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EGF increases expression and activity of PAs in preimplantation rat embryos and their implantation rate
Eliahu D Aflalo, Uriel A Sod-Moriah, Gad Potashnik, Iris Har-Vardi
Reproductive Biology and Endocrinology , 2007, DOI: 10.1186/1477-7827-5-4
Abstract: In the present work we studied the effect of EGF on PAs activity, quantity and embryo implantation.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.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.The major obstacle in IVF treatments is the low embryo implantation rate. The composition of the embryo culture medium supposed to mimic the physiological environment, addition of growth factors to the medium may be vital not only for improving the embryo development but also for increasing the embryo implantation rates in IVF programs. Plasminogen activators are members of one of the main enzyme family that participate in embryo implantation.Plasminogen activators (PAs) and matrix metalloproteinases (MMPs) have been implicated in mammalian gametogenesis [1], ovulation [2,3], fertilization [4,5], early development and embryo implantation [3,6,7]. The PAs a
Epigenetic regulation in mammalian preimplantation embryo development
Lingjun Shi, Ji Wu
Reproductive Biology and Endocrinology , 2009, DOI: 10.1186/1477-7827-7-59
Abstract: Starting from fertilization and ending with implantation, preimplantation embryo development can be divided into several well-orchestrated stages: fertilization, cell cleavage, morula and blastocyst formation. Understanding the stages of preimplantation development and the underlying regulatory molecular mechanisms is of pivotal importance for basic reproductive biology and for practical applications including regenerative medicine. To decipher the regulatory mechanisms, determining the global gene, RNA and protein expression patterns of early embryo is indispensable. Early attempts to do this provided data on protein expression patterns using comparative electrophoretic analysis with radiolabeled tyrosine and lysine [1] and RNA expression patterns were estimated using cDNA library analysis [2]. A series of subsequent studies reported modified or novel methods including polymerase chain reaction (PCR)-based differential display [3] and subtractive cDNA library construction techniques [4]. With the development of microarray technology, microarray analysis soon became one of the most powerful approaches, providing more comprehensive and precise global expression pattern data, especially on gene expression profiles [5-7]. Based on transitions in gene expression patterns in mouse embryos, preimplantation development can also be divided into several phases: phase I from fertilization to the 2-cell stage; phase II from the 4-cell to the 8-cell stage and phase III from the 8-cell embryo to the blastocyst stage. Phases I and II can also be called zygotic genome activation (ZGA) and mid-preimplantation gene activation, respectively [8]. At ZGA, some proteins originally formed in the stage of oogenesis remain after fertilization and contribute to the regulation of the next developmental processes.Preimplantation embryo development is regulated both genetically and epigenetically. Based on the knowledge of genetic profiles, studies proceeded to elucidate epigenetic profiles in
Characterization of Trophoblast and Extraembryonic Endoderm Cell Lineages Derived from Rat Preimplantation Embryos  [PDF]
Ilya Chuykin,Irina Lapidus,Elena Popova,Larisa Vilianovich,Valentina Mosienko,Natalia Alenina,Bert Binas,Guixuan Chai,Michael Bader,Alexander Krivokharchenko
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0009794
Abstract: Previous attempts to isolate pluripotent cell lines from rat preimplantation embryo in mouse embryonic stem (ES) cell culture conditions (serum and LIF) were unsuccessful, however the resulting cells exhibited the expression of such traditional pluripotency markers as SSEA-1 and alkaline phosphatase. We addressed the question, which kind of cell lineages are produced from rat preimplantation embryo under “classical” mouse ES conditions.
