Ann Van Soom

Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos

BackgroundReal-time quantitative PCR is a sensitive and very efficient technique to examine gene transcription patterns in preimplantation embryos, in order to gain information about embryo development and to optimize assisted reproductive technologies. Critical to the succesful application of real-time PCR is careful assay design, reaction optimization and validation to maximize sensitivity and accuracy. In most of the studies published GAPD, ACTB or 18S rRNA have been used as a single reference gene without prior verification of their expression stability. Normalization of the data using unstable controls can result in erroneous conclusions, especially when only one reference gene is used.ResultsIn this study the transcription levels of 8 commonly used reference genes (ACTB, GAPD, Histone H2A, TBP, HPRT1, SDHA, YWHAZ and 18S rRNA) were determined at different preimplantation stages (2-cell, 8-cell, blastocyst and hatched blastocyst) in order to select the most stable genes to normalize quantitative data within different preimplantation embryo stages.ConclusionUsing the geNorm application YWHAZ, GAPD and SDHA were found to be the most stable genes across the examined embryonic stages, while the commonly used ACTB was shown to be highly regulated. We recommend the use of the geometric mean of those 3 reference genes as an accurate normalization factor, which allows small expression differences to be reliably measured.

Relationship between timing of development, morula morphology, and cell allocation to inner cell mass and trophectoderm in in vitro-produced bovine embryos.

Noninvasive measurements of bovine embryo quality, such as timing of cleavage, morula morphology, blastocyst formation, and hatching ability, were linked with the number of inner cell mass (ICM) cells and trophectoderm (TE) cells of the resulting embryos. First, it was confirmed that fast‐cleaving embryos proved to have significantly higher chances to reach advanced developmental stages vs. intermediate and slow cleavers (P = 0.01). They also showed significantly less fragmentation at the morula stage, implying the presence of more excellent morulae among fast‐cleaving embryos (P < 0.05). Second, the quality of hatched blastocysts, resulting from morulae of different morphological grades, was examined by differential staining. The total cell and ICM cell numbers were significantly lower for hatched blastocysts developed from poor morulae compared to hatched blastocysts developed from excellent, good, or fair morulae. However, hatched blastocysts with <10 ICM cells were seen in embryos belonging to all four morphological scores. Finally, it was found that timing of first cleavage was not significantly correlated with timing of blastocyst formation or with cell number of blastocysts. Timing of blastocyst formation, however, was significantly correlated with cell number: day 8 blastocysts had significantly lower total cell and ICM cell numbers than day 6 and day 7 blastocysts (P < 0.001). These results suggest that the quality of in vitro‐produced bovine embryos is very variable and cannot be linked with a single criterion such as embryo morphology and/or hatching ability. Timing of blastocyst formation was the most valuable criterion with regard to embryonic differentiation. Mol. Reprod. Dev. 47:47–56, 1997.

Markers of stemness in equine mesenchymal stem cells: a plea for uniformity.

Mesenchymal stromal cells (MSC) are a very promising subpopulation of adult stem cells for cell-based regenerative therapies in veterinary medicine. Despite major progress in the knowledge on adult stem cells during recent years, a proper identification of MSC remains a challenge. In human medicine, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) recently proposed three criteria to define MSC. Firstly, cells must be plastic-adherent when maintained under standard culture conditions. Secondly, MSC must express CD73, CD90 and CD105, and lack expression of CD34, CD45, CD14 or CD11b, CD79α or CD19 and MHC class II antigens. Thirdly, MSC must be able to differentiate into osteoblasts, adipocytes and chondroblasts in vitro. Successful isolation and differentiation of equine MSC from different sources such as bone marrow, fat tissue, umbilical cord blood, Whartons Jelly or peripheral blood has been widely reported. However, their unequivocal immunophenotyping is hampered by the lack of a single specific marker and the limited availability of monoclonal anti-horse antibodies, which are two major factors complicating successful research on equine MSC. Detection of gene expression on mRNA level is hereby a valuable alternative, although the need still exists to test several antibody clones in search for cross-reactivity. To date, commercial antibodies recognizing equine epitopes are only available for CD13, CD44 and MHC-II. Moreover, as the expression of certain adult stem cell markers may differ between species, it is mandatory to define a set of CD markers which can be uniformly applied for the identification of equine MSC.

