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1
Rodríguez-Farré, Eduardo, Marcel Roberfroid, and Giovanni N. Fracchia. "Research and Development of In Vitro Pharmacotoxicology: A European Perspective." Alternatives to Laboratory Animals 21, no. 2 (April 1993): 285–93. http://dx.doi.org/10.1177/026119299302100224.
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The experts taking part in the Workshop were: E. Rodríguez-Farré ( Coordinator); G.N. Fracchia, (Secretary); M. Adolphe, École des Hautes Études, Paris, France); P.H. Bach (University of East London, UK); M. Baeder (Hoechst Ltd, Hattersheira, Germany); R. Bass (BGA, Berlin, Germany); H.G. Baumgarten (Frei Universität, Berlin, Germany); H. Bazin (DGXII, CEC, Brussels, Belgium); P. Bentley (Ciba-Geigy, Basle, Switzerland); A. Boobis (Royal Postgraduate Medical School, London, UK); J. Castell (Hospital La Fé, Valencia, Spain); J.P. Contzen (DGXII, CEC, Brussels, Belgium); A. Cordier (Sandoz Pharma Ltd, Basle, Switzerland); J. Diezi (Université de Lausanne, Switzerland); L. Dubertret (INSERM U-312, Creteil, France); P.M. Fasella (DGXII, CEC, Brussels, Belgium); J.H. Fentem (FRAME, Nottingham, UK); A. Guillouzo (INSERM U-49, Rennes, France); I. Kimber (Zeneca, Macclesfield, UK); T. Krieg (Universität zu Koln, Germany); A. Mantovani (Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy); K. Miller (BIBRA, Carshalton, UK); J.P. Morin (INSERM U-295, Rouen, France); D. Paul (Fraunhofer Institut für Toxikologie und Aerosolforschung, Hannover, Germany); P.W.J. Peters (Riijkinstituut voor Volksgezondheid en Milieuhygiene, Bilthoven, The Netherlands); J. Picard (Faculté des Sciences, Louvain la Neuve, Belgium); D. Poggiolini (Ministry of Health, Rome, Italy); C.M. Regan (University College, Dublin, Ireland); C.A. Reinhardt (SIAT, Zurich, Switzerland); B. Robaire (McGill University, Montreal, Canada); M. Roberfroid (Université Catholique de Louvain, Brussels, Belgium); V. Rogiers (Vrije Universiteit Brussels, Belgium); J. Rueff (Istituto de Higiene e Medicina Tropical, Lisbon, Portugal); H. Spielmann (ZEBET, Berlin, Germany); H. Stolte (Medizinische Hochschule, Hannover, Germany); J. van Noordwijk (European Pharmacopeia Commission, Bosch en Duin, The Netherlands); E. Walum (University of Stockholm, Sweden); D.C. Williams (Trinity College, Dublin, Ireland); and M. Yaniv (Institut Pasteur, Paris, France), and their contributions are gratefully acknowledged.
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Peters, A. R., L. A. Dwyer, A. Dawson, P. A. Canham, and J. D. Mackinnon. "Effects of Buserelin on day of service or 12 days post service on farrowing rate and litter size to first service in outdoor sows and gilts." Proceedings of the British Society of Animal Science 1999 (1999): 176. http://dx.doi.org/10.1017/s1752756200003318.
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The problem of seasonal infertility in pigs has been recognised for many years. The infertility complex can may be manifested by increased returns to service, prolonged weaning to oestrus intervals and decreased litter size. The purpose of this trial was to evaluate the effects of Buserelin treatment on fertility in sows and gilts during the seasonally infertile period.A total of 1231 mixed parity sows and gilts from five outdoor herds in East Anglia were randomly assigned to one of three treatment groups. Any sows not presented for service at first post weaning oestrus were excluded. All sows and gilts judged to be in adequate health and condition to be kept in a commercial breeding herd were included. Group C sows and gilts were given no treatment. Group R1 sows and gilts were injected i.m. with 8μg Buserelin (2.0ml Receptal; Hoechst Roussel Vet UK Ltd) on the day of service.
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McMillen, S. R., S. M. Rhind, and A. S. McNeilly. "Effect of body condition on ovarian sensitivity to exogenous FSH in ewes in which endogenous gonadotrophin secretion is suppressed." Proceedings of the British Society of Animal Production (1972) 1991 (March 1991): 172. http://dx.doi.org/10.1017/s0308229600021218.
