2014-11-01

Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined.

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

Directory of Open Access Journals (Sweden)

Bernd Rosenbusch

2014-01-01

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

Cell cycle-related fluctuations in transcellular ionic currents and plasma membrane Ca2+/Mg2+ ATPase activity during early cleavages of Lymnaea stagnalis embryos.

Science.gov (United States)

Zivkovic, Danica; Créton, Robbert; Dohmen, René

1991-08-01

During the first four mitotic division cycles of Lymnaea stagnalis embryos, we have detected cell cycle-dependent changes in the pattern of transcellular ionic currents and membrane-bound Ca 2+ -stimulated ATPase activity. Ionic currents ranging from 0.05 to 2.50 μA/cm 2 have been measured using the vibrating probe technique. Enzyme activity was detected using Ando's cytochemical method (Ando et al. 1981) which reveals Ca 2+ /Mg 2+ ATPase localization at the ultrastructural level, and under high-stringency conditions with respect to calcium availability, it reveals Ca 2+ -stimulated ATPase. The ionic currents and Ca 2+ -stimulated ATPase localization have in common that important changes occur during the M-phase of the cell cycles. Minimal outward current at the vegetal pole coincides with metaphase/anaphase. Maximal inward current at the animal pole coincides with the onset of cytokinesis at that pole. Ca 2+ -stimulated ATPase is absent from one half of the embryo at metaphase/anaphase of the two- and four-cell stage, whereas it is present in all cells during the remaining part of the cell cycle. Since fluctuations of cytosolic free calcium concentrations appear to correlate with both karyokinesis and cytokinesis, we speculate that part of the cyclic pattern of Ca 2+ -stimulated ATPase localization and of the transcellular ionic currents reflects the elevation of cytosolic free calcium concentration during the M-phase.

Physiology and culture of the human blastocyst.

Science.gov (United States)

Gardner, David K; Lane, Michelle; Schoolcraft, William B

2002-01-01

The human embryo undergoes many changes in physiology during the first 4 days of life as it develops and differentiates from a fertilized oocyte to the blastocyst stage. Concomitantly, the embryo is exposed to gradients of nutrients within the female reproductive tract and exhibits changes in its own nutrient requirements and utilization. Determining the nature of such nutrient gradients in the female tract and the changing requirements of the embryo has facilitated the formulation of stage-specific culture media designed to support embryo development throughout the preimplantation period. Resultant implantation rates attained with the culture and transfer of human blastocysts are higher than those associated with the transfer of cleavage stage embryos to the uterus. Such increases in implantation rates have facilitated the establishment of high pregnancy rates while reducing the number of embryos transferred. With the introduction of new scoring systems for the blastocyst and the non-invasive assessment of metabolic activity of individual embryos, it should be possible to move to single blastocyst transfer for the majority of patients.

Supplementation of fetal bovine serum alters histone modification H3R26me2 during preimplantation development of in vitro produced bovine embryos

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Daniel R. Arnold

2015-07-01

Full Text Available Abstract In vitro production (IVP of bovine embryos is not only of great economic importance to the cattle industry, but is also an important model for studying embryo development. The aim of this study was to evaluate the histone modification, H3R26me2 during pre-implantation development of IVP bovine embryos cultured with or without serum supplementation and how these in vitro treatments compared to in vivo embryos at the morula stage. After in vitro maturation and fertilization, bovine embryos were cultured with either 0 or 2.5% fetal bovine serum (FBS. Development was evaluated and embryos were collected and fixed at different stages during development (2-, 4-, 8-, 16-cell, morula and blastocyst. Fixed embryos were then used for immunofluorescence utilizing an antibody for H3R26me2. Images of stained embryos were analyzed as a percentage of total DNA. Embryos cultured with 2.5% FBS developed to blastocysts at a greater rate than 0%FBS groups (34.85±5.43% vs. 23.38±2.93%; P<0.05. Levels of H3R26me2 changed for both groups over development. In the 0%FBS group, the greatest amount of H3R26me2 staining was at the 4-cell (P<0.05, 16-cell (P<0.05 and morula (P<0.05 stages. In the 2.5%FBS group, only 4-cell stage embryos were significantly higher than all other stages (P<0.01. Morula stage in vivo embryos had similar levels as the 0%FBS group, and both were significantly higher than the 2.5%FBS group. These results suggest that the histone modification H3R26me2 is regulated during development of pre-implantation bovine embryos, and that culture conditions greatly alter this regulation.

Polycystin-1 Cleavage and the Regulation of Transcriptional Pathways

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Merrick, David; Bertuccio, Claudia A.; Chapin, Hannah C.; Lal, Mark; Chauvet, Veronique; Caplan, Michael J.

2013-01-01

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of end stage renal disease, affecting ~1 in 1,000 people. The disease is characterized by the development of numerous large fluid filled renal cysts over the course of decades. These cysts compress the surrounding renal parenchyma and impair its function. Mutations in two genes are responsible for ADPKD. The protein products of both of these genes, polycystin-1 and polycystin-2, localize to the primary cilium and participate in a wide variety of signaling pathways. Polycystin-1 undergoes several proteolytic cleavages that produce fragments that manifest biological activities. Recent results suggest that the production of polycystin-1 cleavage fragments is necessary and sufficient to account for at least some, although certainly not all, of the physiological functions of the parent protein. PMID:23824180

Automatic segmentation of time-lapse microscopy images depicting a live Dharma embryo.

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Zacharia, Eleni; Bondesson, Maria; Riu, Anne; Ducharme, Nicole A; Gustafsson, Jan-Åke; Kakadiaris, Ioannis A

2011-01-01

Biological inferences about the toxicity of chemicals reached during experiments on the zebrafish Dharma embryo can be greatly affected by the analysis of the time-lapse microscopy images depicting the embryo. Among the stages of image analysis, automatic and accurate segmentation of the Dharma embryo is the most crucial and challenging. In this paper, an accurate and automatic segmentation approach for the segmentation of the Dharma embryo data obtained by fluorescent time-lapse microscopy is proposed. Experiments performed in four stacks of 3D images over time have shown promising results.

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

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

2008-07-01

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

A set of simple cell processes is sufficient to model spiral cleavage.

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Brun-Usan, Miguel; Marín-Riera, Miquel; Grande, Cristina; Truchado-Garcia, Marta; Salazar-Ciudad, Isaac

2017-01-01

During cleavage, different cellular processes cause the zygote to become partitioned into a set of cells with a specific spatial arrangement. These processes include the orientation of cell division according to: an animal-vegetal gradient; the main axis (Hertwig's rule) of the cell; and the contact areas between cells or the perpendicularity between consecutive cell divisions (Sachs' rule). Cell adhesion and cortical rotation have also been proposed to be involved in spiral cleavage. We use a computational model of cell and tissue biomechanics to account for the different existing hypotheses about how the specific spatial arrangement of cells in spiral cleavage arises during development. Cell polarization by an animal-vegetal gradient, a bias to perpendicularity between consecutive cell divisions (Sachs' rule), cortical rotation and cell adhesion, when combined, reproduce the spiral cleavage, whereas other combinations of processes cannot. Specifically, cortical rotation is necessary at the 8-cell stage to direct all micromeres in the same direction. By varying the relative strength of these processes, we reproduce the spatial arrangement of cells in the blastulae of seven different invertebrate species. © 2017. Published by The Company of Biologists Ltd.

Integrated toxic evaluation of sulfamethazine on zebrafish: Including two lifespan stages (embryo-larval and adult) and three exposure periods (exposure, post-exposure and re-exposure).

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Yan, Zhengyu; Yang, Qiulian; Jiang, Weili; Lu, Jilai; Xiang, Zhongrun; Guo, Ruixin; Chen, Jianqiu

2018-03-01

Persistence of antibiotics in aquatic environment may pose a risk to the non-target aquatic organisms. This study provided an integrated evaluation to analyze the toxic stress of sulfamethazine (SMZ) on zebrafish in two lifespan stages (embryo-larval and adult) and three exposure periods (exposure, post-exposure and re-exposure). Zebrafish embryos and adult zebrafish were exposed to SMZ at 0.2, 20 and 2000 μg/L, respectively. The results showed that SMZ at any given concentration inhibited the hatching of embryos at 58-96 hpf (hours post-fertilization). Our result also indicated that two major kinds of the malformation, which was induced by the antibiotic, were edema and spinal curvature. Additionally, the antibiotic stimulated the heartbeat while reduced the body length of the embryo at 72 hpf. Superoxide dismutase (SOD) activities and malondialdehyde (MDA) contents significantly increased at 120 hpf when the embryos were exposed to the lowest concentration (0.2 μg/L) of the antibiotic. On the other hand, the antibiotic induced SOD activities and MDA contents in adult zebrafish in the exposure and re-exposure periods. The MDA contents could recover while SOD activities still increased in 2 d after the exposure. Both SOD activities and MDA contents could recover in 7 d after the exposure. Levels of SOD and MDA in the re-exposure were higher than those in the first exposure. Our results suggested that SMZ had toxic effects on both embryos and adult zebrafish, and provided an integrated evaluation of the toxic effects of SMZ on zebrafish at a new perspective. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

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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.

Analysis of the expression of putatively imprinted genes in bovine peri-implantation embryos

DEFF Research Database (Denmark)

Tveden-Nyborg, Pernille Yde; Alexopoulos, N.I.; Cooney, M.A.

2008-01-01

The application of assisted reproductive technologies (ART) has been shown to induce changes in the methylation of the embryonic genome, leading to aberrant gene expression, including that of imprinted genes. Aberrant methylation and gene expression has been linked to the large offspring syndrome...... (LOS) in bovine embryos resulting in increased embryonic morbidity and mortality. In the bovine, limited numbers of imprinted genes have been studied and studies have primarily been restricted to pre-implantation stages. This study reports original data on the expression pattern of 8 putatively...... imprinted genes (Ata3, Dlk1, Gnas, Grb10, Magel2, Mest-1, Ndn and Sgce) in bovine peri-implantation embryos. Two embryonic developmental stages were examined, Day 14 and Day 21. The gene expression pattern of single embryos was recorded for in vivo, in vitro produced (IVP) and parthenogenetic embryos...

