Dynamics of 5-methylcytosine and 5-hydroxymethylcytosine during pronuclear development in equine zygotes produced by ICSI

Background: Global epigenetic reprogramming is considered to be essential during embryo development to establish totipotency. In the classic model first described in the mouse, the genome-wide DNA demethylation is asymmetric between the paternal and the maternal genome. The paternal genome undergoes ten-eleven translocation (TET)-mediated active DNA demethylation, which is completed before the end of the first cell cycle. Since TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine, the latter is postulated to be an intermediate stage toward DNA demethylation. The maternal genome, on the other hand, is protected from active demethylation and undergoes replication-dependent DNA demethylation. However, several species do not show the asymmetric DNA demethylation process described in this classic model, since 5-methylcytosine and 5-hydroxymethylcytosine are present during the first cell cycle in both parental genomes. In this study, global changes in the levels of 5-methylcytosine and 5-hydroxymethylcytosine throughout pronuclear development in equine zygotes produced in vitro were assessed using immunofluorescent staining. Results: We were able to show that 5-methylcytosine and 5-hydroxymethylcytosine both were explicitly present throughout pronuclear development, with similar intensity levels in both parental genomes, in equine zygotes produced by ICSI. The localization patterns of 5-methylcytosine and 5-hydroxymethylcytosine, however, were different, with 5-hydroxymethylcytosine homogeneously distributed in the DNA, while 5-methylcytosine tended to be clustered in certain regions. Fluorescence quantification showed increased 5-methylcytosine levels in the maternal genome from PN1 to PN2, while no differences were found in PN3 and PN4. No differences were observed in the paternal genome. Normalized levels of 5-hydroxymethylcytosine were preserved throughout all pronuclear stages in both parental genomes. Conclusions: In conclusion, the horse does not seem to follow the classic model of asymmetric demethylation as no evidence of global DNA demethylation of the paternal pronucleus during the first cell cycle was demonstrated. Instead, both parental genomes displayed sustained and similar levels of methylation and hydroxymethylation throughout pronuclear development

Cryopreservation of equine oocytes: looking into the crystal ball

In vitro embryo production has evolved rapidly in the horse over the past decade, but blastocyst rates from vitrified equine oocytes remain quite poor and further research is needed to warrant application. Oocyte vitrification is affected by several technical and biological factors. In the horse, short exposure of immature oocytes to the combination of permeating and non-permeating cryoprotective agents has been associated with the best results so far. High cooling and warming rates are also crucial and can be obtained by using minimal volumes and open cryodevices. Vitrification of in vivo-matured oocytes has yielded better results, but is less practical. The presence of the corona radiata seems to partially protect those factors that are necessary for the construction of the normal spindle and for chromosome alignment, but multiple layers of cumulus cells may impair permeation of cryoprotective agents. In addition to the spindle, the oolemma and mitochondria are also particularly sensitive to vitrification damage, which should be minimised in future vitrification procedures. This review presents promising protocols and novel strategies in equine oocyte vitrification, with a focus on blastocyst development and foal production as most reliable outcome parameters

Bta-miR-10b secreted by bovine embryos negatively impacts preimplantation embryo quality

In a previous study, we found miR-10b to be more abundant in a conditioned culture medium of degenerate embryos compared to that of blastocysts. Here, we show that miR-10b mimics added to the culture medium can be taken up by embryos. This uptake results in an increase in embryonic cell apoptosis and aberrant expression of DNA methyltransferases (DNMTs). Using several algorithms, Homeobox Al (HOXA1) was identified as one of the potential miR-10b target genes and dual-luciferase assay confirmed HOXA1 as a direct target of miR-10b. Microinjection of si-HOXA1 into embryos also resulted in an increase in embryonic cell apoptosis and downregulation of DNMTs. Cell progression analysis using Madin-Darby bovine kidney cells (MDBKs) showed that miR-10b overexpression and HOXA1 knockdown results in suppressed cell cycle progression and decreased cell viability. Overall, this work demonstrates that miR-10b negatively influences embryo quality and might do this through targeting HOXA1 and/or influencing DNA methylation

