Institute of Farm Animal Genetics FLI

Neustadt an der Weinstraße, Germany

Institute of Farm Animal Genetics FLI

Neustadt an der Weinstraße, Germany

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Herrmann D.,Institute of Farm Animal Genetics FLI | Dahl J.A.,University of Oslo | Lucas-Hahn A.,Institute of Farm Animal Genetics FLI | Collas P.,University of Oslo | And 2 more authors.
Epigenetics | Year: 2013

Normal development depends on the precise sequence of changes in the configuration of chromatin; these are primarily related to specific biochemical modifications such as acetylation or methylation of histones and DNA methylation. While the role of DNA methylation during preimplantation development has been studied extensively, little is known about histone modifications related to early embryonic development. Here, we investigated gene-specific histone modifications in in vitro produced bovine blastocysts. Selected genes thought to be critical for bovine preimplantation development were examined and included POU5F1 (OCT4), NANOG, INFT, GAPDH, SLC2A3 and IGF1. We used chromatin immunoprecipitation from pools of bovine blastocysts to unravel several modifications of histone H3 in relation to mRNA expression profiles. We focused on the two cell compartments of the blastocyst, the inner cell mass (ICM) and the trophectoderm (TE). We show that gene expression patterns in the ICM and TE of the bovine blastocyst are consistent with histone modification patterns on the promoter of the corresponding genes. The data show a complex epigenetic pattern of promoter occupancy by transcriptionally permissive and repressive H3 modifications. These results pave the way to in-depth epigenetic studies of preimplantation embryos that are crucial to gain a better understanding of the epigenetic changes frequently observed after use of assisted reproductive technologies. © 2013 Landes Bioscience.


Deshmukh R.S.,Copenhagen University | Ostrup O.,Copenhagen University | Ostrup E.,Copenhagen University | Vejlsted M.,Copenhagen University | And 6 more authors.
Epigenetics | Year: 2011

DNA demethylation and remethylation are crucial for reprogramming of the differentiated parental/somatic genome in the recipient ooplasm upon somatic cell nuclear transfer. Here, we analyzed the DNA methylation dynamics during porcine preimplantation development. Porcine in vivo developed (IV), in vitro fertilized (IVF), somatic cell nuclear transfer (SCNT) and parthenogenetically activated (PA) embryos were evaluated for DNA methylation quantification at different developmental stages. Fertilized (IV and IVF) one-cell stages lacked a substantial active demethylation of the paternal genome. Embryos produced under in vitro conditions had higher levels of DNA methylation than IV. A lineage-specific DNA methylation (hypermethylation of the inner cell mass and hypomethylation of the trophectoderm) was observed in porcine IV late blastocysts, but was absent in PA- and SCNT-derived blastocysts despite the occurrence of de novo methylation in early blastocysts. Comparable levels of DNA methylation were found in IV embryos and in 50 and 14% of SCNT early and late blastocysts, respectively. In conclusion, DNA methylation patterns were adversely affected by in vitro embryo production. © 2011 Landes Bioscience.


Racedo S.E.,Institute of Farm Animal Genetics FLI | Racedo S.E.,Yeshiva University | Rawe V.Y.,REPROTEC | Rawe V.Y.,Crea - Tec | Niemann H.,Institute of Farm Animal Genetics FLI
Reproduction | Year: 2012

