Oxygen-regulated gene expression in bovine blastocysts

Oxygen concentrations used during in vitro embryo culture can influence embryo development, cell numbers, and gene expression. Here we propose that the preimplantation bovine embryo possesses a molecular mechanism for the detection of, and response to, oxygen, mediated by a family of basic helix-loop-helix transcription factors, the hypoxia-inducible factors (HIFs). Day 5 compacting bovine embryos were cultured under different oxygen tensions (2%, 7%, 20%) and the effect on the expression of oxygen-regulated genes, development, and cell number allocation and HIFalpha protein localization were examined. Bovine in vitro-produced embryos responded to variations in oxygen concentration by altering gene expression. GLUT1 expression was higher following 2% oxygen culture compared with 7% and 20% cultured blastocysts. HIF mRNA expression (HIF1alpha, HIF2alpha) was unaltered by oxygen concentration. HIF2alpha protein was predominantly localized to the nucleus of blastocysts. In contrast, HIF1alpha protein was undetectable at any oxygen concentration or in the presence of the HIF protein stabilizer desferrioxamine (DFO), despite being detectable in cumulus cells following normal maturation conditions, acute anoxic culture, or in the presence of DFO. Oxygen concentration also significantly altered inner cell mass cell proportions at the blastocyst stage. These results suggest that oxygen can influence gene expression in the bovine embryo during postcompaction development and that these effects may be mediated by HIF2alpha.

The conceptus regulates tryptophanyl-tRNA synthetase and superoxide dismutase 2 in the sheep caruncular endometrium during early pregnancy

Copyright © 2015 Elsevier Ltd. All rights reserved. This research project was funded by NHS Grampian R&D (project number RG05/019).

The conceptus induces a switch in protein expression and activities of superoxide dismutase 1 and 2 in the sheep endometrium during early pregnancy

Acknowledgements We thank Philippe Bolifraud (INRA, France), Krawiec Angele, Sandra Grange, Laurence Puillet-Anselme (CHU Grenoble, France) and Margaret Fraser (Aberdeen, UK) for their expert technical assistance. The authors also thank the staff of the sheep sheds of Jouy-en-Josas (INRA, France). The authors would also like to thank the anonymous reviewers for their close examination of this article and their useful comments.

Working sheep at 'Glen Shiel'

Working Sheep on 'Glen Shiel' is a key work of the author's exhibition Lightsite, which toured Western Australian galleries from February 2006 to November 2007. It is a five-minute-long exposure photographic image captured inside a purpose-built, room-sized pinhole camera which is demountable and does not have a floor. The work depicts octogenarian Ian Mangan who is both one of the first and last soldier settler farmers in the Gairdner-Jerramungup district in the Great Southern Region of Western Australia. Ian, his son, Stuart and Grandson Jacob, are preparing the last mob of sheep for sale before they move off their farm. The pinhole camera-room is sited amongst the sheep in the farm's sheep yards. Stuart and Jacob are depicted here standing amongst the sheep. The light from this exterior landscape is 'projected' inside the camera-room and illuminates the interior scene which includes that part of the sheep yards upon which the floorless room is erected, along with Ian who is standing motionless inside. The image evokes the temporality of light. Here, light itself is portrayed as the primary medium through which we both perceive and describe landscape. In this way it is through the agency of light that we construct our connectivity to landscape.

Working sheep

Working Sheep expands understandings of the role of photographic media in the representation of landscapes. It does so by combining architectural construction with B&W photographic processing techniques. A purpose-built room-sized camera obscura is first constructed over a portion of the landscape to be recorded. Photosensitive paper is applied to the interior wall surfaces and is exposed to the inverted light entering a small aperture. These photographs are subsequently developed within the camera itself and consequently 'suffer' embellishments and aberrations from the makeshift darkroom conditions. In this way the specificity of both the landscape and the event of its recording are registered in the final image. Many images were destroyed in the process. The idea of the work is to help the viewer reflect on the role media plays in our understanding of landscape and to thus question the means by which they themselves record and interpret landscape representations.