Cell allocation to the inner cell mass and the trophectoderm in bovine embryos cultured in two different media

Data from other laboratories have shown that speed of bovine blastocyst development is higher when Ménézo B2 is used for coculture compared to TCM199. It was our purpose to investigate whether this early blastocyst formation was also indicative of embryo quality by studying the allocation of inner cells in embryos generated by B2‐coculture and by TCM199‐coculture. For this purpose, a differential staining technique was used. General embryo development was similar for TCM199‐ and B2‐embryos expressed as rate of cleavage at day 3 and morula‐blastocyst formation at day 8 (P > 0.05), but significantly different when expressed as number of eight‐cell stages at day 3 and expanded or hatched blastocysts at day 8 (P < 0.01). B2‐embryos cultured until day 5, 6, and 7 post insemination, had total cell numbers of 24, 65, and 109 respectively, which was significantly higher than the cell number of TCM199 embryos cultured over the same time period (18, 41, and 71 respectively, P < 0.001). Morphological differentiation was significantly more advanced for B2‐embryos at day 7 and 8 (P < 0.0001 and P < 0.001, respectively). First presumptive inner cells appeared in eight‐ to 16‐cell stages at day 3. Because the determination of inner cells by differential staining is depending upon the presence of functional tight junctions, we concluded that the establishment of the tight junction seal in B2‐embryos differed from that in TCM199‐embryos: Inner cells appeared 0.56 cell cycle later in B2‐embryos (P < 0.001) and a larger variation existed in the number of ICM‐cells in B2‐blastocysts (P < 0.001). The higher total cell number of B2‐expanded blastocysts was mainly acquired by trophectoderm growth (P < 0.06). These data indicate that the apparent better quality of B2‐embryos (faster cleavage, earlier blastocyst formation) is not reflected in a reliable number of inner cells of B2‐blastocysts.

Structural Aspects of the Zona Pellucida of In Vitro-Produced Bovine Embryos: A Scanning Electron and Confocal Laser Scanning Microscopic Study

Abstract Structural aspects of the bovine zona pellucida (ZP) of in vitro-matured (IVM) oocytes and in vitro-produced (IVP) embryos were studied in two experiments to find a tentative explanation for the zonas barrier function against viral infection. In Experiment 1, the ultrastructure of the outer ZP surface was studied. The diameter (nm) and the number of the outer pores within an area of 5000 μm2 of 10 IVM oocytes, 10 zygotes, 10 8-cell-stage embryos, and 10 morulae were evaluated by scanning electron microscopy. In oocytes and morulae, the ZP surface showed a rough and spongy appearance with numerous pores. In zygotes, the ZP surface was found to have a smooth, melted appearance with only a few pores. In 8-cell-stage embryos, both surface patterns were found. The mean number (per 5000 μm2) and the mean diameter of the outer pores were different between the four stages of development (P < 0.001): 1511 pores in oocytes, 1187 in zygotes, 1658 in 8-cell-stage embryos, and 3259 in morulae, with mean diameters of 182, 223, 203, and 155 nm, respectively. In Experiment 2, the continuity of the meshes (network of pores) towards the embryonic cells was examined by confocal laser scanning microscopy. Therefore, the passage through and the location in the ZP of fluorescent microspheres, with similar dimensions as bovine viral diarrhea virus (BVDV, 40–50 nm) and bovine herpesvirus-1 (BHV-1; 180–200 nm), were evaluated. For all stages, the smallest beads were detected halfway through the thickness of the ZP, whereas the beads with a size of 200 nm were found only within the outer-fourth part of the ZP. It can be concluded that the intact ZP of bovine IVM oocytes and IVP embryos are constructed in such a way that BVDV and BHV-1 should not be able to traverse the ZP and reach the embryonic cells. However, the risk exists that viral particles can be trapped in the outer layers of the ZP.