For successful fertilization by the male gamete, oocyte cytoplasmic organelles such as the Golgi apparatus have to undergo specific changes: the entire process is known as cytoplasmic maturation. The goal of this study was to unravel the dynamics of the Golgi apparatus in bovine oocytes at critical stages of in vitro maturation, i.e. germinal vesicle (GV), GV breakdown (GVBD), metaphase I (MI) and metaphase II, and to investigate the role of various molecules critically involved therein. The cytoplasmic distribution of proteins was assessed by immunocytochemistry and laser confocal microscopy. We applied specific inhibitors, including nocodazole to unravel the functional role of the microtubular elements; sodium orthovanadate, which primarily inhibits cytoplasmic dynein ATPase activity; monastrol which inhibits the kinesin EG5; and roscovitine to inhibit the kinase cyclin-dependent kinase 2A (CDC2A). Prior to GVBD, the Golgi apparatus was translocated from the centre of the cytoplasm to the cortical area in the periphery, where it underwent fragmentation. A second translocation was observed between GVBD and MI stages, when the Golgi apparatuswas moved from the cortex to the centre of the cytoplasm. Incubation with the specific inhibitors revealed that microtubules played an active role in the final localization at GVBD, while CDC2A was essential for Golgi fragmentation at GVBD stage. This partitioning was a precondition for the second movement. In conclusion, for the first time we show basic mechanisms critically involved in the regulation of the dynamic changes of Golgi apparatus during meiosis of the bovine oocyte. © 2012 Society for Reproduction and Fertility.


Petersen S.,Laser Zentrum Hannover e.V. | Barchanski A.,Laser Zentrum Hannover e.V. | Taylor U.,Institute of Farm Animal Genetics FLI | Klein S.,Institute of Farm Animal Genetics FLI | And 2 more authors.
Journal of Physical Chemistry C | Year: 2011

Gold nanoparticles (AuNPs) are promising imaging agents for the long-term visualization and tracing of intracellular functions because they bear outstanding optical properties and are fairly easily bioconjugated. However, the design of such multifunctional nanosystems might be limited by their bioavailability. Cell-penetrating peptides (CPPs) have been shown to be efficient molecular transporters with very few indices of cytotoxicity also in conjunction with nanoparticles. In this context, the current work aims to explore the approach of in situ conjugation during laser ablation in liquids for the design of CPP-NP conjugates at the example of penetratin-conjugated AuNPs. Because penetratin is positively charged at neutral pH, the conjugation process most likely differs from the previously reported coupling of oligonucleotides with their negatively charged phosphate backbone. Results reveal that penetratin is more efficiently bound to AuNPs, increasing the pH value of the ablation media, whereas the size and morphology of the bioconjugates function in terms of the penetratin concentration during the laser process. Probable underlying processes such as size quenching, aggregation, and laser-induced partial melting are assessed by the means of transmission electron microscopy and UV-vis spectroscopy. In a preliminary biological study, laser scanning confocal and transmission electron microscopy revealed a successful uptake of penetratin-conjugated AuNPs for the first time in up to 100% of coincubated cells within 2 h. © 2010 American Chemical Society.


Niemann H.,Institute of Farm Animal Genetics FLI | Niemann H.,Leibniz University of Hanover | Carnwath J.W.,Institute of Farm Animal Genetics FLI | Herrmann D.,Institute of Farm Animal Genetics FLI | And 4 more authors.
Cellular Reprogramming | Year: 2010

To understand the epigenetic alterations associated with assisted reproduction technology (ART) and the reprogramming of gene expression that follows somatic cell nuclear transfer (SCNT), we screened a panel of 41 amplicons representing 25 developmentally important genes on 15 different chromosomes (a total of 1079 CpG sites). Methylation analysis was performed on DNA from pools of 80 blastocysts representing three classes of embryos. This revealed a subset of amplicons that distinguish between embryos developing in vivo, produced in vitro, or reconstructed by SCNT. Following SCNT, we observed massive epigenetic reprogramming evidenced by reduced levels of methylation in the resultant embryos. Analysis of data from the 28 most informative amplicons (hotspot loci), representing more than 523 individual CpG sites, we discovered subsets of amplicons with methylation patterns that were unique to each class of embryo and may indicate metastable epialleles. Analysis of eight genes with respect to mRNA expression did not reveal a direct correlation with DNA methylation levels. In conclusion, this approach revealed a subset of amplicons that can be used to evaluate blastocyst quality and reprogramming following SCNT, and can also be employed for the localization of the epigenetic control regions within individual genes and for more general studies of stem cell differentiation. © 2010, Mary Ann Liebert, Inc.