A modified cementing technique using BoneSource to augment fixation of the acetabulum in a sheep model

Background and purpose Our aim was to prove in an animal model that the use of HA paste at the cement-bone interface in the acetabulum would improve fixation. We examined, in sheep, the effect of interposing a layer of hydroxyapatite cement around the periphery of a polyethylene socket prior to fixing it using polymethylemethacrylate (PMMA). Methods We made a randomized study involving 22 sheep to test whether the application of BoneSource hydroxyapatite material to the surface of the ovine acetabulum prior to cementing a polyethylene cup at hip arthroplasty improved the fixation and the nature of the interface. We studied the gross radiographical appearance of the implant-bone interface and the histological appearance at the interface. Results There were more radiolucencies evident in the control group. Histologically, only sheep randomized into the BoneSource group exhibited a fully osseointegrated interface. Use of the hydroxyapatite material did not confer any detrimental effects. In some cases the material appeared to have been fully resorbed. When the material was evident on histological section, it was incorporated into an osseointegrated interface. There was no giant cell reaction present in any case. There was no evidence of migration of BoneSource to the articulation. Interpretation The application of HA material prior to cementation of a socket produced an improved interface. The technique may be useful in man with to extend the longevity of the cemented implant by protecting the socket interface from the effect of hydrodynamic fluid flow and particulate debris.

The severity of chorioamnionitis in pregnant sheep is associated with in vivo variation of the surface-exposed multiple-banded antigen/gene of ureaplasma parvum

Ureaplasma species are the bacteria most frequently isolated from human amniotic fluid in asymptomatic pregnancies and placental infections. Ureaplasma parvum serovars 3 and 6 are the most prevalent serovars isolated from men and women. We hypothesized that the effects on the fetus and chorioamnion of chronic ureaplasma infection in amniotic fluid are dependent on the serovar, dose, and variation of the ureaplasma multiple banded antigen (MBA) and mba gene. We injected high- or low dose U. parvum serovar 3, serovar 6, or vehicle intra-amniotically into pregnant ewes at 55 days of gestation (term = 150 days) and examined the chorioamnion, amniotic fluid, and fetal lung tissue of animals delivered by cesarean section at 125 days of gestation. Variation of the multiple banded antigen/mba generated by serovar 3 and serovar 6 ureaplasmas in vivo were compared by PCR assay and Western blot. Ureaplasma inoculums demonstrated only one (serovar 3) or two (serovar 6) MBA variants in vitro, but numerous antigenic variants were generated in vivo: serovar 6 passage 1 amniotic fluid cultures contained more MBA size variants than serovar 3 (P = 0.005),and ureaplasma titers were inversely related to the number of variants (P = 0.025). The severity of chorioamnionitis varied between animals. Low numbers of mba size variants (five or fewer) within amniotic fluid were associated with severe inflammation, whereas the chorioamnion from animals with nine or more mba variants showed little or no inflammation. These differences in chorioamnion inflammation may explain why not all women with in utero Ureaplasma spp. experience adverse pregnancy outcomes.

Ureaplasma colonization of amniotic fluid and efficacy of antenatal corticosteroids for preterm lung maturation in sheep

Objective: To assess the efficacy of maternal betamethasone for improving preterm lung function, in the presence of inflammation induced by amniotic fluid ureaplasma colonization. ----- ----- Study design: Ewes bearing single fetuses were randomized to receive an intra-amniotic injection of Ureaplasma parvum (serovar 6; 2×107 colony forming units) or vehicle at 86±2 days of pregnancy (mean±SD: term is 150d), followed by maternal intramuscular betamethasone (0.5mg/kg) or saline, either 2 or 7 days before delivery of lambs at 123±1d. ----- ----- Results: Amniotic fluid IL-8 was elevated by ureaplasmas (p=0.049) but unaffected by betamethasone. Lung inflammation induced by ureaplasmas was not affected by betamethasone. Lung compliance was increased by ureaplasma colonization (p=0.009) and betamethasone (p=0.042), and effects were additive. Lung surfactant was increased by ureaplasma colonization (p<0.001) and betamethasone 7 days (p=0.001), but not 2 days, before delivery. ----- ----- Conclusion: Inflammation improves preterm lung function due to increases in surfactant. Antenatal corticosteroids further augment lung function, through an apparently independent mechanism.

