Katrien Smits

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

BackgroundApplication of reverse transcription quantitative real-time polymerase chain reaction is very well suited to reveal differences in gene expression between in vivo and in vitro 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 in vivo and fresh and frozen-thawed in vitro embryos.FindingsThe expression stability of 8 candidate reference genes (ACTB, GAPDH, H2A/I, HPRT1, RPL32, SDHA, TUBA4A, UBC) was determined in 3 populations of equine blastocysts (fresh in vivo, fresh and frozen-thawed in vitro embryos). Application of geNorm indicated UBC, GAPDH, ACTB and HPRT1 as the most stable genes in the in vivo embryos and UBC, RPL32, GAPDH and ACTB in both in vitro populations. When in vivo and in vitro embryos were combined, UBC, ACTB, RPL32 and GAPDH were found to be the most stable. SDHA and H2A/I appeared to be highly regulated.ConclusionsBased on these results, the geometric mean of UBC, ACTB, RPL32 and GAPDH is to be recommended for accurate normalization of quantitative real-time PCR data in equine in vivo and in vitro produced blastocysts.

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

IntroductionMesenchymal 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.MethodsEquine 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.ResultsWhile 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 (TGFβ). In contrast, no mRNA expression was found for CD86, indoleamine 2,3-dioxygenase (IDO), and tumor necrosis factor-alpha (TNFα).ConclusionsWhereas 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.

Breeding or Assisted Reproduction? Relevance of the Horse Model Applied to the Conservation of Endangered Equids

Many wild equids are at present endangered in the wild. Concurrently, increased mechanization has pushed back the numbers of some old native horse breeds to levels that are no longer compatible with survival of the breed. Strong concerns arose in the last decade to preserve animal biodiversity, including that of rare horse breeds. Genome Resource Banking refers to the cryostorage of genetic material and is an approach for ex situ conservation, which should be applied in combination with in situ conservation programmes. In this review, we propose that, owing to the great reproductive similarity among the different members of the genus Equus, the domestic horse can be used to optimize cryopreservation and embryo production protocols for future application in wild equids. We will give this hypothesis a scientific underpinning by listing successful applications of epididymal sperm freezing, embryo freezing, intracytoplasmic sperm injection, oocyte vitrification and somatic cell nuclear transfer in domestic horses. Some ART fertilization methods may be performed with semen of very low quality or with oocytes obtained after the death of the mare.

Gene expression profiling of pluripotency and differentiation-related markers in cat oocytes and preimplantation embryos.

During mammalian preimplantation development, two successive differentiation events lead to the establishment of three committed lineages with separate fates: the trophectoderm, the primitive endoderm and the pluripotent epiblast. In the mouse embryo, the molecular mechanisms underlying these two cell fate decisions have been studied extensively, leading to the identification of lineage-specific transcription factors. Species-specific differences in expression patterns of key regulatory genes have been reported, raising questions regarding their role in different species. The aim of the present study was to characterise the gene expression patterns of pluripotency (OCT4, SOX2, NANOG) and differentiation (CDX2, GATA6)-related markers during feline early development using reverse transcription-quantitative polymerase chain reaction. In addition, we assessed the impact of in vitro development on gene expression by comparing transcript levels of the genes investigated between in vitro and in vivo blastocysts. To normalise quantitative data within different preimplantation embryo stages, we first validated a set of stable reference genes. Transcript levels of all genes investigated were present and changed over the course of preimplantation development; a highly significant embryo-stage effect on gene expression was observed. Transcript levels of OCT4 were significantly reduced in in vitro blastocysts compared with their in vivo counterparts. None of the other genes investigated showed altered expression under in vitro conditions. The different gene expression patterns of OCT4, SOX2, CDX2 and GATA6 in cat embryos resembled those described in mouse embryos, indicative of a preserved role for these genes during early segregation. However, because of the absence of any upregulation of NANOG transcription levels after embryonic genome activation, it is unlikely that NANOG is a key regular of lineage segregation. Such results support the hypothesis that the behaviour of early lineage markers can be species specific. The present study also revealed a pool of maternal NANOG mRNA transcripts, the role of which remains to be elucidated. Comparing transcription levels of these genes between in vivo and in vitro blastocysts revealed low levels of OCT4 mRNA in the latter, which may contribute to the reduced developmental competence of embryos under suboptimal conditions.

A pilot comparison of laser-assisted vs piezo drill ICSI for the in vitro production of horse embryos.

Intracytoplasmic sperm injection (ICSI) is the method of choice for the in vitro production (IVP) of equine embryos. However, conventional ICSI has been associated with mechanical damage to the oocyte caused by the deformation of the zona pellucida (ZP) and exposure of the oolemma to negative pressure during injection. Introduction of the less traumatic and more efficient piezo drill-assisted ICSI (PDAI) yielded higher cleavage rates and more consistent results. Nevertheless, PDAI is also associated with disadvantages such as the use of mercury and possible DNA damage. This led us to explore an alternative method avoiding oocyte trauma, namely laser-assisted ICSI (LAI), which involves creating a hole in the ZP prior to ICSI. In this pilot study, PDAI and LAI were compared for ICSI in the horse. No significant influences on subsequent embryonic development were observed.