Truong C.V.C.,Institute of Farm Animal Genetics FLI | Groeneveld L.F.,Institute of Farm Animal Genetics FLI | Groeneveld L.F.,Max Planck Institute for Evolutionary Anthropology | Morgenstern B.,University of Gottingen | Groeneveld E.,Institute of Farm Animal Genetics FLI
BMC Bioinformatics | Year: 2011

Background: Long-term sample storage, tracing of data flow and data export for subsequent analyses are of great importance in genetics studies. Therefore, molecular labs do need a proper information system to handle an increasing amount of data from different projects.Results: We have developed a molecular labs information management system (MolabIS). It was implemented as a web-based system allowing the users to capture original data at each step of their workflow. MolabIS provides essential functionality for managing information on individuals, tracking samples and storage locations, capturing raw files, importing final data from external files, searching results, accessing and modifying data. Further important features are options to generate ready-to-print reports and convert sequence and microsatellite data into various data formats, which can be used as input files in subsequent analyses. Moreover, MolabIS also provides a tool for data migration.Conclusions: MolabIS is designed for small-to-medium sized labs conducting Sanger sequencing and microsatellite genotyping to store and efficiently handle a relative large amount of data. MolabIS not only helps to avoid time consuming tasks but also ensures the availability of data for further analyses. The software is packaged as a virtual appliance which can run on different platforms (e.g. Linux, Windows). MolabIS can be distributed to a wide range of molecular genetics labs since it was developed according to a general data model. Released under GPL, MolabIS is freely available at http://www.molabis.org. © 2011 Truong et al; licensee BioMed Central Ltd.


Heinzmann J.,Institute of Farm Animal Genetics FLI | Mattern F.,University of Würzburg | Aldag P.,Institute of Farm Animal Genetics FLI | Bernal-Ulloa S.M.,Institute of Farm Animal Genetics FLI | And 3 more authors.
Molecular Human Reproduction | Year: 2015

To mimic post-ovulatory ageing, we have extended the in vitro maturation (IVM) phase to 48 h and examined effects on (i) developmental potential, (ii) expression of a panel of developmentally important genes and (iii) gene-specific epigenetic marks. Results were compared with the 24 h IVM protocol (control) usually employed for bovine oocytes. Cleavage rates and blastocyst yields were significantly reduced in oocytes after extended IVM. No significant differences were observed in the methylation of entire alleles in oocytes for the genes bH19, bSNRPN, bZAR1, bOct4 and bDNMT3A. However, we found differentially methylated CpG sites in the bDNMT3Ls locus in oocytes after extended IVM and in embryos derived from them compared with controls. Moreover, embryos derived from the 48 h matured oocyte group were significantly less methylated at CpG5 and CpG7 compared with the 24 h group. CpG7 was significantly hypermethylated in embryos produced from the control oocytes, but not in oocytes matured for 48 h. Furthermore, methylation for CpG5-CpG8 of bDNMT3Ls was significantly lower in oocytes of the 24 h group compared with embryos derived therefrom, whereas no such difference was found for oocytes and embryos of the in vitro aged group. Expression of most of the selected genes was not affected by duration of IVM. However, transcript abundance for the imprinted gene bIGF2R was significantly reduced in oocytes analyzed after extended IVM compared with control oocytes. Transcript levels for bPRDX1, bDNMT3A and bBCLXL were significantly reduced in 4-to 8-cell embryos derived from in vitro aged oocytes. These results indicate that extended IVM leads to ageing-like alterations and demonstrate that epigenetic mechanisms are critically involved in ageing of bovine oocytes, which warrants further studies into epigenetic mechanisms involved in ageing of female germ cells, including humans. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.