Cumulus contributions during bovine fertilization in vitro

A mandatory step in performing micromanipulation techniques, studying sperm-oocyte interactions and evaluating morphological aspects of oocyte quality is the removal of cumulus cells from oocytes or zygotes at various stages. In cattle, cumulus removal shortly before fertilization in vitro strongly decreases sperm penetration rates. This study was conducted to evaluate the function of the cumulus oophorus during bovine fertilization in vitro. The importance of cumulus secretions during IVF was investigated by inseminating cumulus-denuded oocytes (CDOs) in fertilization medium supplemented with individual cumulus secretions, such as progesterone or hyaluronic acid. None of these substances increased the fertilization rate of CDOs. However, fertilizing CDOs in cumulus-conditioned medium or on a cumulus monolayer partially restored the reduction in fertilization rate (P<0.05). The fertilization rate of CDOs inseminated on a cumulus monolayer further increased when physical contact between the gametes and the monolayer was prevented by fertilizing them inside a culture plate insert placed on the monolayer (P<0.05). Finally, the importance of reactive oxygen species (ROS) generation and O(2) concentration during IVF was studied. Luminol-dependent chemiluminescence revealed a higher ROS load in conditioned medium of cumulus-enclosed oocytes (CEOs) than in that of CDOs after sperm-oocyte co-incubation (P<0.05). Furthermore, lowering the external O(2) concentration from 20 to 5% decreased the fertilization rate of both CEOs and CDOs, but had a higher impact on CEOs (P<0.05). In conclusion, this study provides evidence that the cumulus oophorus benefits the fertilizing ability of penetrating spermatozoa by creating a complex microenvironment of both cumulus secretions and metabolic products around the oocyte. Gap junctional communication between the oocyte and corona cells as well as sperm trapping by the cumulus oophorus seem to be essential factors in supporting fertilization.

Effect of technical settings on canine semen motility parameters measured by the Hamilton-Thorne analyzer

Computerized measuring devices are needed to assess canine semen quality objectively both for research and practical purposes. As internal image settings may influence the results considerably, the effect of different technical settings and semen processing on the parameters assessed by the Hamilton-Thorne Ceros 12.1 semen analyzer (HTR Ceros 12.1) was investigated. The frame rate (15, 30 or 60 frames/s) significantly (P<0.05) influenced most of the measured motility characteristics in experiment 1 while no differences in the motility parameters were found using a different sampling duration (0.5 or 1 s, i.e. 30 or 60 frames scanned) in experiment 2. In experiment 3, an increase in sperm velocity (VAP, VSL, VCL), in linearity and in the percentage of motile and rapidly moving spermatozoa was observed with increasing sperm concentrations (25 x 10(6), 50 x 10(6) or 100 x 10(6) ml(-1)). In experiment 4, a clear effect of the diluent used was visible with higher velocity parameters (VAP, VSL, VCL) and higher percentages of motile, progressive and rapid spermatozoa for semen samples diluted in Hepes-TALP or prostatic fluid in comparison with physiological saline or egg-yolk-Tris extender. In experiment 5, significant (P<0.01) and high correlations were found between the conventional dog semen analysis methods and HTR Ceros 12.1 measurements (n=97 semen samples) for the sperm concentration (r=0.91), the motility (r=0.74) and the progressive motility (r=0.84). In experiment 6, the ejaculates from 21 proven, fertile dogs were compared with the ejaculates of a population (N: 11) of young beagles (1.5 years) but no significant differences in HTR Ceros 12.1 measurements were found between the two groups. Based on our results, diluting dog semen samples to 50 x 10(6) ml(-1) with physiological saline solution and scanning 30 frames at a frame rate of 60 frames/s (i.e. a scanning time of 0.5 s), are the set-up parameters proposed to obtain objective and standardized canine semen motility results using the HTR Ceros 12.1.