Influence of the uterine environment on the development of in vitro produced equine embryos

The necessity for early interaction between the embryo and the oviductal and/or uterine environment in the horse is reflected by several striking differences between equine embryos that develop in vivo and those produced in vitro. Better understanding of the salient interactions may help to improve the efficiency of in vitro equine embryo production. In an initial experiment, cleavage-stage in vitro-produced (IVP) equine embryos were transferred into the uterus of recipient mares that had ovulated recently to determine whether premature placement in this in vivo environment would improve subsequent development. In a second experiment, an important element of the uterine environment was mimicked by adding uterocalin, a major component of the endometrial secretions during early pregnancy, to the culture medium. Intrauterine transfer of cleavage-stage IVP equine embryos yielded neither ultrasonographically detectable pregnancies nor day 7 blastocysts, indicating that the uterus is not a suitable environment for pre-compact morula stage horse embryos. By contrast, exposure to uterocalin during IVP improved capsule formation, although it did not measurably affect the development or expression of a panel of genes known to differ between in vivo and in vitro embryos. Further studies are required to evaluate whether uterocalin serves purely as a carrier protein or more directly promotes improved capsule development.

In vivo-derived horse blastocysts show transcriptional upregulation of developmentally important genes compared with in vitro-produced horse blastocysts

In vitro-produced (IVP) equine blastocysts can give rise to successful pregnancies, but their morphology and developmental rate differ from those of in vivo-derived equine blastocysts. The aim of the present study was to evaluate this difference at the genetic level. Suppression subtractive hybridisation (SSH) was used to construct a cDNA library enriched for transcripts preferentially expressed in in vivo-derived equine blastocysts compared with IVP blastocysts. Of the 62 different genes identified in this way, six genes involved in embryonic development (BEX2, FABP3, HSP90AA1, MOBKL3, MCM7 and ODC) were selected to confirm this differential expression by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). Using RT-qPCR, five genes were confirmed to be significantly upregulated in in vivo-derived blastocysts (i.e. FABP3, HSP90AA1 (both P<0.05), ODC, MOBKL3 and BEX2 (P<0.005 for all three)), confirming the results of the SSH. There was no significant difference in MCM7 expression between IVP and in vivo-derived blastocysts. In conclusion, five genes that are transcriptionally upregulated in in vivo-derived equine blastocysts compared with IVP blastocysts have been identified. Because of their possible importance in embryonic development, the expression of these genes can be used as a marker to evaluate in vitro embryo production systems in the horse.

Procaine Induces Cytokinesis in Horse Oocytes via a pH-Dependent Mechanism

ABSTRACT Coincubating equine gametes in the presence of procaine has been reported to facilitate in vitro fertilization, with cleavage rates exceeding 60%. We report that while procaine does trigger sperm hyperactivation, it independently induces cleavage of equine oocytes. First, we found that procaine (1–5 mM) did not facilitate stallion sperm penetration of equine oocytes but instead induced sperm-independent oocyte cytokinesis in the absence of the second polar body extrusion. Indeed, 56 ± 4% of oocytes cleaved within 2.5 days of exposure to 2.5 mM procaine regardless of sperm presence. However, the cleaved oocytes did not develop beyond 8 to 16 cells, and the daughter cells either lacked nuclei or contained aberrant, condensed DNA fragments. By contrast, intracytoplasmic sperm injection (ICSI) was followed by second polar body extrusion and formation of normal blastocysts. Moreover, neither the calcium oscillations detectable using fura-2 AM staining nor the cortical granule reaction visualized by LCA-FITC staining, after oocyte activation induced by ICSI or ionomycin treatment, were detected after exposing oocytes to 2.5 mM procaine. Instead, procaine initiated an ooplasmic alkalinization, detectable by BCECF-AM staining that was not observed after other treatments. This alkalinization was followed, after an additional 18 h of incubation, by cortical F-actin depolymerization, as demonstrated by reduced actin phalloidin-FITC staining intensity, that resembled preparation for cytokinesis in ICSI-fertilized zygotes. Overall, we conclude that procaine induces cytokinesis in equine oocytes accompanied by aberrant chromatin condensation and division; this explains why embryos produced after exposing equine oocytes to procaine fail to develop beyond the 8- to 16-cell stage.

An Abortion of Monozygotic Twins in a Warmblood Mare

Naturally occurring monozygotic twins are extremely rare in the horse. This paper describes an abortion in a mare after 260 days of pregnancy with monozygotic twins, one a fresh foal and the other a mummified foal.

Acute in vivo interactions of Helicobacter equorum with its equine host

REASONS FOR PERFORMING STUDY A novel urease-negative Helicobacter species has been isolated from faecal samples of clinically healthy horses, but no information is available about the main sites of colonisation in the equine gastrointestinal tract nor is the pathogenic potential of this microorganism known. An experimental infection in horses was therefore carried out. METHODS Four horses were infected with H. equorum strain CCUG 52199T and subjected to euthanasia at 10 (n = 2) and 30 days (n = 2) post inoculation. A fifth animal was inoculated with phosphate buffered saline and used as control. Gastrointestinal samples were examined histologically and bacteriologically. These samples, as well as faecal material collected at regular intervals, were also subjected to PCR analysis. RESULTS All horses remained clinically healthy and no specific macroscopic lesions were identified, nor were there any microscopic changes. H. equorum-DNA was detected in the faeces during the whole experiment in all infected animals but not in the negative control. Sites of colonisation were caecum, colon and rectum. CONCLUSIONS H. equorum is able to colonise the equine lower bowel and is excreted in faeces without apparent pathology. No association between the presence of the organism and gastrointestinal disease was demonstrated.