Characterization of a diverse secretome generated by the mouse preimplantation embryo in vitro
Amanda J Beardsley, Yan Li, Chris O'Neill
Reproductive Biology and Endocrinology , 2010, DOI: 10.1186/1477-7827-8-71
Abstract: A number of lines of evidence indicate that the early embryo secretes an interesting array of chemically defined, biologically active mediators. The first such evidence was the observation that early pregnancy in a number of species is associated with a detectable peripheral thrombocytopenia [1]. This was caused by the de novo synthesis and release by the preimplantation embryo of a potent ether phospholipid, Paf (1-o-alkyl-2-acetyl-sn-glycero-3-phosphocholine) [2,3]. The quantity of lipid released by the human embryo in vitro showed a significant association with the viability of embryos following embryo transfer, indicating that the released factor played an important role in embryo development [4,5].A role for a peptide/protein secretome was inferred from studies showing that the early embryo expressed a range of putative trophic ligands and also their receptors [6,7]. Evidence that the addition of one or more of these ligands to culture media caused an improvement in the rate of embryo development in vitro was indicative of a functional role for their secretion by the embryo [8,9]. Some studies have detected the expression of trophic protein ligands in spent embryo culture media (for example, IGF-II [10]). Another example of the release of protein is the immunological mediator, HLA-G. Soluble HLA-G is detected in spent human embryo culture media, and the level of its expression is reported to be positively associated with the pregnancy potential of these embryos [11,12]. The HLA-G major histocompatibility antigen is thought to be a homologue of the mouse Qa-2 antigen. This antigen is coded for by the Ped gene, and its expression influences the rate and success of mouse embryo development in vitro [13,14].The development in recent years of mass spectrometry-based proteomic techniques offer the possibility for high throughput, cost-effective and sensitive methods for such analysis. One of the most accessible approaches for non-specialist in the field is the surfac
Simulated Microgravity Influences Bovine Oocyte in vitro Fertilization and Preimplantation Embryo Development
Song-yi Jung,Susan D. Bowers,Scott T. Willarda
Journal of Animal and Veterinary Advances , 2012,
Abstract: The aim of this study was to investigate, whether in vitro fertilization and preimplantation embryos exposed to a simulated microgravity environment in vitro would improve, or be deleterious to their fertilization and embryonic development. A Rotating Cell Culture System (RCCS) bioreactor with a High Aspect Ratio Vessel (HARV) was used to simulate a microgravity environment. In vitro Fertilization (IVF) and Culture (IVC) were conducted in standard microdrop culture method conditions (Control) and simulated microgravity conditions; HARV rotated at 34 rpm (high speed) and at 3.7 rpm (Low speed) on a horizontal axis. Embryonic development rates were determined during IVF (experiment 1), during IVC at presumptive zygote stage (experiment 2) and IVC at 2-8 cell stages of embryo development (experiment 3). For IVF studies (experiment 1), 77.3% of bovine oocytes were fertilized in the Control group; however, bovine oocytes and sperm fertilization did not occur in high and low speed groups. Moreover, none of the presumptive zygotes (experiment 2) and 2-8 cell stage embryos (experiment 3) cultured in high and low speed groups were able to develop to the further stages. These results indicate that simulated microgravity environments have a negative impact on bovine In vitro fertilization and preimplantation embryo development.
Preimplantation embryotoxicity after mouse embryo exposition to reactive oxygen species
Cebral,Elisa; Carrasco,Isabel; Vantman,David; Smith,Rosita;
Biocell , 2007,
Abstract: exposure of either gametes or embryos to conditions and/or factors that generate oxidative stress has been associated with impaired early embryogenesis. the effects of reactive oxygen species (ros) on mouse preimplantation development, depending of the ros-concentration and time of exposition, were studied. two-cell embryos were incubated with 5, 10, 25 and 50 μm of hydrogen peroxide (h2o2) for 30 and 60 minutes of exposition and allowed to develop for 72 h to study the quality of development. the incubation with 50 μm2ho2 for 30 or 60 minutes, strongly inhibited the 2-cell embryo development as compared to the control (p<0.001). twenty-five μm2ho2 produced inhibition of blastocyst formation (p<0.001) and 10 μm h2o2 significantly decreased the percentages of expanded and hatched blastocysts, which resulted morphologically altered (p<0.05 and p<0.01, respectively). the higher h2o2 concentrations were able to elicit necrotic morphology in the 2-cell arrested embryos, while 10 μm2ho2 induced moderate damage with the arrested embryos partially fragmented. in conclusion, important causes for defective preimplantation development and for early embryo losses may be due to oxidative stress because early mouse embryos exposed to ros for short times arrested at the first cellular cycle (2-cell) and/or impaired embryo differentiation and morphogenesis, being these effects ros-concentration-dependent.