In Search for Cross-Reactivity to Immunophenotype Equine Mesenchymal Stromal Cells by Multicolor Flow Cytometry

During recent years, cell‐based therapies using mesenchymal stem cells (MSC) are reported in equine veterinary medicine with increasing frequency. In most cases, the isolation and in vitro differentiation of equine MSC are described, but their proper immunophenotypic characterization is rarely performed. The lack of a single marker specific for MSC and the limited availability of monoclonal antibodies (mAbs) for equine MSC in particular, strongly hamper this research. In this study, 30 commercial mAbs were screened with flow cytometry for recognizing equine epitopes using the appropriate positive controls to confirm their specificity. Cross‐reactivity was found and confirmed by confocal microscopy for CD45, CD73, CD79α, CD90, CD105, MHC‐II, a monocyte marker, and two clones tested for CD29 and CD44. Unfortunately, none of the evaluated CD34 clones recognized the equine epitopes on positive control endothelial cells. Subsequently, umbilical cord blood‐derived undifferentiated equine MSC of the fourth passage of six horses were characterized using multicolor flow cytometry based on the selected nine‐marker panel of both cell surface antigens and intracytoplasmatic proteins. In addition, appropriate positive and negative controls were included, and the viable single cell population was analyzed by excluding dead cells using 7‐aminoactinomycin D. Isolated equine MSC of the fourth passage were found to be CD29, CD44, CD90 positive and CD45, CD79α, MHC‐II, and a monocyte marker negative. A variable expression was found for CD73 and CD105. Successful differentiation towards the osteogenic, chondrogenic, and adipogenic lineage was used as additional validation. We suggest that this selected nine‐marker panel can be used for the adequate immunophenotyping of equine MSC.

Assessment of mammalian embryo quality: what can we learn from embryo morphology?

Embryo morphology assessment, however imperfect it may be, is at present the most popular method for embryo selection prior to transfer, both in human and bovine assisted reproduction. A major difference between human and bovine embryos is the fact that in the latter, assessment of morphology is jeopardized by the opacity of the blastomeres, which is caused by lipid droplet accumulation. This opacity makes it difficult to assess nuclear and nucleolar morphology, aspects which can easily be evaluated in human zygotes or early cleaving embryos. However, recent research which focused on correlation between bovine embryo morphology and embryonic ultrastructure, gene expression and cryoresistance, has provided evidence that much more can be deduced from mere embryo morphology than previously thought. Morphological features such as colour of the blastomeres, the extent of compaction, timing of blastocyst formation and expansion and diameter of the embryo at hatching can be linked with embryo quality. On the other hand, cattle embryos of deviant chromosomal constitution or with aberrant genetic make-up cannot be selected against by means of the current morphological techniques. Possible solutions include the visualization of bovine pronuclei at the zygote stage by means of ultracentrifugation or multiphoton laser scanning microscopy, and adjustment of genetic analysis in order to reconstruct embryo genetic make-up starting from the biopsy material.

Sperm Binding to Epithelial Oviduct Explants in Bulls with Different Nonreturn Rates Investigated with a New In Vitro Model

Abstract A new in vitro method was developed for analyzing the capacity of sperm to bind to oviductal epithelium to determine whether this binding capacity could be used to predict nonreturn rates (NRR). Sperm binding was evaluated by counting 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1)-labeled spermatozoa attached to oviductal epithelium and by measuring the surface area of the oviduct explants by means of an image analysis program. Hepes + Tyrode albumin lactate pyruvate (TALP) was a more useful medium than in vitro fertilization (IVF)-TALP, TCM-199 medium + 10% fetal calf serum, and TCM-199 medium alone for the investigation of sperm binding to oviductal explants. Oviduct explants with a surface area of ;lt20 000 μm2 provided more consistent results than did explants with a surface area of >100 000 μm2. A positive association was found between the loge transformed number of spermatozoa bound to 0.1 mm2 oviductal epithelium and the NRR of the respective sires after 24 h of coincubation, provided that the membrane integrity of the sperm sample was >60%. Determination of the capacity of sperm to bind to oviductal explants could become a reliable in vitro method for predicting the NRR of a given sire.