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The results of previous experiments indicate that the effect of body condition (BC) on ovulation rate is mediated through changes in the number of large, potentially ovulatory, ovarian follicles. However, these differences in follicle populations were not associated with consistent differences in gonadotrophin profiles. It was therefore postulated that effects of level of BC on follicle development and ovulation rate are due to differential ovarian sensitivity of FSH.Two groups of 20 Scottish Blackface ewes were fed differentially so that they achieved mean (±s.e.m.) body condition scores of 1.78±0.017 (Low; L) and 2.94±0.031 (High; H) by 2 weeks before sample collection. They were then fed to maintain these body conditions throughout the remainder of the experiment. Four weeks before study, Alzet minipumps (Charles Rivers UK Ltd.) containing GnRH agonist (buserilin; Hoechst AG; Frankfurt, F.R.G.) were inserted, subcutaneously, in all ewes. This treatment partially suppresses FSH secretion and totally inhibits LH pulses so that growth of ovarian follicles >2.5 mm in diameter is inhibited.
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Silva, Danielle Fabiola Pereira, Dalmo Lopes Siqueira, Rosana Gonçalves Pires Matias, Sílvia Paula Oliveira, Leila Cristina Rosa de Lins, and Luiz Carlos Chamhum Salomão. "Desempenho de filmes comestíveis em comparação ao filme de policloreto de vinila na qualidade pós-colheita de mexericas 'Poncã'." Ciência Rural 42, no. 10 (August 28, 2012): 1770–73. http://dx.doi.org/10.1590/s0103-84782012005000076.
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O objetivo do trabalho foi avaliar o efeito de diferentes concentrações de fécula de mandioca na vida útil pós-colheita de frutos 'Mexerica Poncã' durante o armazenamento em temperatura ambiente em substituição ao filme de policloreto de vinila (PVC). Os frutos foram imersos em suspensões aquosas de fécula de mandioca a 0; 1; 2 e 3% (m:v) acrescidas de 0,5mL L-1 de óleo mineral Assist (Bayer Cropscience, da empresa Hoechst Schering AgrEvo UK Ltd. - Inglaterra) ou recobertos com PVC de 14µm de espessura, e armazenados a temperatura ambiente. As amostragens foram realizadas no tempo zero (início do experimento) e a cada dois dias, por oito dias. Foram avaliadas a perda de massa fresca, rendimento de suco, sólidos solúveis (SS), acidez titulável (AT), relação SS/AT e teor de ácido ascórbico. A perda de massa fresca aumentou durante o armazenamento, sendo mais acentuada na dose de 2%. A redução no teor de ácido ascórbico foi maior nos frutos recobertos com filme de PVC. A concentração de fécula de mandioca a 1% foi a que proporcionou melhores resultados quanto à manutenção da qualidade físico-química de frutos de 'Mexerica Poncã' durante oito dias de armazenamento.
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Kirby, P. S., N. A. Watson, D. G. Rennie, and T. O. Jones. "Timing of fish meal supplementation for finishing beef cattle offered grass silage." Proceedings of the British Society of Animal Production (1972) 1986 (March 1986): 113. http://dx.doi.org/10.1017/s0308229600016196.
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Results from three previous experiments with finishing cattle on an 18-month beef system indicate that the major increases in daily live-weight gain (DLG) occur within the first 50 to 60 days of supplementation with fish meal. Hence, it may be possible to remove fish meal from the diet after the initial two months of the finishing winter without any subsequent effect on animal performance.For the last nine weeks at grass the experimental cattle were given 1.4-kg/head/day dried sugar beet pulp nuts. On housing this allowance was increased to 3.0 kg and the 48 British Friesian steers were offered grass silage ad libitum (round bale silage for one week and precision-chopped clamp silage thereafter). The 3.0-kg dried sugar beet pulp was given for five days and after a 10-day changeover period the nuts were replaced by 15-kg potatoes. Cattle were offered the basal diet of precision-chopped silage and potatoes for 12 days before starting the experiment.Steers were implanted with 300-mg trenbolone acetate (Finaplix, Hoechst UK Ltd, Milton Keynes) and 36-mg zeranol (Ralgro, Crown Chemical Company Ltd, Lamberhurst) 20 days before randomisation.