Fertiliteitsbehandelingen bij het paard: toepassingsmogelijkheden en beperkingen

Recent developments in the assisted reproduction in horses allow to breed foals from sub- and infertile mares, as well as from recently deceased mares or stallions. Oocytes can be obtained from live donor mares by ovum pick-up (OPU), by flushing oocytes from follicles using a transvaginal or transabdominal approach. Post mortem oocytes can be obtained by scraping the follicles. After oocyte maturation, the oocytes can be fertilized in vitro or can be transferred to the oviduct of an inseminated recipient mare (in vivo). Since conventional in vitro fertilization (IVF) is very unsuccessful in the horse, fertilization is performed by intracytoplasmic sperm injection (ICSI). After ICSI, the fertilized oocytes can be transferred to the oviduct of a synchronized recipient mare or further cultured in vitro up to the blastocyst stage. Subsequently, obtained blastocyts can be transferred to the uterus of a recipient mare. In this article, in vitro embryo production in the horse is highlighted, and possible advantages and disadvantages and clinical and scientific applications are reviewed

Twisting of mummified remains around the umbilical cord as a potential risk of delayed twin reduction in the mare

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Characterization and profiling of immunomodulatory genes of equine mesenchymal stromal cells from non-invasive sources

Introduction: Mesenchymal stromal cells (MSCs) have been extensively studied for their promising capabilities in regenerative medicine. Although bone marrow is the best-known source for isolating equine MSCs, non-invasive alternative sources such as umbilical cord blood (UCB), umbilical cord matrix (UCM), and peripheral blood (PB) have also been reported. Methods: Equine MSCs from three non-invasive alternative sources were isolated from six individual mares (PB) and their foals (UCB and UCM) at parturition. To minimize inter-horse variability, the samples from the three sources were matched within the same mare and for UCB and UCM even within the same foal from that specific mare. The following parameters were analyzed: (i) success rate of isolation, (ii) proliferation capacity, (iii) tri-lineage differentiation ability, (iv) immunophenotypical protein, and (v) immunomodulatory mRNA profiles. Linear regression models were fit to determine the association between the source of MSCs (UCB, UCM, PB) and (i) the moment of first observation, (ii) the moment of first passage, (iii) cell proliferation data, (iv) the expression of markers related to cell immunogenicity, and (v) the mRNA profile of immunomodulatory factors, except for hepatocyte growth factor (HGF) as no normal distribution could be obtained for the latter variable. To evaluate the association between the source of MSCs and the mRNA expression of HGF, the non-parametric Kruskal-Wallis test was performed instead. Results: While equine MSCs could be isolated from all the UCB and PB samples, isolation from UCM was successful in only two samples because of contamination issues. Proliferation data showed that equine MSCs from all three sources could be easily expanded, although UCB-derived MSCs appeared significantly faster in culture than PB- or UCM-derived MSCs. Equine MSCs from both UCB and PB could be differentiated toward the osteo-, chondro-, and adipogenic lineage, in contrast to UCM-derived MSCs in which only chondro-and adipogenic differentiation could be confirmed. Regardless of the source, equine MSCs expressed the immunomodulatory genes CD40, CD80, HGF, and transforming growth factor-beta (TGF beta). In contrast, no mRNA expression was found for CD86, indoleamine 2,3-dioxygenase (IDO), and tumor necrosis factor-alpha (TNF alpha). Conclusions: Whereas UCM seems less feasible because of the high contamination risks and low isolation success rates, UCB seems a promising alternative MSC source, especially when considering allogeneic MSC use

Ovarian teratoma in the mare: a review and two cases

A 4-year-old Belgian Warmblood mare suffering from pelvic flexure impaction was diagnosed with a teratoma of the left ovary. The enlarged ovary was identified several days after treatment of the impaction. Surgical removal of the affected ovary was performed and histo pathological examination confirmed the presence of a teratoma. The features of another ovarian teratoma, which was an accidental finding at slaughter, are also described

Maternal recognition of pregnancy in the horse : are MicroRNAs the secret messengers?