Preimplantation embryotoxicity after mouse embryo exposition to reactive oxygen species
Elisa Cebral,Isabel Carrasco,David Vantman,Rosita Smith
Biocell , 2007,
Abstract: Exposure of either gametes or embryos to conditions and/or factors that generate oxidative stress has been associated with impaired early embryogenesis. The effects of reactive oxygen species (ROS) on mouse preimplantation development, depending of the ROS-concentration and time of exposition, were studied. Two-cell embryos were incubated with 5, 10, 25 and 50 μM of hydrogen peroxide (H2O2) for 30 and 60 minutes of exposition and allowed to develop for 72 h to study the quality of development. The incubation with 50 μM2HO2 for 30 or 60 minutes, strongly inhibited the 2-cell embryo development as compared to the control (p<0.001). Twenty-five μM2HO2 produced inhibition of blastocyst formation (p<0.001) and 10 μM H2O2 significantly decreased the percentages of expanded and hatched blastocysts, which resulted morphologically altered (p<0.05 and p<0.01, respectively). The higher H2O2 concentrations were able to elicit necrotic morphology in the 2-cell arrested embryos, while 10 μM2HO2 induced moderate damage with the arrested embryos partially fragmented. In conclusion, important causes for defective preimplantation development and for early embryo losses may be due to oxidative stress because early mouse embryos exposed to ROS for short times arrested at the first cellular cycle (2-cell) and/or impaired embryo differentiation and morphogenesis, being these effects ROS-concentration-dependent.
Transcription-Independent Heritability of Induced Histone Modifications in the Mouse Preimplantation Embryo  [PDF]
Matthew D. VerMilyea, Laura P. O'Neill, Bryan M. Turner
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0006086
Abstract: Enzyme-catalyzed, post-translational modifications of core histones have been implicated in the complex changes in gene expression that drive early mammalian development. However, until recently the small number of cells available from the preimplantation embryo itself has prevented quantitative analysis of histone modifications at key regulator genes. The possible involvement of histone modifications in the embryo's response to extracellular signals, or as determinants of cell fate or lineage progression, remains unclear. Here we describe the use of a recently-developed chromatin immunoprecipitation technique (CChIP) to assay histone modification levels at key regulator genes (Pou5f1, Nanog, Cdx2, Hoxb1, Hoxb9) as mouse embryos progress from 8-cell to blastocyst in culture. Only by the blastocyst stage, when the embryonic (Inner Cell Mass) and extra-embryonic (Trophoblast) lineages are compared, do we see the expected association between histone modifications previously linked to active and silent chromatin, and transcriptional state. To explore responses to an environmental signal, we exposed embryos to the histone deacetylase inhibitor, anti-epileptic and known teratogen valproic acid (VPA), during progression from 8-cell to morula stage. Such treatment increased H4 acetylation and H3 lysine 4 methylation at the promoters of Hoxb1 and Hoxb9, but not the promoters of Pou5f1, Nanog,Cdx2 or the housekeeping gene Gapdh. Despite the absence of detectable Hoxb transcription, these VPA-induced changes were heritable, following removal of the inhibitor, at least until the blastocyst stage. The selective hyperacetylation of Hoxb promoters in response to a histone deacetylase inhibitor, suggests that Hox genes have a higher turnover of histone acetates than other genes in the preimplantation embryo. To explain the heritability, through mitosis, of VPA-induced changes in histone modification at Hoxb promoters, we describe how an epigenetic feed-forward loop, based on cross-talk between H3 acetylation and H3K4 methylation, might generate a persistently increased steady-state level of histone acetylation in response to a transient signal.