Niemann H.,Institute of Farm Animal Genetics FLI
Theriogenology | Year: 2016

The birth of "Dolly," the first mammal cloned from an adult mammary epithelial cell, abolished the decades-old scientific dogma implying that a terminally differentiated cell cannot be reprogrammed into a pluripotent embryonic state. The most dramatic epigenetic reprogramming occurs in SCNT when the expression profile of a differentiated cell is abolished and a new embryo-specific expression profile, involving 10,000 to 12,000 genes, and thus, most genes of the entire genome is established, which drives embryonic and fetal development. The initial release from somatic cell epigenetic constraints is followed by establishment of post-zygotic expression patterns, X-chromosome inactivation, and adjustment of telomere length. Somatic cell nuclear transfer may be associated with a variety of pathologic changes of the fetal and placental phenotype in a proportion of cloned offspring, specifically in ruminants, that are thought to be caused by aberrant epigenetic reprogramming. Improvements in our understanding of this dramatic epigenetic reprogramming event will be instrumental in realizing the great potential of SCNT for basic research and for important agricultural and biomedical applications. Here, current knowledge on epigenetic reprogramming after use of SCNT in livestock is reviewed, with emphasis on gene-specific and global DNA methylation, imprinting, X-chromosome inactivation, and telomere length restoration in early development. © 2016 Elsevier Inc.


PubMed | Institute of Farm Animal Genetics FLI
Type: Journal Article | Journal: Theriogenology | Year: 2016

The birth of Dolly, the first mammal cloned from an adult mammary epithelial cell, abolished the decades-old scientific dogma implying that a terminally differentiated cell cannot be reprogrammed into a pluripotent embryonic state. The most dramatic epigenetic reprogramming occurs in SCNT when the expression profile of a differentiated cell is abolished and a new embryo-specific expression profile, involving 10,000 to 12,000 genes, and thus, most genes of the entire genome is established, which drives embryonic and fetal development. The initial release from somatic cell epigenetic constraints is followed by establishment of post-zygotic expression patterns, X-chromosome inactivation, and adjustment of telomere length. Somatic cell nuclear transfer may be associated with a variety of pathologic changes of the fetal and placental phenotype in a proportion of cloned offspring, specifically in ruminants, that are thought to be caused by aberrant epigenetic reprogramming. Improvements in our understanding of this dramatic epigenetic reprogramming event will be instrumental in realizing the great potential of SCNT for basic research and for important agricultural and biomedical applications. Here, current knowledge on epigenetic reprogramming after use of SCNT in livestock is reviewed, with emphasis on gene-specific and global DNA methylation, imprinting, X-chromosome inactivation, and telomere length restoration in early development.


Nowak-Imialek M.,Institute of Farm Animal Genetics FLI | Kues W.,Institute of Farm Animal Genetics FLI | Carnwath J.W.,Institute of Farm Animal Genetics FLI | Niemann H.,Institute of Farm Animal Genetics FLI
Microscopy and Microanalysis | Year: 2011

Pluripotent cells are unique because of their ability to differentiate into the cell lineages forming the entire organism. True pluripotent stem cells with germ line contribution have been reported for mice and rats. Human pluripotent cells share numerous features of pluripotentiality, but confirmation of their in vivo capacity for germ line contribution is impossible due to ethical and legal restrictions. Progress toward derivation of embryonic stem cells from domestic species has been made, but the derived cells were not able to produce germ line chimeras and thus are termed embryonic stem-like cells. However, domestic animals, in particular the domestic pig (Sus scrofa), are excellent large animals models, in which the clinical potential of stem cell therapies can be studied. Reprogramming technologies for somatic cells, including somatic cell nuclear transfer, cell fusion, in vitro culture in the presence of cell extracts, in vitro conversion of adult unipotent spermatogonial stem cells into germ line derived pluripotent stem cells, and transduction with reprogramming factors have been developed with the goal of obtaining pluripotent, germ line competent stem cells from domestic animals. This review summarizes the present state of the art in the derivation and maintenance of pluripotent stem cells in domestic animals. © Microscopy Society of America 2011.

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