Developmental potential of bovine hand-made clone embryos reconstructed by aggregation or fusion with distinct cytoplasmic volumes.

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Ribeiro, Eduardo de Souza; Gerger, Renato Pereira da Costa; Ohlweiler, Lain Uriel; Ortigari, Ivens; Mezzalira, Joana Cláudia; Forell, Fabiana; Bertolini, Luciana Relly; Rodrigues, José Luiz; Ambrósio, Carlos Eduardo; Miglino, Maria Angélica; Mezzalira, Alceu; Bertolini, Marcelo

2009-09-01

Animal cloning has been associated with developmental abnormalities, with the level of heteroplasmy caused by the procedure being one of its potential limiting factors. The aim of this study was to determine the effect of the fusion of hemicytoplasts or aggregation of hemiembryos, varying the final cytoplasmic volume, on development and cell density of embryos produced by hand-made cloning (HMC), parthenogenesis or by in vitro fertilization (IVF). One or two enucleated hemicytoplasts were paired and fused with one skin somatic cell. Activated clone and zona-free parthenote embryos and hemiembryos were in vitro cultured in the well-of-the-well (WOW) system, being allocated to one of six experimental groups, on a per WOW basis: single clone or parthenote hemiembryos (1 x 50%); aggregation of two (2 x 50%), three (3 x 50%), or four (4 x 50%) clone or parthenote hemiembryos; single clone or parthenote embryos (1 x 100%); or aggregation of two clone or parthenote embryos (2 x 100%). Control zona-intact parthenote or IVF embryos were in vitro cultured in four-well dishes. Results indicated that the increase in the number of aggregated structures within each WOW was followed by a linear increase in cleavage, blastocyst rate, and cell density. The increase in cytoplasmic volume, either by fusion or by aggregation, had a positive effect on embryo development, supporting the establishment of pregnancies and the birth of a viable clone calf after transfer to recipients. However, embryo aggregation did not improve development on a hemicytoplast basis, except for the aggregation of two clone embryos.

Neural network classification of sweet potato embryos

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Molto, Enrique; Harrell, Roy C.

1993-05-01

Somatic embryogenesis is a process that allows for the in vitro propagation of thousands of plants in sub-liter size vessels and has been successfully applied to many significant species. The heterogeneity of maturity and quality of embryos produced with this technique requires sorting to obtain a uniform product. An automated harvester is being developed at the University of Florida to sort embryos in vitro at different stages of maturation in a suspension culture. The system utilizes machine vision to characterize embryo morphology and a fluidic based separation device to isolate embryos associated with a pre-defined, targeted morphology. Two different backpropagation neural networks (BNN) were used to classify embryos based on information extracted from the vision system. One network utilized geometric features such as embryo area, length, and symmetry as inputs. The alternative network utilized polar coordinates of an embryo's perimeter with respect to its centroid as inputs. The performances of both techniques were compared with each other and with an embryo classification method based on linear discriminant analysis (LDA). Similar results were obtained with all three techniques. Classification efficiency was improved by reducing the dimension of the feature vector trough a forward stepwise analysis by LDA. In order to enhance the purity of the sample selected as harvestable, a reject to classify option was introduced in the model and analyzed. The best classifier performances (76% overall correct classifications, 75% harvestable objects properly classified, homogeneity improvement ratio 1.5) were obtained using 8 features in a BNN.

/sup 31/P nuclear-magnetic-resonance studies an the developing embryos of Xenopus laevis

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Gadian, D G [Oxford Univ. (UK). Dept. of Biochemistry; Colman, A [Oxford Univ. (UK). Dept. of Zoology

1976-01-01

The concentrations of nucleoside triphosphate, inorganic phosphate and yolk proteins, phosvitin and lipovitellin, have been monitored in living embryos of Xenopus laevis by /sup 31/P nuclear magnetic resonance (NMR) spectroscopy. The nucleoside triphosphate levels remain relatively constant at about 3.5 - 4.5 nmol/embryo at least until the 'spontaneous movement' stage of development. By the swimming tadpole stage an inorganic phosphate resonance representing about 30 nmol/embryo becomes evident in the NMR spectrum. Computer manipulation also shows such a resonance, although smaller, to be present at a somewhat earlier developmental stage; these findings are confirmed biochemically. The major contribution to the NMR spectrum of oocytes, unfertilized eggs and early embryos is the yolk phosphoprotein resonance. On isolation of the yolk from the embryos it is possible to quantify the contribution to the NMR spectrum from the lipid-phosphate and protein-phosphate moieties of the yolk proteins. During development, as the yolk is used up, it is found that the protein-phosphate resonance disappears at a greater rate than the lipid-phosphate peak. The total phosphorus content of the embryo (ca. 200 nmol/embryo) is shown biochemically to remain constant during development; however, the total amount of phosphorus observed by NMR decreases by about 40% during development. From the resonance positions of their ..cap alpha.., ..beta.. and ..gamma.. phosphate groups is is deduced that the nucleoside triphosphate molecules are liganded in vivo to a divalent cation which is not manganese, but could be either magnesium or calcium. From the position of the inorganic phosphate resonance it is deduced that the internal pH of embryos where this resonance is evident is 6.8 +- 0.2.

Embryo development and corresponding factors affecting in vitro germination of Cymbidium faberi × C. sinense hybrid seeds

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Li Fengtong

2016-01-01

Full Text Available A better understanding of embryo development would provide insights into seed quality and subsequent germination events in the interspecific hybridization of Cymbidium faberi ‘Jiepeimei’ × C. sinense ‘Qijianheimo’. At the mature stage, 26.1% of the ovules were abnormal. Most of the hybrid embryos could develop normally. Abortions mainly occurred at the zygote (9.5% and 2-4-celled embryo (15.1% stages. No germination was observed at 90 and 105 days after pollination (DAP, when the embryo was at the early globular stage, with abundant organelles but no storage materials. During 110-130 DAP, the globular embryo was formed and the starch grains began to accumulate in plastids. The hybrid seeds collected at 120 DAP showed initiation of germination. Germination significantly increased at 135 DAP and was maximal at 150 DAP, during which period the hybrid embryos developed into the late globular stage. The storage materials, i.e. lipid and protein bodies, began to accumulate and the filamentary structures derived from suspensor cells still persisted. After the seeds matured (160 DAP, the germination percentage declined sharply. Safranin staining revealed that the outer seed coat was totally cuticularized and the inner seed coat appeared as a cuticle layer enclosing the embryo proper tightly, which may be the main factor inhibiting the subsequent germination of hybrid seeds. In conclusion, 150 DAP should be the opportune time for the in vitro germination of C. faberi ‘Jiepeimei’ × C. sinense ‘Qijianheimo’ hybrid seeds.

["The first stages of the human egg" by Auguste d'Eternod published one hundred years ago in the Comptes Rendus de l'Association des Anatomistes].

Science.gov (United States)

Catala, M

2014-06-01

The development of the embryo and foetus fascinates, but its study in humans is difficult because of both technical and ethical problems. Auguste d'Eternod, Swiss embryologist, published in 1913 an article entitled "The early stages of the human egg" in the Comptes Rendus de l'Association des Anatomistes, the ancestor of the journal Morphologie. This work is focused not only on the early stages of development: fertilization, cleavage of the egg, blastocyst formation, gastrulation, but also on the extra-embryonic processes characteristic of mammals. On the occasion of the centenary of the publication of this work, I propose a critical review by placing the data published in the literature and historical context of the time. Finally, I try to extract from these observations the concepts that are still used today by embryologists. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

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

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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.

Irreversible barrier to the reprogramming of donor cells in cloning with mouse embryos and embryonic stem cells.

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Ono, Yukiko; Kono, Tomohiro

2006-08-01

Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. However, a fundamental question in this context is whether the developmental abnormalities reported to date are specific to somatic cloning. The aim of this study was to determine the stage of nuclear differentiation during development that leads to developmental abnormalities associated with embryo cloning. In order to address this issue, we reconstructed cloned embryos using four- and eight-cell embryos, morula embryos, inner cell mass (ICM) cells, and embryonic stem cells as donor nuclei and determined the occurrence of abnormalities such as developmental arrest and placentomegaly, which are common characteristics of all mouse somatic cell clones. The present analysis revealed that an acute decline in the full-term developmental competence of cloned embryos occurred with the use of four- and eight-cell donor nuclei (22.7% vs. 1.8%) in cases of standard embryo cloning and with morula and ICM donor nuclei (11.4% vs. 6.6%) in serial nuclear transfer. Histological observation showed abnormal differentiation and proliferation of trophoblastic giant cells in the placentae of cloned concepti derived from four-cell to ICM cell donor nuclei. Enlargement of placenta along with excessive proliferation of the spongiotrophoblast layer and glycogen cells was observed in the clones derived from morula embryos and ICM cells. These results revealed that irreversible epigenetic events had already started to occur at the four-cell stage. In addition, the expression of genes involved in placentomegaly is regulated at the blastocyst stage by irreversible epigenetic events, and it could not be reprogrammed by the fusion of nuclei with unfertilized oocytes. Hence, developmental abnormalities such as placentomegaly as

SOMATIC EMBRYOGENESIS AND MORPHOANATOMY OF Ocotea porosa SOMATIC EMBRYOS

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Luciana Luiza Pelegrini

2013-01-01

Full Text Available Ocotea porosa seeds have strong tegument dormancy, recalcitrant behavior, low and irregular germination and that makes its natural propagation difficult. The aim of this study was to establish a protocol of regeneration of Ocotea porosa from somatic embryogenesis. Immature embryonic axes were inoculated on WPM culture medium supplemented with 2.4-D (200 μM combined or not with hydrolyzed casein or glutamine (0.5 or 1 g l-1, during 90 days. The repetitive embryogenesis was induced on medium with 2.4-D (22.62 μM combined with 2-iP (2.46 μM followed by transfer to culture medium with hydrolyzed casein or glutamine (1 g l-1 during 90 days. The maturation of somatic embryos was tested in culture medium containing NAA (0.5 μM and 2-iP (5; 10 and 20 μM. The highest percentage of somatic embryos induction (8.3% was observed in WPM culture medium containing 200 μM 2.4-D and 1 g L-1 hydrolyzed casein and the development of somatic embryos occurred indirectly. Repetitive somatic embryogenesis was promoted in WPM medium containing hydrolyzed casein or glutamine. However, the culture medium containing hydrolyzed casein promoted the maintenance of embryogenic capacity for more than two years. During the maturity phase, there was a low progression of globular embryos to cordiform and torpedo stages. The different ontogenetic stages of somatic embryos of Ocotea porosa were characterized by histological studies.