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Norton, Lord Kings. "Extract from A Wrack Behind: Myself, when old." Aeronautical Journal 103, no. 1022 (April 1999): 221. http://dx.doi.org/10.1017/s0001924000096573.
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I am not quite sure when I became old, but I think I must date it in my seventies when I retired from a variety of activities and settled into a more sedentary style of life. I retired from Dowty Rotol and Bergers (and so Hoechst UK) in 1975, from the Royal Institution in 1976, from Ricardo’s and British Printing Corporation in 1977 and sold Withers Estates, very profitably for the Withers family, in 1981. I was left with the chairmanships of two tiny companies, Land-speed and Cotswold Research. While Landspeed has not served its purpose, Cotswold Research has its place in history. Both companies are concerned with the inventions of Professor Eric Laithwaite, who demonstrated in model and prototype form tracked hovercraft powered by the linear motor. Landspeed failed to develop this, through lack of funds, to a commercially viable transport system, though the Japanese appear to have done so. Cotswold Research, however, made a success of an adaptation of the linear motors to the separation of aluminium from scrap material and for their work, which ended in profitable industrial apparatus and a big saving in manpower, Mr A.P. Bird and I received the Prince of Wales Award in 1986.
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Drew, S. B., and A. R. Peters. "The effect of treatment with a gonadotrophin releasing hormone on the fertility of dairy cows." Proceedings of the British Society of Animal Production (1972) 1991 (March 1991): 171. http://dx.doi.org/10.1017/s0308229600021206.
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Work in New Zealand has suggested that treatment of dairy cows with buserelin an analogue of GnRH between days 11 and 13 after insemination can increase pregnancy rate by reducing embryo loss (MacMillan, Taufa and Day, 1986). This effect is probably mediated via a luteoprotective mechanism (MacMillan, Thatcher & Drost 1989; Thatcher, MacMillan, Hansen & Drost, 1989). The present study was carried out to attempt to confirm this effect of buserelin under UK conditions.A total of 660 Holstein/Friesian cows on 10 commercial dairy farms were used. The cows were paired according to date, of previous calving and allocated at random to control or treated group on the day of service. Only cows in the parity range 1-5 with no obvious reproductive disorders were used. Bulls and inseminators were used on the same number of cows in each group. Milk samples were taken on the day of service for progesterone assay. Cows not in oestrus were removed from the study. Cows in treated groups were given an intramuscular injection of 10 ug buserelin [2.5 ml Receptal, Hoechst] 12 days after first insemination. Cows not observed to return to oestrus before Day 24 were re-sampled for progesterone assay. Pregnancy was confirned by palpation or ultrasonic scanning at six weeks.
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Eshel, Alex, Najim A. Abdulwahid, N. Adjeidu Armar, Judith M. Adams, and Howard S. Jacobs. "Pulsatile luteinizing hormone–releasing hormone therapy in women with polycystic ovary syndrome**Supported in part by grant from Birthright to Dr. N. A. Armar and by Hoechst UK." Fertility and Sterility 49, no. 6 (June 1988): 956–60. http://dx.doi.org/10.1016/s0015-0282(16)59943-9.
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Romano, Vito, Mohit Parekh, Alessandro Ruzza, Colin E. Willoughby, Stefano Ferrari, Diego Ponzin, Stephen B. Kaye, and Hannah J. Levis. "Comparison of preservation and transportation protocols for preloaded Descemet membrane endothelial keratoplasty." British Journal of Ophthalmology 102, no. 4 (November 13, 2017): 549–55. http://dx.doi.org/10.1136/bjophthalmol-2017-310906.