Instability prolongs the chondral phase during bone healing in sheep

In this sheep study, we investigated the influence of fixation stability on the temporal and spatial distribution of tissues in the fracture callus. As the initial mechanical conditions have been cited as being especially important for the healing outcome, it was hypothesized that differences in the path of healing would be seen as early as the initial phase of healing. ----- ----- Sixty-four sheep underwent a mid-shaft tibial osteotomy that was treated with either a rigid or a semi-rigid external fixator. Animals were sacrificed at 2, 3, 6 and 9 weeks postoperatively and the fracture calluses were analyzed using radiological, biomechanical and histological techniques. Statistical comparison between the groups was performed using the Mann–Whitney U test for unpaired non-parametric data. ----- ----- In the callus of the tibia treated with semi-rigid fixation, remnants of the fracture haematoma remained present for longer, although new periosteal bone formation during early healing was similar in both groups. The mechanical competence of the healing callus at 6 weeks was inferior compared to tibiae treated with rigid fixation. Semi-rigid fixation resulted in a larger cartilage component of the callus, which persisted longer. Remodeling processes were initiated earlier in the rigid group, while new bone formation continued throughout the entire investigated period in the semi-rigid group. ----- ----- In this study, evidence is provided that less rigid fixation increased the time required for healing. The process of intramembranous ossification appeared during the initial stages of healing to be independent of mechanical stability. However, the delay in healing was related to a prolonged chondral phase.

The mechanical heterogeneity of the hard callus influences local tissue strains during bone healing : a finite element study based on sheep experiments

During secondary fracture healing, various tissue types including new bone are formed. The local mechanical strains play an important role in tissue proliferation and differentiation. To further our mechanobiological understanding of fracture healing, a precise assessment of local strains is mandatory. Until now, static analyses using Finite Elements (FE) have assumed homogenous material properties. With the recent quantification of both the spatial tissue patterns (Vetter et al., 2010) and the development of elastic modulus of newly formed bone during healing (Manjubala et al., 2009), it is now possible to incorporate this heterogeneity. Therefore, the aim of this study is to investigate the effect of this heterogeneity on the strain patterns at six successive healing stages. The input data of the present work stemmed from a comprehensive cross-sectional study of sheep with a tibial osteotomy (Epari et al., 2006). In our FE model, each element containing bone was described by a bulk elastic modulus, which depended on both the local area fraction and the local elastic modulus of the bone material. The obtained strains were compared with the results of hypothetical FE models assuming homogeneous material properties. The differences in the spatial distributions of the strains between the heterogeneous and homogeneous FE models were interpreted using a current mechanobiological theory (Isakson et al., 2006). This interpretation showed that considering the heterogeneity of the hard callus is most important at the intermediate stages of healing, when cartilage transforms to bone via endochondral ossification.

Temporal tissue patterns in bone healing of sheep

Secondary fracture healing in long bones leads to the successive formation of intricate patterns of tissues in the newly formed callus. The main aim of this work was to quantitatively describe the topology of these tissue patterns at different stages of the healing process and to generate averaged images of tissue distribution. This averaging procedure was based on stained histological sections (2, 3, 6, and 9 weeks post-operatively) of 64 sheep with a 3 mm tibial mid-shaft osteotomy, stabilized either with a rigid or a semi-rigid external fixator. Before averaging, histological images were sorted for topology according to six identified tissue patterns. The averaged images were obtained for both fixation types and the lateral and medial side separately. For each case, the result of the averaging procedure was a collection of six images characterizing quantitatively the progression of the healing process. In addition, quantified descriptions of the newly formed cartilage and the bone area fractions (BA/TA) of the bony callus are presented. For all cases, a linear increase in the BA/TA of the bony callus was observed. The slope was greatest in the case of the most rigid stabilization and lowest in the case of the least stiff. This topological description of the progression of bone healing will allow quantitative validation (or falsification) of current mechano-biological theories.

Size and habit of mineral particles in bone and mineralized callus during bone healing in sheep

Bone healing is known to occur through the successive formation and resorption of various tissues with different structural and mechanical properties. To get a better insight into this sequence of events, we used environmental scanning electron microscopy (ESEM) together with scanning small-angle X-ray scattering (sSAXS) to reveal the size and orientation of bone mineral particles within the regenerating callus tissues at different healing stages (2, 3, 6, and 9 weeks). Sections of 200 µm were cut from embedded blocks of midshaft tibial samples in a sheep osteotomy model with an external fixator. Regions of interest on the medial side of the proximal fragment were chosen to be the periosteal callus, middle callus, intercortical callus, and cortex. Mean thickness (T parameter), degree of alignment (ρ parameter), and predominant orientation (ψ parameter) of mineral particles were deduced from resulting sSAXS patterns with a spatial resolution of 200 µm. 2D maps of T and ρ overlapping with ESEM images revealed that the callus formation occurred in two waves of bone formation, whereby a highly disordered mineralized tissue was deposited first, followed by a bony tissue with more lamellar appearance in the ESEM and where the mineral particles were more aligned, as revealed by sSAXS. As a consequence, degree of alignment and mineral particle size within the callus increased with healing time, whereas at any given moment there were structural gradients, for example, from periosteal toward the middle callus.