SOMATIC EMBRYOGENESIS AND MORPHOANATOMY OF Ocotea porosa SOMATIC EMBRYOS

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Luciana Luiza Pelegrini

2013-12-01

Full Text Available http://dx.doi.org/10.5902/1980509812343Ocotea porosa seeds have strong tegument dormancy, recalcitrant behavior, low and irregular germinationand that makes its natural propagation difficult. The aim of this study was to establish a protocol ofregeneration of Ocotea porosa from somatic embryogenesis. Immature embryonic axes were inoculatedon WPM culture medium supplemented with 2.4-D (200 μM combined or not with hydrolyzed casein orglutamine (0.5 or 1 g l-1, during 90 days. The repetitive embryogenesis was induced on medium with 2.4-D(22.62 μM combined with 2-iP (2.46 μM followed by transfer to culture medium with hydrolyzed caseinor glutamine (1 g l-1 during 90 days. The maturation of somatic embryos was tested in culture mediumcontaining NAA (0.5 μM and 2-iP (5; 10 and 20 μM. The highest percentage of somatic embryos induction(8.3% was observed in WPM culture medium containing 200 μM 2.4-D and 1 g L-1 hydrolyzed casein andthe development of somatic embryos occurred indirectly. Repetitive somatic embryogenesis was promotedin WPM medium containing hydrolyzed casein or glutamine. However, the culture medium containinghydrolyzed casein promoted the maintenance of embryogenic capacity for more than two years. Duringthe maturity phase, there was a low progression of globular embryos to cordiform and torpedo stages.The different ontogenetic stages of somatic embryos of Ocotea porosa were characterized by histologicalstudies.

Feel, smell and see in an egg: emergence of perception and learning in an immature invertebrate, the cuttlefish embryo.

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Romagny, Sébastien; Darmaillacq, Anne-Sophie; Guibé, Mathieu; Bellanger, Cécile; Dickel, Ludovic

2012-12-01

It is now well established that prenatal sensory experience affects development itself and has long-term consequences in terms of postnatal behavior. This study focused on the functionality of the sensory system in cuttlefish in ovo. Embryos of stage 23, 25 and 30 received a tactile, chemical or visual stimulus. An increase of mantle contraction rhythm was taken to indicate a behavioral response to the stimulus. We clearly demonstrated that tactile and chemical systems are functional from stage 23, whereas the visual system is functional only from stage 25. At stage 25 and 30, embryos were also exposed to a repeated light stimulus. Stage 30 embryos were capable of habituation, showing a progressive decrease in contractions across stimulations. This process was not due to fatigue as we observed response recovery after a dishabituation tactile stimulus. This study is the first to show that cuttlefish embryos behaviorally respond to stimuli of different modalities and that the visual system is the last to become functional during embryonic development, as in vertebrate embryos. It also provides new evidence that the memory system develops in ovo in cuttlefish.

The Effects of Progesterone on Oocyte Maturation and Embryo Development

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Saeed Zavareh

2013-01-01

Full Text Available Oocyte maturation and embryo development are controlled by intra-ovarian factors suchas steroid hormones. Progesterone (P4 exists in the follicular fluid that contributes tonormal mammalian ovarian function and has several critical functions during embryodevelopment and implantation, including endometrial receptivity, embryonic survivalduring gestation and transformation of the endometrial stromal cells to decidual cells.It is well known that the physiological effects of P4 during the pre-implantation stages ofsome mammal’s embryos are mediated by P4 receptors and their gene expression is determined.The effects of P4 on oocytes and embryo development have been assessed bysome investigations, with contradictory results. P4, a dominant steroid in follicular fluidat approximately 18 hours after the luteinizing hormone (LH surge may have a criticalrole in maturation of oocytes at the germinal stage. However, it has been shown that differentconcentrations of P4 could not improve in vitro maturation rates of germinal vesicles(GV in cumulus oocyte complexes (COCs and cumulus denuded oocytes (CDOs.Culture media supplemented with P4 significantly improved mouse embryo development.In addition, an in vivo experimental design has shown high blastocyst survival andimplantation rates in P4-treated mice.In this review we explain some of the findings that pertain to the effects of P4 onoocyte maturation and embryo development both in vitro and in vivo.

Culture media for human pre-implantation embryos in assisted reproductive technology cycles.

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Youssef, Mohamed M A; Mantikou, Eleni; van Wely, Madelon; Van der Veen, Fulco; Al-Inany, Hesham G; Repping, Sjoerd; Mastenbroek, Sebastiaan

2015-11-20

.Six studies reported clinical pregnancy rate. One of these found a difference between the media compared, suggesting that for cleavage-stage embryo transfer, Quinn's Advantage was associated with higher clinical pregnancy rates than G5 (odds ratio (OR) 1.56; 95% confidence interval (CI) 1.12 to 2.16; 692 women). This study was available only as an abstract and the quality of the evidence was low.With regards to adverse effects, three studies reported multiple pregnancies and six studies reported miscarriage. None of them found any evidence of a difference between the culture media used. None of the studies reported on the health of offspring.Most studies (22/32) failed to report their source of funding and none described their methodology in adequate detail. The overall quality of the evidence was rated as very low for nearly all comparisons, the main limitations being imprecision and poor reporting of study methods. An optimal embryo culture medium is important for embryonic development and subsequently the success of IVF or ICSI treatment. There has been much controversy about the most appropriate embryo culture medium. Numerous studies have been performed, but no two studies compared the same culture media and none of them found any evidence of a difference between the culture media used. We conclude that there is insufficient evidence to support or refute the use of any specific culture medium. Properly designed and executed randomised trials are necessary.

Use of methanol as cryoprotectant and its effect on sox genes and proteins in chilled zebrafish embryos.

Science.gov (United States)

Desai, Kunjan; Spikings, Emma; Zhang, Tiantian

2015-08-01

Methanol is a widely used cryoprotectant (CPA) in cryopreservation of fish embryos, however little is known about its effect at the molecular level. This study investigated the effect of methanol on sox gene and protein expression in zebrafish embryos (50% epiboly) when they were chilled for 3 h and subsequently warmed and cultured to the hatching stages. Initial experiments were carried out to evaluate the chilling tolerance of 50% epiboly embryos which showed no significant differences in hatching rates for up to 6 h chilling in methanol (0.2-, 0.5- and 1 M). Subsequent experiments in embryos that had been chilled for 3 h in 1 M methanol and warmed and cultured up to the hatching stages found that sox2 and sox3 gene expression were increased significantly in hatched embryos that had been chilled compared to non-chilled controls. Sox19a gene expression also remained above control levels in the chilled embryos at all developmental stages tested. Whilst stable sox2 protein expression was observed between non-chilled controls and embryos chilled for 3 h with or without MeOH, a surge in sox19a protein expression was observed in embryos chilled for 3 h in the presence of 1 M MeOH compared to non-chilled controls and then returned to control levels by the hatching stage. The protective effect of MeOH was increased with increasing concentrations. Effect of methanol at molecular level during chilling was reported here first time which could add new parameter in selection of cryoprotectant while designing cryopreservation protocol. Copyright © 2015. Published by Elsevier Inc.

Treatment of porcine donor cells and reconstructed embryos with the antioxidant melatonin enhances cloning efficiency.

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Pang, Yun-Wei; An, Lei; Wang, Peng; Yu, Yong; Yin, Qiu-Dan; Wang, Xiao-Hong; Xin-Zhang; Qian-Zhang; Yang, Mei-Ling; Min-Guo; Wu, Zhong-Hong; Tian, Jian-Hui

2013-05-01

This study was conducted to investigate the effect of melatonin during the culture of donor cells and cloned embryos on the in vitro developmental competence and quality of cloned porcine embryos. At concentrations of 10(-6 )M or 10(-8) M, melatonin significantly enhanced the proliferation of porcine fetal fibroblasts (PFFs), and the blastocyst rate was significantly increased in the 10(-10) M melatonin-treated donor cell group. Cloned embryo development was also improved in embryo culture medium that was supplemented with 10(-9) M or 10(-12) M melatonin. When both donor cells and cloned embryos were treated with melatonin, the cleavage rate and total cell number of blastocysts were not significantly affected; however, the blastocyst rate was increased significantly (20.0% versus 11.7%). TUNEL assays showed that combined melatonin treatment reduced the rate of apoptotic nuclei (3.6% versus 6.1%). Gene expression analysis of the apoptosis-related genes BAX, BCL2L1, and p53 showed that the expression of BCL2L1 was significantly elevated 2.7-fold relative to the control group, while the expression of BAX and p53 was significantly decreased by 3.7-fold and 23.2-fold, respectively. In addition, we detected the expression of two melatonin receptors (MT1 and MT2) in PFFs but not in porcine cloned embryos. We conclude that exogenous melatonin enhances the development of porcine cloned embryos and improves embryo quality by inhibiting p53-mediated apoptotic pathway. The proliferation of PFFs may be mediated by receptor binding, but the beneficial effects of melatonin on embryonic development may be receptor-independent, possibly through melatonin's ability to directly scavenge free radicals. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

Using CR1aa versus KSOM as the culture medium for in vitro embryo production of cattle

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Endang Triwulaninngsih

2002-03-01

Full Text Available This research has been conducted at the laboratory of in vitro fertilization in the Department of Animal Science University of Wisconsin, USA. These embryos can be used for improving genetic value of Indonesian cattle. Oocytes were matured in TCM- 199 medium (in 5% CO2 incubator and at 390C enriched with follicle stimulating hormone (FSH 10 μl/ml, oestradiol 17 β 1μl/ml and 10% Fetal Calf Serum (FCS. The oocytes were fertilized in vitro with motile sperm and incubation between sperm and oocytes in fertilization medium Tyroide Albumin Lactate Pyruvate (TALP for 20 hours. All zygotes were cultured in CR1aa (n=1549 medium versus modification of protein-free pottasium simplex optimized medium (KSOM (n=675 up to blastocyst stage and were fed FCS 5 μl/50 μl medium on day 6, as treatment A and B respectively. Data were analyzed by completely randomized design with SAS program. Percentages of cleavage, morula, blastocyst, expanded blastocyst, unfertilized and degenerated ova in this study were 91.4% vs 75.6 %; 75.6% vs 58.9%; 61.5% vs 38.5%; 31.2% vs 5.1%, 8.6% vs 24.4%, 15.7% vs 8% which were significantly different (P<0.01 for treatment CR1aa and KSOM respectively. Based on this study, CR1aa medium is better culture medium than KSOM for efficient in vitro production (IVP of bovine embryos.