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Background/aimsDescemet membrane endothelial keratoplasty (DMEK) preparation is technically demanding and is a limiting factor for uptake of this kind of surgery. Supply methods that simplify the procedure for surgeons are key to increasing uptake. This study compares two different shipping protocols for DMEK.MethodsAn 8.5 mm DMEK graft was punched, marked and loaded for transportation in two different conditions: (A) endothelium trifolded inwards in organ culture conditions (n=7) and (B) endothelium rolled outwards in hypothermic conditions (n=7). Tissues were shipped from Italy to the UK, then analysed for orientation, endothelial cell density, denuded areas, cell mortality, triple viability staining (Hoechst/ethidium homodimer/calcein AM (HEC)), immunolocalisation of ZO-1 and Na/K-ATPase proteins, visualisation of actin filaments using phalloidin and histological analysis using H&E on paraffin-embedded sections.ResultsAll tissues clearly showed the mark used for graft orientation. After shipping in condition A, there was an increase in cell mortality of 8.1% and in denuded areas of 22.4%, whereas for condition B there was an increase in cell mortality of 14.2% and in denuded areas of 34.3% after shipping. HEC staining revealed areas of viable cells and apoptotic cells, with large denuded areas found in the periphery for condition B and within folds for condition A.ConclusionsPrestripped preloaded DMEK grafts retained sufficient viable cells for transplantation, with condition A (endothelium-in) offering the advantage of greater flexibility of use due to a longer shelf-life. HEC analysis provides further detailed information as to the status of DMEK grafts and should be used in future similar studies.
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Bentick, Bernard, Robert W. Shaw, Claire A. Iffland, Graham Burford, and Albert Bernard. "A randomized comparative study of purified follicle stimulating hormone and human menopausal gonadotropin after pituitary desensitization with Buserelin**Buserelin, Hoechst UK Ltd., Pharmaceutical Division, Hounslow, Middlesex, United Kingdom. for superovulation and in vitro fertilization." Fertility and Sterility 50, no. 1 (July 1988): 79–84. http://dx.doi.org/10.1016/s0015-0282(16)60012-2.
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Hall, V., D. Compton, P. Stojkovic, M. Nesbitt, M. Herbert, A. Murdoch, and M. Stojkovic. "36 DEVELOPMENTAL COMPETENCE OF HUMAN AGED OOCYTES AS HOST CELLS FOR NUCLEAR TRANSFER." Reproduction, Fertility and Development 18, no. 2 (2006): 126. http://dx.doi.org/10.1071/rdv18n2ab36.
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The use of aged metaphase II oocytes (cultured in vitro for more than 14 h) for somatic cell nuclear transfer (SCNT) in varying species has resulted in lower developmental outcomes compared with non-aged in vitro- or in vivo-matured oocytes. However, due to limited resources of fresh oocytes for the derivation of nuclear transfer stem cell lines, further investigation in using spare oocytes is required. Aged spare oocytes (48 h post oocyte retrieval) were consigned for research (under HFEA and local ethics approval) by couples undergoing either in vitro fertilization (failed IVF oocytes, f-IVF) or intracytoplasmic sperm injection (failed-ICSI oocytes, f-ICSI) treatments. Aged oocytes were randomly assigned for double-labeling immunocytochemical analysis (f-IVF, n = 10; f-ICSI, n = 7) for the microtubule markers, NuMA and �-tubulin, or parthenogenetic activation. Immunocytochemical analysis was performed as previously described (Chatzimeletiou et al. 2005 Hum. Reprod. 20, 672-682) using primary anti-rabbit NuMA (gift from D. Compton, Dartmouth Medical School, Hanover, NH, USA) and anti-mouse DM1-�. Secondary antibodies were donkey anti-rabbit and anti-mouse immunoglobulins. Oocytes were counterstained with Hoechst 33342. Negative controls were performed as above with blocking solution substituting for primary antibodies. Parthenogenetic activation was performed for 4 h using 10 �M calcium ionophore (5 min) and 2 mM 6-dimethylaminopurine (Ca-I/DMAP) for f-IVF (n = 10) and f-ICSI oocytes (n = 11) or 10 �g/mL puromycin (Ca-I/Pur) for f-IVF (n = 12) and f-ICSI oocytes (n = 10) (4 h). Activated oocytes were cultured in a biphasic system, G1.3" and G2.3" (Vitrolife UK, Ltd., Ediburgh, Lothian, UK) for 5 days at 37 �C in 5% CO2 in humidified air. NuMA was localized to the metaphase spindle in 6/10 (60%) and 7/7 (100%) oocytes for f-IVF and f-ICSI, respectively, and/or in cytoplasmic cytasters. One f-IVF oocyte and four f-ICSI oocytes had visible tetrapolar spindles. Unusual patterns of diffuse NuMA staining containing dense foci within these regions, but not associated with the cytasters or metaphase spindle, were also observed in two f-IVF oocytes. The majority of oocytes displayed ring-like staining of DM1-�, which was aberrant in two f-ICSI oocytes. Parthenogenetic development was poor for both treatments. Cleavage rates were 17% and 20% for f-IVF using Ca-I/PUR and Ca-I/DMAP, respectively, and 40% and 45% for f-ICSI using Ca-I/PUR and Ca-I/DMAP, respectively. Fragmentation rates were high across all treatments. No parthenogenetic embryos developed beyond the 6-cell stage. Thus, the use of aged human oocytes for SCNT may be difficult due to their incapacity to artificially activate using current activation protocols and, in addition, due to the microtubule abnormalities observed in many of these aged oocytes.