The signal for maternal recognition of pregnancy (MRP) has still not been identified in the horse. High-throughput molecular biology at the embryo-maternal interface has substantially contributed to the knowledge on pathways affected during MRP, but an integrated study in which proteomics, transcriptomics and miRNA expression can be linked directly is currently lacking. The aim of this study was to provide such analysis. Endometrial biopsies, uterine fluid, embryonic tissues, and yolk sac fluid were collected 13 days after ovulation during pregnant and control cycles from the same mares. Micro-RNA-Sequencing was performed on all collected samples, mRNA-Sequencing on the same tissue samples and mass spectrometry was conducted previously on the same fluid samples. Differential expression of miRNA, mRNA and proteins showed high conformity with literature and confirmed involvement in pregnancy establishment, embryo quality, steroid synthesis and prostaglandin regulation, but the link between differential miRNAs and their targets was limited and did not indicate the identity of an unequivocal signal for MRP in the horse. Differential expression at the embryo-maternal interface was prominent, highlighting a potential role of miRNAs in embryo-maternal communication during early pregnancy in the horse. These data provide a strong basis for future targeted studies

Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts

<p>Abstract</p> <p>Background</p> <p>Application of reverse transcription quantitative real-time polymerase chain reaction is very well suited to reveal differences in gene expression between <it>in vivo </it>and <it>in vitro </it>produced embryos. Ultimately, this may lead to optimized equine assisted reproductive techniques. However, for a correct interpretation of the real-time PCR results, all data must be normalized, which is most reliably achieved by calculating the geometric mean of the most stable reference genes. In this study a set of reliable reference genes was identified for equine <it>in vivo </it>and fresh and frozen-thawed <it>in vitro </it>embryos.</p> <p>Findings</p> <p>The expression stability of 8 candidate reference genes (<it>ACTB</it>, <it>GAPDH</it>, <it>H2A/I</it>, <it>HPRT1</it>, <it>RPL32</it>, <it>SDHA</it>, <it>TUBA4A</it>, <it>UBC</it>) was determined in 3 populations of equine blastocysts (fresh <it>in vivo</it>, fresh and frozen-thawed <it>in vitro </it>embryos). Application of geNorm indicated <it>UBC</it>, <it>GAPDH</it>, <it>ACTB </it>and <it>HPRT1 </it>as the most stable genes in the <it>in vivo </it>embryos and <it>UBC</it>, <it>RPL32</it>, <it>GAPDH </it>and <it>ACTB </it>in both <it>in vitro </it>populations. When <it>in vivo </it>and <it>in vitro </it>embryos were combined, <it>UBC</it>, <it>ACTB</it>, <it>RPL32 </it>and <it>GAPDH </it>were found to be the most stable. <it>SDHA </it>and <it>H2A/I </it>appeared to be highly regulated.</p> <p>Conclusions</p> <p>Based on these results, the geometric mean of <it>UBC</it>, <it>ACTB</it>, <it>RPL32 </it>and <it>GAPDH </it>is to be recommended for accurate normalization of quantitative real-time PCR data in equine <it>in vivo </it>and <it>in vitro </it>produced blastocysts.</p

Tumorbank@uza: A Collection of Tissue, Fluid Samples and Associated Data of Oncology Patients for the Use in Translational Research

Tumorbank@UZA is an academic hospital integrated biobank that collects tissue, blood and urine samples from oncology patients. We work according to a quality management system and have established SOPs for all work procedures in the biobank. Tumorbank@UZA is funded by the National Cancer Plan, an initiative from the Belgian government since 2009. Samples from our biobank are available for both academic as well as commercial researchers, through a well-established access procedure. Currently the collection consists of more than 85.000 samples of more than 8000 patients. Funding statement: Tumorbank@UZA is funded by the National Cancer Plan (initiative 27) from the Ministry of Health of the Belgian Federal Government.</p