DNA apurinization and apyridinization in gamma-irradiated Bombyx mori embryos at various stages of development

International Nuclear Information System (INIS)

Agaev, F.A.; Vasil'ev, S.P.; Gaziev, A.I.

1993-01-01

A study was made of the formation and repair of apurine-apyridine sites in DNA of gamma-irradiated 3- and 7-day embryos of Bombix mori differing drastically in radiosensitivity. The kinetics of the postirradiation recovery of AP sites in DNA of 3- and 7-day Bombix mori embryos was heterogeneous and varied significantly

Runx expression is mitogenic and mutually linked to Wnt activity in blastula-stage sea urchin embryos.

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Anthony J Robertson

Full Text Available The Runt homology domain (Runx defines a metazoan family of sequence-specific transcriptional regulatory proteins that are critical for animal development and causally associated with a variety of mammalian cancers. The sea urchin Runx gene SpRunt-1 is expressed throughout the blastula stage embryo, and is required globally during embryogenesis for cell survival and differentiation.Depletion of SpRunt-1 by morpholino antisense-mediated knockdown causes a blastula stage deficit in cell proliferation, as shown by bromodeoxyuridine (BrdU incorporation and direct cell counts. Reverse transcription coupled polymerase chain reaction (RT-PCR studies show that the cell proliferation deficit is presaged by a deficit in the expression of several zygotic wnt genes, including wnt8, a key regulator of endomesoderm development. In addition, SpRunt-1-depleted blastulae underexpress cyclinD, an effector of mitogenic Wnt signaling. Blastula stage cell proliferation is also impeded by knockdown of either wnt8 or cyclinD. Chromatin immunoprecipitation (ChIP indicates that Runx target sites within 5' sequences flanking cyclinD, wnt6 and wnt8 are directly bound by SpRunt-1 protein at late blastula stage. Furthermore, experiments using a green fluorescent protein (GFP reporter transgene show that the blastula-stage operation of a cis-regulatory module previously shown to be required for wnt8 expression (Minokawa et al., Dev. Biol. 288: 545-558, 2005 is dependent on its direct sequence-specific interaction with SpRunt-1. Finally, inhibitor studies and immunoblot analysis show that SpRunt-1 protein levels are negatively regulated by glycogen synthase kinase (GSK-3.These results suggest that Runx expression and Wnt signaling are mutually linked in a feedback circuit that controls cell proliferation during development.

Gene Coexpression and Evolutionary Conservation Analysis of the Human Preimplantation Embryos

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Tiancheng Liu

2015-01-01

Full Text Available Evolutionary developmental biology (EVO-DEVO tries to decode evolutionary constraints on the stages of embryonic development. Two models—the “funnel-like” model and the “hourglass” model—have been proposed by investigators to illustrate the fluctuation of selective pressure on these stages. However, selective indices of stages corresponding to mammalian preimplantation embryonic development (PED were undetected in previous studies. Based on single cell RNA sequencing of stages during human PED, we used coexpression method to identify gene modules activated in each of these stages. Through measuring the evolutionary indices of gene modules belonging to each stage, we observed change pattern of selective constraints on PED for the first time. The selective pressure decreases from the zygote stage to the 4-cell stage and increases at the 8-cell stage and then decreases again from 8-cell stage to the late blastocyst stages. Previous EVO-DEVO studies concerning the whole embryo development neglected the fluctuation of selective pressure in these earlier stages, and the fluctuation was potentially correlated with events of earlier stages, such as zygote genome activation (ZGA. Such oscillation in an earlier stage would further affect models of the evolutionary constraints on whole embryo development. Therefore, these earlier stages should be measured intensively in future EVO-DEVO studies.

Perkembangan Praimplantasi Embrio Mencit dengan Materi Genetik yang Berasal dari Parental, Maternal, dan Inti Sel Somatik (PRE-IMPLANTATION DEVELOPMENT OF MOUSE EMBRYO WITH GENETIC MATERIAL DERIVED FROM PARENTAL, MATERNAL AND SOMATIC CELL NUCLEUS

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Harry Murti

2014-05-01

Full Text Available Cloned embryo and parthenogenetic embryo are a potential source of stem cells for regenerativemedicine. Stem cells derived from those embryos are expected to overcome the ethical issues to the use offertilization embryos for therapeutic purposes. The pre-implantation development is a critical step fordeveloping embryos reach the blastocyst stage. The objectives in vivo of this research are to produce mousecloned embryo, parthenogenetic embryo, and fertilized embryo and to study stages of  in vitro pre-implantation development culture. In vivo fertilized embryos, mouse oocytes, and cumulus cells were usedin this study. Treatment was performed on female mice superovulated with PMSG and hCG injections.Two-cell stage of in vivo fertilized embryos were collected on the second day post hCG injection. Clonedembryos were produced through Somatic Cell Nuclear Transfer (SCNT, which included enucleation, nucleartransfer and artificial activation. Parthenogenetic embryos were produced with artificial activationtechnique. The result of the research indicated that SCNT application was able to produce cloned embryos which could develop to blastocyst stage (3,2%. In addition, artificial activation of oocytes could produceparthenogenetic embryos which were able to develop up to the blastocyst stage (8,6%. In conclusion,efficiency level of parthenogenetic embryos that is able to reach the blastocyst stage was higher than in thecloned embryos. Fertilized embryos shows a better development and more efficient compared to in vitrocloned embryos and parthenogenetic embryos cultures.

Cultures of preimplantation mouse embryos

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Streffer, C.; Molls, M.

1987-01-01

In the preimplantation mouse embryos the chromosomal damage develops through several postradiation cell cycles and mitoses. New chromosome aberrations are seen during the second and third postradiation mitoses. Also, more micronuclei appear during later postradiation interphases. This is in agreement with the assumption that unrepaired chromosomal radiation damage develops during the cell generation cycle to such a form (i.e. double-strand breaks in DNA) that chromosomal breaks occur. This proposition is strengthened by the observation that radiation-induced damage is more rapidly expressed after neutron exposure (first or second postradiation mitosis) than after exposure to X rays at the one- or two-cell stage. The preimplantation mouse embryo culture is an inviting system for additional studies at the molecular level, especially now that within the last few years more sensitive methods have been developed for study of DNA and protein structure, regulation, and synthesis. The results from these studies of cultures of preimplantation mouse embryos present a favorable case for the study of complex biological systems under very defined conditions in vitro for extrapolation to effects in vivo

Effect of abscisic acid on the linoleic acid metabolism in developing maize embryos

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Abian, J.; Gelpi, E.; Pages, M.

1991-01-01

Partially purified protein extracts from maize (Zea mays L.) embryos, whether treated or not with abscisic acid (ABA), were incubated with linoleic acid (LA) and 1-[ 14 C]LA. The resulting LA metabolites were monitored by high performance liquid chromatography with a radioactivity detector and identified by gas chromatography-mass spectrometry. α- and γ-ketol metabolites arising from 9-lipoxygenase activity were the more abundant compounds detected in the incubates, although the corresponding metabolites produced by 13-lipoxygenase were also present in the samples. In addition, a group of stereoisomers originating form two isomeric trihydroxy acids (9,12,13-trihydroxy-10-octadecenoic and 9,10,13-trihydroxy-11-octadecenoic acids) are described. Important variations in the relative proportions of the LA metabolites were observed depending on the embryo developmental stage and on ABA treatment. Two new ABA-induced compounds have been detected. These compounds are present in embryos at all developmental stages, being more abundant in old (60 days) embryos. Furthermore, ABA induction of these compounds is maximum at very young development stages, decreasing as maturation progresses. A tentative structure for these compounds (10-oxo-9,13-dihydroxy-11-octadecenoic acid and 12-oxo-9,13-dihydroxy-10-octadecenoic acid) is also provided. This study revealed an early stage in maize embryogenesis characterized by a higher relative sensitivity to ABA. The physiological importance of ABA on LA metabolism is discussed

Deprenyl Enhances the Teratogenicity of Hydroxyurea in Organogenesis Stage Mouse Embryos

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Schlisser, Ava E.; Hales, Barbara F.