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Fouladi-Nashta, A. A., R. Alberio, B. Nicholas, K. H. S. Campbell, and R. Webb. "151A SIMPLE AND FAST METHOD FOR CONCURRENT DIFFERENTIAL STAINING AND TUNEL LABELLING OF BOVINE BLASTOCYSTS." Reproduction, Fertility and Development 16, no. 2 (2004): 197. http://dx.doi.org/10.1071/rdv16n1ab151.
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Currently techniques of TUNEL labelling for detection of apoptosis and differential staining for counting the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells are used separately for assessment of embryo quality in different species. The majority of these techniques are antibody-based, and time-consuming, frequently giving inconsistent results. Here we report on the development of a simple and fast method for simultaneous differential staining and TUNEL labelling of bovine embryos. Cleaved embryos produced from in vitro-matured and fertilized oocytes were cultured to the blastocyst stage in synthetic oviductal fluid culture medium (SOF) supplemented with 4mgmL−1 BSA and 5% FCS. Embryos were partially permeabilized in 0.5% Triton X-100 solution containing 2μMmL−1 TOTO-3 dye (Molecular Probes, Eugene, OR, USA) for 30s. TOTO-3 is a cell-impermeant nucleic acid dye; thus only permeabilized cells are stained red. The embryos were then quickly washed in PBS containing 3mgmL−1 PVA, fixed for 15min at RT in 4% paraformaldehyde containing 10μgmL−1 Hoescht, and TUNEL-labelled using a Cell Death Kit (Roche Applied Science, East Sussex, UI) for 30min at 37°C in a humid chamber. The embryos were then treated with RNase A (50UmL−1) for 30min at 37°C, washed and mounted in a small drop of glycerol on a glass slide. RQ1-DNase (3UmL−1)-treated embryos were used as a positive control. After three-dimensional reconstruction using a Leica TCS SP2 confocal microscope, we determined the number of ICM (blue), TE (red) and apoptotic nuclei (green). Only peripheral cells of the blastocysts were labelled red, indicating that TE cells were permeabilized by the short exposure to the detergent Triton. ICM cells were consistently stained blue by the cell permeant dye Hoechst. Apoptotic nuclei were found in both types of cells. More consistent differential staining was observed in hatched blastocysts (n=30) than in zona-enclosed blastocysts (n=35); also, more apoptotic nuclei were observed. No differences were found in the consistency of the technique for embryos grown with or without FCS. When compared to dual staining without Tunel, no differences in cell number (74±22) , and ICM/TE ratio (0.28±0.06) were detected, indicating that the Tunel procedure does not affect the labeling of the DNA. Preliminary observations also indicate that this method can be successfully applied to porcine and ovine embryos. This technique has the advantage of being fast and can be applied for assessment of embryo quality. It can also be used to determine the time and origin of ICM and TE differentiation while monitoring the degree of apoptosis in different culture systems and in different species. This work was in part supported by Department of Environment Food and Rural Affairs (Defra) UK.
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Schauser, K., T. S. Sundkvist, M. Vejlsted, and P. Maddox-Hyttel. "153 IMMUNOCYTOCHEMICAL STUDIES OF OCT-4, SOX-2, AND β-TUBULIN III EXPRESSION IN EARLY PORCINE EMBRYOS." Reproduction, Fertility and Development 19, no. 1 (2007): 194. http://dx.doi.org/10.1071/rdv19n1ab153.