Dynamics of DNA Methylation during Early Development of the Preimplantation Bovine Embryo  [PDF]
Kyle B. Dobbs, Marlon Rodriguez, Mateus J. Sudano, M. Sofia Ortega, Peter J. Hansen
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0066230
Abstract: There is species divergence in control of DNA methylation during preimplantation development. The exact pattern of methylation in the bovine embryo has not been established nor has its regulation by gender or maternal signals that regulate development such as colony stimulating factor 2 (CSF2). Using immunofluorescent labeling with anti-5-methylcytosine and embryos produced with X-chromosome sorted sperm, it was demonstrated that methylation decreased from the 2-cell stage to the 6–8 cell stage and then increased thereafter up to the blastocyst stage. In a second experiment, embryos of specific genders were produced by fertilization with X- or Y-sorted sperm. The developmental pattern was similar to the first experiment, but there was stage × gender interaction. Methylation was greater for females at the 8-cell stage but greater for males at the blastocyst stage. Treatment with CSF2 had no effect on labeling for DNA methylation in blastocysts. Methylation was lower for inner cell mass cells (i.e., cells that did not label with anti-CDX2) than for trophectoderm (CDX2-positive). The possible role for DNMT3B in developmental changes in methylation was evaluated by determining gene expression and degree of methylation. Steady-state mRNA for DNMT3B decreased from the 2-cell stage to a nadir for D 5 embryos >16 cells and then increased at the blastocyst stage. High resolution melting analysis was used to assess methylation of a CpG rich region in an intronic region of DNMT3B. Methylation percent decreased between the 6–8 cell and the blastocyst stage but there was no difference in methylation between ICM and TE. Results indicate that DNA methylation undergoes dynamic changes during the preimplantation period in a manner that is dependent upon gender and cell lineage. Developmental changes in expression of DNMT3B are indicative of a possible role in changes in methylation. Moreover, DNMT3B itself appears to be under epigenetic control by methylation.
MATER protein expression and intracellular localization throughout folliculogenesis and preimplantation embryo development in the bovine
Sophie Pennetier, Christine Perreau, Svetlana Uzbekova, Aurore Thélie, Bernadette Delaleu, Pascal Mermillod, Rozenn Dalbiès-Tran
BMC Developmental Biology , 2006, DOI: 10.1186/1471-213x-6-26
Abstract: Here we have analyzed gene expression during folliculogenesis and preimplantation embryo development. In situ hybridization and immunohistochemistry on bovine ovarian section revealed that both the transcript and protein are restricted to the oocyte from primary follicles onwards, and accumulate in the oocyte cytoplasm during follicle growth. In immature oocytes, cytoplasmic, and more precisely cytosolic localization of MATER was confirmed by immunohistochemistry coupled with confocal microscopy and immunogold electron microscopy. By real-time PCR, MATER messenger RNA was observed to decrease strongly during maturation, and progressively during the embryo cleavage stages; it was hardly detected in morulae and blastocysts. The protein persisted after fertilization up until the blastocyst stage, and was mostly degraded after hatching. A similar predominantly cytoplasmic localization was observed in blastomeres from embryos up to 8-cells, with an apparent concentration near the nuclear membrane.Altogether, these expression patterns are consistent with bovine MATER protein being an oocyte specific maternal effect factor as in mouse.Preimplantation embryo development is largely dependent on maternal transcripts and proteins synthesized during oogenesis. Maternal factors are able to support the first cleavages, while blastocyst formation involves both maternal and embryonic factors. Over the last years, oocyte-restricted maternal effect genes have been the focus of much attention due to their specific expression profile and crucial function in early embryo development. They are predominantly expressed in oocyte, remain present in early embryos and then are degraded at the time of maternal-to-embryo transition (MET), without compensation by embryonic transcription. Functional studies based on knock-out mouse models have demonstrated their essential role in preimplantation embryo development, whereas functions in the oocyte itself have not been elucidated until this day.Mat
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