2013-01-01

Hydroxyurea, an antineoplastic drug, is a model teratogen. The administration of hydroxyurea to CD1 mice on gestation day 9 induces oxidative stress, increasing the formation of 4-hydroxy-2-nonenal adducts to redox-sensitive proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the caudal region of the embryo. GAPDH catalytic activity is reduced, and its translocation into the nucleus is increased. Because the nuclear translocation of GAPDH is associated with oxidative stress–induced cell death, we hypothesized that this translocation plays a role in mediating the teratogenicity of hydroxyurea. Deprenyl (also known as selegiline), a drug used as a neuroprotectant in Parkinson’s disease, inhibits the nuclear translocation of GAPDH. Hence, timed pregnant CD1 mice were treated with deprenyl (10mg/kg) on gestation day 9 followed by the administration of hydroxyurea (400 or 600mg/kg). Deprenyl treatment significantly decreased the hydroxyurea-induced nuclear translocation of GAPDH in the caudal lumbosacral somites. Deprenyl enhanced hydroxyurea-mediated caudal malformations, inducing specifically limb reduction, digit anomalies, tail defects, and lumbosacral vertebral abnormalities. Deprenyl did not augment the hydroxyurea-induced inhibition of glycolysis or alter the ratio of oxidized to reduced glutathione. However, it did dramatically increase cleaved caspase-3 in embryos. These data suggest that nuclear GAPDH plays an important, region-specific, role in teratogen-exposed embryos. Deprenyl exacerbated the developmental outcome of hydroxyurea exposure by a mechanism that is independent of oxidative stress. Although the administration of deprenyl alone did not affect pregnancy outcome, this drug may have adverse consequences when combined with exposures that increase the risk of malformations. PMID:23696560

Teratogenic Effects of Diatom Metabolites on Sea Urchin Paracentrotus lividus Embryos

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Giovanna Romano

2010-03-01

Full Text Available The diatom-derived polyunsaturated aldehydes (PUAs, 2-trans,4-trans-decadienal, 2-trans,4-trans-octadienal, 2-trans,4-trans,7-octatrienal, 2-trans,4-trans-heptadienal, as well as tridecanal were tested on early and later larval development in the sea urchin Paracentrotus lividus. We also tested the effect of some of the more abundant diatom polyunsaturated fatty acids (PUFAs on development, in particular 5,8,11,14,17-eicosapentaenoic acid (EPA, one of the main precursors of diatom PUAs, as well as 4,7,10,13,16,19-docosahexaenoic acid (DHA, 6,9,12,15-octadecatetraenoic acid (stearidonic acid, 6,9,12-octadecatrienoic acid (γ-linolenic acid and 9,12-octadecadienoic acid (linoleic acid. PUAs blocked sea urchin cell cleavage in a dose dependent manner and with increasing chain length from C7 to C10 PUAs, with arrest occurring at 27.27 µM with heptadienal, 16.13 µM with octadienal, 11.47 µM with octatrienal and 5.26 mM with decadienal. Of the PUFAs tested, only EPA and stearidonic acid blocked cleavage, but at much higher concentrations compared to PUAs (331 μM for EPA and 181 μM for stearidonic acid. Sub-lethal concentrations of decadienal (1.32–5.26 μM delayed development of embryos and larvae which showed various degrees of malformations depending on the concentrations tested. Sub-lethal concentrations also increased the proportion of TUNEL-positive cells indicating imminent death in embryos and larvae. Using decadienal as a model PUA, we show that this aldehyde can be detected spectrophotometrically for up to 14 days in f/2 medium.

Viability of bovine demi embryo after splitting of fresh and frozen thawed embryo derived from in vitro embryo production

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M Imron

2007-06-01

Full Text Available In vivo embryo production was limited by number of donor, wide variability respond due to superovulation program and also immunoactifity of superovulation hormone (FSH. Splitting technology could be an alternative to increase the number of transferrable embryos into recipien cows. Splitting is done with cutting embryo becoming two equal pieces (called demi embrio base on ICM orientation. The objective of this research was to determine the viability of demi embryo obtained from embryo splitting of fresh and frozen thawed embryo. The results showed that demi embryos which performed blastocoel reexpansion 3 hours after embryo splitting using fresh and frozen thawed embryos were 76.9 and 76.2% respectively. Base on existention of inner cell mass (ICM, the number of demi embryos developed with ICM from fresh and frozen thawed embryos were not significantly different (90.6 and 85.7% respectively. The cell number of demi embryo from fresh embryos splitting was not different compared with those from frozen thawed embryos (36.1 and 35.9 respectively. These finding indicated that embryo splitting can be applied to frozen thawed embryos with certain condition as well as fresh embryos.

Birth of rats following nuclear exchange at the 2-cell stage.

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Roh, Sangho; Guo, Jitong; Malakooti, Nakisa; Morrison, John R; Trounson, Alan O; Du, Zhong Tao

2003-11-01

We report full-term development of nuclear transfer embryos following nuclear exchange at the 2-cell stage. Nuclei from 2-cell rat embryos were transferred into enucleated 2-cell embryos and developed to term after transfer to recipients (NT2). Pronuclear exchange in zygotes was used for comparison (NT1). Zygotes and 2-cell embryos were harvested from 4-week-old female Sprague-Dawley rats. Nuclear transfer was performed by transferring the pronuclei or karyoplasts into the perivitelline space of recipient embryos followed by electrofusion to reconstruct embryos. Fused couplets were cultured for 4 or 24 h before being transferred into day 1 pseudopregnant recipients (Hooded Wistar) at the 1- or 2-cell stage. In vitro culture was also carried out to check the developmental competence of the embryos. In vitro development to the blastocyst stage was not significantly different between the two groups (NT1, 34.3%; NT2, 45.0%). Two of three recipients from NT1 and two of five recipients from NT2 became pregnant. Six pups (3 from NT1, 3 from NT2) were delivered from the four foster mothers. Three female pups survived; 2 from NT1 and 1 from NT2. At 2 months of age these pups appeared healthy, and were mated with Sprague-Dawley males. One rat derived from NT1 delivered 15 pups (5 males, 10 females) as did the rat from NT2 (7 males, 8 females). Our results show that by using karyoplasts from 2-cell stage embryos as nuclear donors and reconstructing them with enucleated 2-cell embryos, healthy rats can be produced.

Hand-made cloned buffalo (Bubalus bubalis) embryos: comparison of different media and culture systems.

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Shah, Riaz A; George, Aman; Singh, Manoj K; Kumar, Dharmendra; Chauhan, Manmohan S; Manik, Radhaysham; Palta, Prabhat; Singla, Suresh K

2008-12-01

Hand-made cloning (HMC) has proved to be an efficient alternative to the conventional micromanipulator-based technique in some domestic animal species. This study reports the development of an effective culture system for in vitro culture of zona-free cloned buffalo (Bubalus bubalis) embryos reconstructed using adult skin fibroblast cells as nucleus donor. Cleavage and blastocyst rates observed were 52 and 0% in modified Charles Rosenkrans 2 (mCR2), 61 and 4.6% in modified Synthetic Oviductal Fluid (mSOF), and 82 and 40.3% in Research Vitro Cleave (RVCL; Cook, Australia) medium, respectively. Similarly, higher blastocyst rates (24.5 +/- 4.1%) were observed when zona-free parthenotes were cultured in RVCL medium. Culturing zona-free cloned buffalo embryos on flat surfaces (FS) yielded significantly higher (p WOW) or microdrops (MD). Furthermore, development in WOW was found to be significantly better than MD culture. The quality of HMC blastocysts was examined using differential staining. This study establishes the application of zona-free nuclear transfer procedures for the production of hand-made cloned buffalo embryos and the development of efficient culture system and appropriate media requirements for enhancing their preimplantation development.

Thermal effect on heart rate and hemodynamics in vitelline arteries of stage 18 chicken embryos.

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Lee, Jung Yeop; Lee, Sang Joon

2010-12-01

We investigated the thermal effects on heart rate, hemodynamics, and response of vitelline arteries of stage-18 chicken embryos. Heart rate was monitored by a high-speed imaging method, while hemodynamic quantities were evaluated using a particle image velocimetry (PIV) technique. Experiments were carried out at seven different temperatures (36-42 °C with 1 °C interval) after 1h of incubation to stabilize the heart rate. The heart rate increased in a linear manner (r = 0.992). Due to the increased cardiac output (or heart rate), the hemodynamic quantities such as mean velocity (U(mean)), velocity fluctuation (U(fluc)), and peak velocity (U(peak)) also increased with respect to the Womersley number (Ω) in the manner r = 0.599, 0.693, and 0.725, respectively. This indicates that the mechanical force exerting on the vessel walls increases. However, the active response (or regulation) of the vitelline arteries was not observed in this study. Copyright © 2010 Elsevier Ltd. All rights reserved.

Inhibition of Apoptosis Overcomes Stage-Related Compatibility Barriers to Chimera Formation in Mouse Embryos.

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Masaki, Hideki; Kato-Itoh, Megumi; Takahashi, Yusuke; Umino, Ayumi; Sato, Hideyuki; Ito, Keiichi; Yanagida, Ayaka; Nishimura, Toshinobu; Yamaguchi, Tomoyuki; Hirabayashi, Masumi; Era, Takumi; Loh, Kyle M; Wu, Sean M; Weissman, Irving L; Nakauchi, Hiromitsu

2016-11-03

Cell types more advanced in development than embryonic stem cells, such as EpiSCs, fail to contribute to chimeras when injected into pre-implantation-stage blastocysts, apparently because the injected cells undergo apoptosis. Here we show that transient promotion of cell survival through expression of the anti-apoptotic gene BCL2 enables EpiSCs and Sox17 + endoderm progenitors to integrate into blastocysts and contribute to chimeric embryos. Upon injection into blastocyst, BCL2-expressing EpiSCs contributed to all bodily tissues in chimeric animals while Sox17 + endoderm progenitors specifically contributed in a region-specific fashion to endodermal tissues. In addition, BCL2 expression enabled rat EpiSCs to contribute to mouse embryonic chimeras, thereby forming interspecies chimeras that could survive to adulthood. Our system therefore provides a method to overcome cellular compatibility issues that typically restrict chimera formation. Application of this type of approach could broaden the use of embryonic chimeras, including region-specific chimeras, for basic developmental biology research and regenerative medicine. Copyright © 2016 Elsevier Inc. All rights reserved.

Global gene expression profiling of individual human oocytes and embryos demonstrates heterogeneity in early development.

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Lisa Shaw

Full Text Available Early development in humans is characterised by low and variable embryonic viability, reflected in low fecundity and high rates of miscarriage, relative to other mammals. Data from assisted reproduction programmes provides additional evidence that this is largely mediated at the level of embryonic competence and is highly heterogeneous among embryos. Understanding the basis of this heterogeneity has important implications in a number of areas including: the regulation of early human development, disorders of pregnancy, assisted reproduction programmes, the long term health of children which may be programmed in early development, and the molecular basis of pluripotency in human stem cell populations. We have therefore investigated global gene expression profiles using polyAPCR amplification and microarray technology applied to individual human oocytes and 4-cell and blastocyst stage embryos. In order to explore the basis of any variability in detail, each developmental stage is replicated in triplicate. Our data show that although transcript profiles are highly stage-specific, within each stage they are relatively variable. We describe expression of a number of gene families and pathways including apoptosis, cell cycle and amino acid metabolism, which are variably expressed and may be reflective of embryonic developmental competence. Overall, our data suggest that heterogeneity in human embryo developmental competence is reflected in global transcript profiles, and that the vast majority of existing human embryo gene expression data based on pooled oocytes and embryos need to be reinterpreted.

Dynamic distribution of spindlin in nucleoli, nucleoplasm and spindle from primary oocytes to mature eggs and its critical function for oocyte-to-embryo transition in gibel carp.

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Sun, Min; Li, Zhi; Gui, Jian-Fang

2010-10-01

Spindlin (Spin) was thought as a maternal-effect factor associated with meiotic spindle. Its role for the oocyte-to-embryo transition was suggested in mouse, but its direct evidence for the function had been not obtained in other vertebrates. In this study, we used the CagSpin-specific antibody to investigate CagSpin expression pattern and distribution during oogenesis of gibel carp (Carassius auratus gibelio). First, the oocyte-specific expression pattern and dynamic distribution was revealed in nucleoli, nucleoplasm, and spindle from primary oocytes to mature eggs by immunofluorescence localization. In primary oocytes and growth stage oocytes, CagSpin accumulates in nucleoli in increasing numbers along with the oocyte growth, and its disassembly occurs in vitellogenic oocytes, which implicates that CagSpin may be a major component of a large number of nucleoli in fish growth oocytes. Then, co-localization of CagSpin and β-tubulin was revealed in meiotic spindle of mature egg, indicating that CagSpin is one spindle-associated factor. Moreover, microinjection of CagSpin-specific antibody into the fertilized eggs blocked the first cleavage, and found that the CagSpin depletion resulted in spindle assembly disturbance. Thereby, our study provided the first direct evidence for the critical oocyte-to-embryo transition function of Spin in vertebrates, and confirmed that Spin is one important maternal-effect factor that participates in oocyte growth, oocyte maturation, and oocyte-to-embryo transition.

Serotonin has early, cilia-independent roles in Xenopus left-right patterning

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Laura N. Vandenberg

2013-01-01

Consistent left-right (LR patterning of the heart and viscera is a crucial part of normal embryogenesis. Because errors of laterality form a common class of birth defects, it is important to understand the molecular mechanisms and stage at which LR asymmetry is initiated. Frog embryos are a system uniquely suited to analysis of the mechanisms involved in orientation of the LR axis because of the many genetic and pharmacological tools available for use and the fate-map and accessibility of early blastomeres. Two major models exist for the origin of LR asymmetry and both implicate pre-nervous serotonergic signaling. In the first, the charged serotonin molecule is instructive for LR patterning; it is redistributed asymmetrically along the LR axis and signals intracellularly on the right side at cleavage stages. A second model suggests that serotonin is a permissive factor required to specify the dorsal region of the embryo containing chiral cilia that generate asymmetric fluid flow during neurulation, a much later process. We performed theory-neutral experiments designed to distinguish between these models. The results uniformly support a role for serotonin in the cleavage-stage embryo, long before the appearance of cilia, in ventral right blastomeres that do not contribute to the ciliated organ.

Expression of microRNAs in bovine and human pre-implantation embryo culture media

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Kropp, Jenna; Salih, Sana M.; Khatib, Hasan

2014-01-01

MicroRNAs (miRNA) are short non-coding RNAs which act to regulate expression of genes driving numerous cellular processes. These RNAs are secreted within exosomes from cells into the extracellular environment where they may act as signaling molecules. In addition, they are relatively stable and are specifically expressed in association to certain cancers making them strong candidates as biological markers. Moreover, miRNAs have been detected in body fluids including urine, milk, saliva, semen, and blood plasma. However, it is unknown whether they are secreted by embryonic cells into the culture media. Given that miRNAs are expressed throughout embryonic cellular divisions and embryonic genome activation, we hypothesized that they are secreted from the embryo into the extracellular environment and may play a role in the developmental competence of bovine embryos. To test this hypothesis, bovine embryos were cultured individually from day 5 to day 8 of development in an in vitro fertilization system and gene expression of 5 miRNAs was analyzed in both embryos and culture media. Differential miRNA gene expression was observed between embryos that developed to the blastocyst stage and those that failed to develop from the morula to blastocyst stage, deemed degenerate embryos. MiR-25, miR-302c, miR-196a2, and miR-181a expression was found to be higher in degenerate embryos compared to blastocyst embryos. Interestingly, these miRNAs were also found to be expressed in the culture media of both bovine and human pre-implantation embryos. Overall, our results show for the first time that miRNAs are secreted from pre-implantation embryos into culture media and that miRNA expression may correlate with developmental competence of the embryo. Expression of miRNAs in in vitro culture media could allow for the development of biological markers for selection of better quality embryos and for subsequent successful pregnancy. PMID:24795753

Cucumber (Cucumis sativus L.) embryo development in situ after pollination with irradiated pollen

International Nuclear Information System (INIS)

Faris, N.M.; Niemirowicz-Szczytt, K.

1999-01-01

Embryological studies were undertaken to compare the normal development of cucumber endosperm and embryo with that observed after pollination with gamma-irradiated pollen (0.1 and 0.3 kGy). Delayed penetration of the pollen tube occurred at both irradiation doses. Endosperm and embryo development was also delayed, but was initiated within 6 days after pollination in 100% of embryo sacs at 0.1 kGy and in 70-80% at 0.3 kGy. Various abnormalities in endosperm and embryo cell structure confirmed progressive degeneration, which occurred earlier with the higher dose of irradiation. Degeneration increased dramatically; only 30-40% of the embryos reached the globular stage 15 days after pollination. (author)

Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

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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.

[Assisted reproductive technologies and the embryo status].

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Englert, Y

The status of the human embryo has always be a subject of philosophical and theological thoughts with major social consequences, but, until the 19th century, it has been mainly an abstraction. The arrival of the human embryo in vitro, materialized by Louise Brown's birth in 1978 and above all by the supernumerary embryos produced by the Australian team of Trounson and Wood following the introduction of ovarian stimulation, will turn theoretical thoughts into a reality. Nobody may ignore the hidden intentions behind the debate, as to recognise a status to a few days old embryo will immediately have a major impact on the status of a few weeks old foetus and therefore on the abortion rights. We will see that the embryo status, essentially based as well on a vision on the good and evil as on social order, cannot be based on a scientific analysis of the reproduction process but comes from a society's choice, by essence " arbitrary " and always disputable. This does not preclude the collectivity right and legitimacy to give a precise status and it is remarkable to observe the law is careful not to specify which status to give to the human embryo. It is more thru handling procedures and functioning rules that the law designed the embryo position, neither with a status of a person, nor of a thing. It nevertheless remains true that there is a constant risk that the legislation gives the embryo a status that would call into question it's unique characteristic of early reproductive stage, jeopardizing at once the hard-won reproductive freedom (reproductive choice) as well as freedom of research on embryonic stem cells, one of the most promising field of medical research.

Protein Expression Landscape of Mouse Embryos during Pre-implantation Development

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Yawei Gao

2017-12-01

Full Text Available Pre-implantation embryo development is an intricate and precisely regulated process orchestrated by maternally inherited proteins and newly synthesized proteins following zygotic genome activation. Although genomic and transcriptomic studies have enriched our understanding of the genetic programs underlying this process, the protein expression landscape remains unexplored. Using quantitative mass spectrometry, we identified nearly 5,000 proteins from 8,000 mouse embryos of each stage (zygote, 2-cell, 4-cell, 8-cell, morula, and blastocyst. We found that protein expression in zygotes, morulas, and blastocysts is distinct from 2- to 8-cell embryos. Analysis of protein phosphorylation identified critical kinases and signal transduction pathways. We highlight key factors and their important roles in embryo development. Combined analysis of transcriptomic and proteomic data reveals coordinated control of RNA degradation, transcription, and translation and identifies previously undefined exon-junction-derived peptides. Our study provides an invaluable resource for further mechanistic studies and suggests core factors regulating pre-implantation embryo development.

An examination of surface epithelium structures of the embryo across the genus Poeciliopsis (Poeciliidae).

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Panhuis, Tami M; Fris, Megan; Tuhela, Laura; Kwan, Lucia

2017-12-01

In viviparous, teleost fish, with postfertilization maternal nutrient provisioning, embryonic structures that facilitate maternal-fetal nutrient transfer are predicted to be present. For the family Poeciliidae, only a handful of morphological studies have explored these embryonic specializations. Here, we present a comparative morphological study in the viviparous poeciliid genus, Poeciliopsis. Using microscopy techniques, we examine the embryonic surface epidermis of Poeciliopsis species that vary in their level of postfertilization maternal nutrient provisioning and placentation across two phylogenetic clades and three independent evolutionary origins of placentation. We focus on surface features of the embryo that may facilitate maternal-fetal nutrient transfer. Specifically, we studied cell apical-surface morphology associated with the superficial epithelium that covers the body and sac (yolk and pericardial) of embryos at different developmental stages. Scanning electron microscopy revealed common surface epithelial cells across species, including pavement cells with apical-surface microridges or microvilli and presumed ionocytes and/or mucus-secreting cells. For three species, in the mid-stage embryos, the surface of the body and sac were covered in microvillus epithelium. The remaining species did not display microvillus epithelium at any of the stages examined. Instead, their epithelium of the body and sac were composed of cells with apical-surface microridges. For all species, in the late stage embryos, the surface of the body proper was composed of apical-surface microridges in a "fingerprint-like arrangement." Despite the differences in the surface epithelium of embryos across Poeciliopsis species and embryonic developmental stages, this variation was not associated with the level of postfertilization maternal nutrient provisioning. We discuss these results in light of previous morphological studies of matrotrophic, teleost fish, phylogenetic

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

Science.gov (United States)

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

1992-10-01

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

Polar body array CGH for prediction of the status of the corresponding oocyte. Part I: clinical results

NARCIS (Netherlands)

Geraedts, Joep; Montag, Markus; Magli, M. Cristina; Repping, Sjoerd; Handyside, Alan; Staessen, Catherine; Harper, Joyce; Schmutzler, Andreas; Collins, John; Goossens, Veerle; van der Ven, Hans; Vesela, Katerina; Gianaroli, Luca

2011-01-01

Several randomized controlled trials have not shown a benefit from preimplantation genetic screening (PGS) biopsy of cleavage-stage embryos and assessment of up to 10 chromosomes for aneuploidy. Therefore, a proof-of-principle study was planned to determine the reliability of alternative form of

Commonly used fertility drugs, a diet supplement, and stress force AMPK-dependent block of stemness and development in cultured mammalian embryos.

Science.gov (United States)

Bolnick, Alan; Abdulhasan, Mohammed; Kilburn, Brian; Xie, Yufen; Howard, Mindie; Andresen, Paul; Shamir, Alexandra M; Dai, Jing; Puscheck, Elizabeth E; Rappolee, Daniel A

2016-08-01

The purpose of the present study is to test whether metformin, aspirin, or diet supplement (DS) BioResponse-3,3'-Diindolylmethane (BR-DIM) can induce AMP-activated protein kinase (AMPK)-dependent potency loss in cultured embryos and whether metformin (Met) + Aspirin (Asa) or BR-DIM causes an AMPK-dependent decrease in embryonic development. The methods used were as follows: culture post-thaw mouse zygotes to the two-cell embryo stage and test effects after 1-h AMPK agonists' (e.g., Met, Asa, BR-DIM, control hyperosmotic stress) exposure on AMPK-dependent loss of Oct4 and/or Rex1 nuclear potency factors, confirm AMPK dependence by reversing potency loss in two-cell-stage embryos with AMPK inhibitor compound C (CC), test whether Met + Asa (i.e., co-added) or DS BR-DIM decreases development of two-cell to blastocyst stage in an AMPK-dependent (CC-sensitive) manner, and evaluate the level of Rex1 and Oct4 nuclear fluorescence in two-cell-stage embryos and rate of two-cell-stage embryo development to blastocysts. Met, Asa, BR-DIM, or hyperosmotic sorbitol stress induces rapid ~50-85 % Rex1 and/or Oct4 protein loss in two-cell embryos. This loss is ~60-90 % reversible by co-culture with AMPK inhibitor CC. Embryo development from two-cell to blastocyst stage is decreased in culture with either Met + Asa or BR-DIM, and this is either >90 or ~60 % reversible with CC, respectively. These experimental designs here showed that Met-, Asa-, BR-DIM-, or sorbitol stress-induced rapid potency loss in two-cell embryos is AMPK dependent as suggested by inhibition of Rex1 and/or Oct4 protein loss with an AMPK inhibitor. The DS BR-DIM or fertility drugs (e.g., Met + Asa) that are used to enhance maternal metabolism to support fertility can also chronically slow embryo growth and block development in an AMPK-dependent manner.

Movement of the external ear in human embryo.

Science.gov (United States)

Kagurasho, Miho; Yamada, Shigehito; Uwabe, Chigako; Kose, Katsumi; Takakuwa, Tetsuya

2012-02-01

External ears, one of the major face components, show an interesting movement during craniofacial morphogenesis in human embryo. The present study was performed to see if movement of the external ears in a human embryo could be explained by differential growth. In all, 171 samples between Carnegie stage (CS) 17 and CS 23 were selected from MR image datasets of human embryos obtained from the Kyoto Collection of Human Embryos. The three-dimensional absolute position of 13 representative anatomical landmarks, including external and internal ears, from MRI data was traced to evaluate the movement between the different stages with identical magnification. Two different sets of reference axes were selected for evaluation and comparison of the movements. When the pituitary gland and the first cervical vertebra were selected as a reference axis, the 13 anatomical landmarks of the face spread out within the same region as the embryo enlarged and changed shape. The external ear did move mainly laterally, but not cranially. The distance between the external and internal ear stayed approximately constant. Three-dimensionally, the external ear located in the caudal ventral parts of the internal ear in CS 17, moved mainly laterally until CS 23. When surface landmarks eyes and mouth were selected as a reference axis, external ears moved from the caudal lateral ventral region to the position between eyes and mouth during development. The results indicate that movement of all anatomical landmarks, including external and internal ears, can be explained by differential growth. Also, when the external ear is recognized as one of the facial landmarks and having a relative position to other landmarks such as the eyes and mouth, the external ears seem to move cranially. © 2012 Kagurasho et al; licensee BioMed Central Ltd.

Formation and reparation of the AP-sites into DNA from the gamma-irradiated embryo of bombyx mori

International Nuclear Information System (INIS)

Agaev, F.A.; Gaziyev, A.I.

2002-01-01

Full text: It is well known that radiosensitivity of an organism is in dependence on the DNA reparation systems functioning into cells. Sharp difference in the radioresistance of silkworm embryo at different stage of growth showed by us earlier (Agaev F.A. et al, 1991) can provide to suggest that DNA reparation system into cells of 3-daily embryo (more radiosensitive stage) and 7-daily embryo (more radioresistance stage) may be functioning with various efficiency. It was shown that quantity of the AP-sites (i.e. apurine and apirimidine sites) registered into DNA of 3-daily embryo is 1,2 - 1,4 time more, that into DNA of 7-daily embryo g-irradiated at the same dozes. The increasing of the difference between the registered AP-sites into DNA of 3- and 7-daily embryo has been observed also at the increasing of the radiation doze. At the postradiation incubation of the 3- and 7- daily embryo the lowering of AP-sites quantity into DNA was observed. This fact allowed to testify that the reparation system of damages, such as DNA-apurinization and apirimidinization are functioned into these embryo cells. At the same time the rate of the AP-sites reparation into embryo cells is varied. For example, the remanent quantity of AP-sites into DNA of 7-daily embryo after 45 min of postradiation period consists of 30% those registered immediately after embryo irradiation. The remanent quantity of AP-sites into DNA of 3-daily embryo is lowered on 50%. The difference in the rate of cells reparation is keeping at the constant level irrespective of g-irradiation doze. The binding reaction between the /14 centigrade/-methoxyamine and AP-sites in DNA in vitro has been showed that the reparation time of the 50% AP-sites for 3-daily embryo is 45 min and for 7-daily embryo is 30 min in respective to registered value at once after 100 Gy irradiation doze. In spite of essential difference in the both AP-sites formation into DNA of 3- and 7-daily embryo at once after irradiation and the

Intra- and interobserver analysis in the morphological assessment of early stage embryos during an IVF procedure: a multicentre study

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Devroe Johanna

2011-09-01

Full Text Available Abstract Background Quality control programs are necessary to maintain good clinical practice. Embryo grading has been described as one of the external quality assurance schemes. Although the evaluation of embryos is based on the assessment of morphological characteristics, considerable intra- and inter-observer variability has been described. In this multicentre study, the variability in the embryo evaluation has been evaluated using morphological characteristics on day 1, day 2 and day 3 of embryo development. Methods Five embryologists of four different IVF centers participated in this study. Multilevel images of embryos were presented on a website at different time points to evaluate intra-and inter-observer agreement in the assessment of embryo morphology. The embryos were evaluated on day 1, day 2 and day 3 of their development and each embryologist had to decide if the embryo had to be transferred, cryopreserved or discarded. Results Both intra-observer agreement and inter-observer agreement were good to excellent for the position of the pronuclei on day 1, the number of blastomeres on day 2 and day 3 and the clinical decision (transfer, cryopreservation, discard. For all other characteristics (size of pronuclei, presence of cytoplasomic halo, degree of fragmentation and size of blastomeres the intra- and inter-observer agreement was moderate to very poor. Conclusions Mono- or multicentre quality control on embryo scoring by morphological assessment can easily be performed through the design of a simple website. In the future the website design can be adapted to generate statistical feedback upon scoring and can even include a training module.

Fertilization capacity with rainbow trout DNA-damaged sperm and embryo developmental success.

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Pérez-Cerezales, S; Martínez-Páramo, S; Beirão, J; Herráez, M P

2010-06-01

Mammalian spermatozoa undergo a strong selection process along the female tract to guarantee fertilization by good quality cells, but risks of fertilization with DNA-damaged spermatozoa have been reported. In contrast, most external fertilizers such as fish seem to have weaker selection procedures. This fact, together with their high prolificacy and external embryo development, indicates that fish could be useful for the study of the effects of sperm DNA damage on embryo development. We cryopreserved sperm from rainbow trout using egg yolk and low-density lipoprotein as additives to promote different rates of DNA damage. DNA fragmentation and oxidization were analyzed using comet assay with and without digestion with restriction enzymes, and fertilization trials were performed. Some embryo batches were treated with 3-aminobenzamide (3AB) to inhibit DNA repair by the poly (ADP-ribose) polymerase, which is an enzyme of the base excision repair pathway. Results showed that all the spermatozoa cryopreserved with egg yolk carried more than 10% fragmented DNA, maintaining fertilization rates of 61.1+/-2.3 but a high rate of abortions, especially during gastrulation, and only 14.5+/-4.4 hatching success. Furthermore, after 3AB treatment, hatching dropped to 3.2+/-2.2, showing that at least 10% DNA fragmentation was repaired. We conclude that trout sperm maintains its ability to fertilize in spite of having DNA damage, but that embryo survival is affected. Damage is partially repaired by the oocyte during the first cleavage. Important advantages of using rainbow trout for the study of processes related to DNA damage and repair during development have been reported.

Impact of the Z potential technique on reducing the sperm DNA fragmentation index, fertilization rate and embryo development.

Science.gov (United States)

Duarte, Carlos; Núñez, Víctor; Wong, Yat; Vivar, Carlos; Benites, Elder; Rodriguez, Urso; Vergara, Carlos; Ponce, Jorge

2017-12-01

In assisted reproduction procedures, we need to develop and enhance new protocols to optimize sperm selection. The aim of this study is to evaluate the ability of the Z potential technique to select sperm with intact DNA in non-normospermic patients and evaluate the impact of this selection on embryonic development. We analyzed a total of 174 human seminal samples with at least one altered parameter. We measured basal, post density gradients, and post density gradients + Z potential DNA fragmentation index. To evaluate the impact of this technique on embryo development, 54 cases were selected. The embryo development parameters evaluated were fertilization rate, cleavage rate, top quality embryos at the third day and blastocysts rate. We found significant differences in the study groups when we compared the sperm fragmentation index by adding the Z potential technique to density gradient selection vs. density gradients alone. Furthermore, there was no significant difference in the embryo development parameters between the low sperm fragmentation index group vs. the moderate and high sperm fragmentation index groups, when selecting sperms with this new technique. The Z potential technique is a very useful tool for sperm selection; it significantly reduces the DNA fragmentation index and improves the parameters of embryo development. This technique could be considered routine for its simplicity and low cost.

Cleavage and creep fracture of rock salt

International Nuclear Information System (INIS)

Chan, K.S.; Munson, D.E.; Bodner, S.R.

1996-01-01

The dominant failure mechanism in rock salt at ambient temperature is either cleavage or creep fracture. Since the transition of creep fracture to cleavage in a compressive stress field is not well understood, failure of rock salt by cleavage and creep fracture is analyzed in this paper to elucidate the effect of stress state on the competition between these two fracture mechanisms. For cleavage fracture, a shear crack is assumed to cause the formation and growth of a symmetric pair of wing cracks in a predominantly compressive stress field. The conditions for wing-crack instability are derived and presented as the cleavage fracture boundary in the fracture mechanism map. Using an existing creep fracture model, stress conditions for the onset of creep fracture and isochronous failure curves of specified times-to-rupture are calculated and incorporated into the fracture mechanism map. The regimes of dominance by cleavage and creep fracture are established and compared with experimental data. The result indicates that unstable propagation of cleavage cracks occurs only in the presence of tensile stress. The onset of creep fracture is promoted by a tensile stress, but can be totally suppressed by a high confining pressure. Transition of creep fracture to cleavage occurs when critical conditions of stress difference and tensile stress for crack instability are exceeded

Dose estimation in embryo or fetus in external fields; Estimacion de dosis en embrion o feto

Energy Technology Data Exchange (ETDEWEB)

Gregori, Beatriz N [Autoridad Regulatoria Nuclear, Buenos Aires (Argentina)

2001-07-01

The embryo or the fetus can be irradiated as result of radiological procedures of diagnosis of therapy in where the beam effects directly on the same one or in tissues or peripherical organs. Some authors have suggested that in the first stages of the pregnancy the dose in ovaries can be the good estimated of the dose in embryo or fetus. In advanced conditions of the development, probably also in the early stage, is more appropriated to specify the dose in the embryo or fetus equal of the uterus. The dose in the uterus is a good estimated so much for external irradiation as for radionuclides incorporation.

Freeze-all policy: fresh vs. frozen-thawed embryo transfer.

Science.gov (United States)

Roque, Matheus; Valle, Marcello; Guimarães, Fernando; Sampaio, Marcos; Geber, Selmo

2015-05-01

To compare in vitro fertilization (IVF) outcomes between fresh embryo transfer (ET) and frozen-thawed ET (the "freeze-all" policy), with fresh ET performed only in cases without progesterone (P) elevation. Prospective, observational, cohort study. Private IVF center. A total of 530 patients submitted to controlled ovarian stimulation (COS) with a gonadotropin-releasing hormone-antagonist protocol, and cleavage-stage, day-3 ET. None. Ongoing pregnancy rates. A total of 530 cycles were included in the analysis: 351 in the fresh ET group (when P levels were ≤1.5 ng/mL on the trigger day); and 179 cycles in the freeze-all group (ET performed after endometrial priming with estradiol valerate, at 6 mg/d, taken orally). For the fresh ET group vs. the freeze-all group, respectively, the implantation rate was 19.9% and 26.5%; clinical pregnancy rate was 35.9% and 46.4%; and ongoing pregnancy rate was 31.1% and 39.7%. The IVF outcomes were significantly better in the group using the freeze-all policy, compared with the group using fresh ET. These results suggest that even in a select group of patients that underwent fresh ET (P levels ≤1.5 ng/mL), endometrial receptivity may have been impaired by COS, and outcomes may be improved by using the freeze-all policy. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Analysis of chlorophyll fluorescence reveals stage specific patterns of chloroplast-containing cells during Arabidopsis embryogenesis

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RICARDO I TEJOS

2010-01-01

Full Text Available The basic body plan of a plant is established early in embryogenesis when cells differentiate, giving rise to the apical and basal regions of the embryo. Using chlorophyll fluorescence as a marker for chloroplasts, we have detected specific patterns of chloroplast-containing cells at specific stages of embryogenesis. Non-randomly distributed chloroplast-containing cells are seen as early as the globular stage of embryogenesis in Arabidopsis. In the heart stage of embryogenesis, chloroplast containing cells are detected in epidermal cells as well as a central region of the heart stage embryo, forming a triangular septum of chloroplast-containing cells that divides the embryo into three equal sectors. Torpedo stage embryos have chloroplast-containing epidermal cells and a central band of chloroplast-containing cells in the cortex layer, just below the shoot apical meristem. In the walking-stick stage of embryogenesis, chloroplasts are present in the epidermal, cortex and endodermal cells. The chloroplasts appear reduced or absent from the provascular and columella cells of walking-stick stage embryos. These results suggest that there is a tight regulation of plastid differentiation during embryogenesis that generates specific patterns of chloroplast-containing cells in specific cell layers at specific stages of embryogenesis.

Description of Phaseolus vulgaris L. aborting embryos from ethyl methanesulfonate (EMS mutagenized plants

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Silué, S.

2013-01-01

Full Text Available The aim of this study was to describe the embryos abortion process and the inheritance of the embryos abortion trait in Phaseolus vulgaris plants deficient in seed development. These plants were isolated within the second generation of an ethyl methanesulfonate (EMS TILLING population of P. vulgaris cv. 'BAT93'. Mutant embryos show abnormalities mainly in suspensors, shoot apical meristem (SAM and cotyledons from the globular to the cotyledon stages and abort before maturity compared to those observed in wild-type samples. Mutant embryos show also hyperhydricity and contain low amount of chlorophyll. Genetic analyses of F1, F2 and F3 populations from the crosses carried out between the mutagenized plants with aborting embryos and the wild-type plants indicated that the embryo abortion phenotype is maternally inherited and controlled by a single recessive gene. These Phaseolus mutant plants with aborting embryos constitute a valuable material for plant embryogenesis studies.

The BMP antagonist follistatin-like 1 is required for skeletal and lung organogenesis

NARCIS (Netherlands)

Sylva, M.; Li, V.S.; Buffing, A.A.; van Es, J.H.; van den Born, M.M.W.; van der Velden, S.; Gunst, Q.; Koolstra, J.H.; Moorman, A.F.; Clevers, H.; van den Hoff, M.J.

2011-01-01

Follistatin-like 1 (Fstl1) is a secreted protein of the BMP inhibitor class. During development, expression of Fstl1 is already found in cleavage stage embryos and becomes gradually restricted to mesenchymal elements of most organs during subsequent development. Knock down experiments in chicken and

Autoradiographic study of protein synthesis recovery in root cells of Zea mays embryos during early stages of germination

International Nuclear Information System (INIS)

Deltour, Roger

1977-01-01

Recovery of protein synthesis was studied in primary root of germinating Zea mays embryos. [H 3 ] leucine or [H 3 ] lysine was provided for two hours at 16 0 C to embryos excised from kernels at various times after the beginning of germination. Protein synthesis (probably dependent on long-lived mRNA stocked in dormant embryo root cells) resumed during the first two hours of seed imbibition [fr

Autoradiographic study of protein synthesis recovery in root cells of Zea mays embryos during early stages of germination

Energy Technology Data Exchange (ETDEWEB)

Deltour, R [Liege Univ. (Belgium)

1977-05-02

Recovery of protein synthesis was studied in primary root of germinating Zea mays embryos. (H/sup 3/) leucine or (H/sup 3/) lysine was provided for two hours at 16/sup 0/C to embryos excised from kernels at various times after the beginning of germination. Protein synthesis (probably dependent on long-lived mRNA stocked in dormant embryo root cells) resumed during the first two hours of seed imbibition.

Culture of bovine embryos on a polydimethylsiloxane (PDMS) microwell plate.

Science.gov (United States)

Akagi, Satoshi; Hosoe, Misa; Matsukawa, Kazutsugu; Ichikawa, Akihiko; Tanikawa, Tamio; Takahashi, Seiya

2010-08-01

We fabricated a polydimethylsiloxane (PDMS)-based microwell plate (PDMS-MP) containing 100 microwells with a rounded bottom and examined whether it can be used for culture of individual in vitro fertilized (IVF) embryos or parthenogenetically activated zona-free embryos in cattle. In Experiment 1, we examined the in vitro developmental ability of IVF embryos cultured individually on PDMS-MP. After IVF, 20 embryos were transferred into 100 microl drops on PDMS-MP and cultured individually in each well of PDMS-MP (PDMS group). After 7 days of culture, the embryos in the PDMS group developed to the blastocyst stage at the same rate of those in the control group cultured in a group of 20 embryos without PDMS-MP. There were no differences in total number of cells and the ratio of inner cell mass to total cells between the PDMS and control groups. In Experiment 2, we examined the in vitro developmental ability of parthenogenetically activated zona-free bovine embryos cultured individually on PDMS-MP. The zona-free embryos were cultured individually in each well of a PDMS-MP or in each well produced by pressing a darning needle onto the bottom of a culture dish (WOW group). After 7 days of culture, the blastocyst formation rate and cell number of blastocysts in the PDMS group did not differ from those of the zona-intact embryos in the control group. Also, there were no differences in the blastocyst formation rate and cell number of blastocysts between the WOW and PDMS groups. These results suggest that the culture system using PDMS-MP is useful for individual embryos or zona-free embryos in cattle.