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In the mouse, the transcription factor SOX-2 is known to have at least 2 roles: (1) it acts as a co-factor of the transcription factor OCT-4, the key regulator of pluripotency essential for the development of the inner cell mass/epiblast; and (2) it is involved in the direction of neural development. In this study, we elucidate the localization of SOX-2 in early porcine embryos in relation to that of OCT-4 and the early neuronal marker β-tubulin III. Embryos were flushed from uteri, fixed in 4% paraformaldehyde, and processed for paraffin sectioning. Sections (5 �m) were stained with anti-OCT-3/4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using the ABC-AEC-method and counterstained with hematoxylin, or processed for double immunofluorescent staining using antibodies against SOX-2 (R&D Systems Europa, Ltd., Abington, Oxon, UK) and β-tubulin III (Sigma-Aldrich Denmark A/S, Copenhagen, Denmark) and counterstaining with Hoechst. The embryos were classified as pre-streak (n = 8), primitive streak (n = 4), neural groove (n = 5), and somite (n = 4) stage (Vejlsted et al. 2006 Mol. Reprod. Dev. 73, 709–718). At the early pre-streak stage, SOX-2 and OCT-4 staining was found in the nuclei and a weak β-tubulin III staining in the cytoplasm of all epiblast cells. At the late pre-streak and the primitive streak stage, SOX-2 staining became polarized to the nuclei in the anterior epiblast region, whereas OCT-4 staining was found in all nuclei of the epiblast and of the forming meso- and endoderm. The β-tubulin III staining was restricted to the epiblast and showed no anterior-posterior polarization. At the primitive streak, when cells were involuting to form the meso- and endoderm, SOX-2 staining of nuclei was absent. At the neural groove stage, the SOX-2 and β-tubulin III staining was localized to nuclei and cytoplasm, respectively, of the same cells and observed in the neural plate and groove. A polarization in SOX-2 staining was observed in an anterior-posterior direction. At the somite stage, the SOX-2 and β-tubulin III staining was again localized to the same cells and observed in the neuropores and neural tube. The SOX-2 staining of the neural tube was polarized in a dorso-ventral direction. At the neural groove and somite stage, the OCT-4 staining gradually disappeared from the epiblast, mesoderm, and endoderm except from scattered cells, presumably primordial germ cells, localized in the endoderm. Our results suggest that also in the porcine embryo SOX-2 plays a dual role, being involved in regulation of both pluripotency and neural development.
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Abazari-kia, A. H., A. Mohammadi-Sangcheshmeh, M. Salehi, and M. Zhandi. "309 ADDITION OF BRAIN-DERIVED NEUROTROPHIC FACTOR IN MATURATION MEDIUM IMPROVED IN VITRO MEIOTIC COMPETENCE OF OVINE OOCYTES AND SUBSEQUENT PARTHENOGENETIC DEVELOPMENT." Reproduction, Fertility and Development 27, no. 1 (2015): 243. http://dx.doi.org/10.1071/rdv27n1ab309.
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Overall efficiency of in vitro embryo production has remained low despite extensive effort to understand the effects of culture conditions, media composition, and supplementation. Brain-derived neurotrophic factor (BDNF), which is a physiologically important neurotrophin, has been used to enhance oocyte maturation in some previous studies (Lee et al. 2007; Zhang et al. 2010). However, the efficacy of BDNF to improve oocyte competence has not been fully established especially in ovine. Therefore, the present study aimed to evaluate the effect of BDNF during in vitro maturation (IVM) on maturation rate, intracellular glutathione (GSH) content, and embryonic development in sheep oocytes. Cumulus-oocyte complexes (COC) were obtained from ovaries of ewes. The COC were placed in maturation medium supplemented with either 10 (IVM-B10) or 100 (IVM-B100) ng mL–1 of BDNF (PeproTech, London, UK). Oocytes in control group were incubated in the same maturation medium without BDNF. The IVM was performed in a humidified atmosphere containing 5% CO2, 5% O2, and 90% N2 at 38.5°C for 24 h. After IVM, several oocytes from the IVM-B10 (n = 110), IVM-B100 (n = 124), and control (n = 110) groups were stained with Hoechst and were evaluated in relation to their metaphase-II rate. To measure GSH content, several oocytes from the IVM-B10 (n = 28), IVM-B100 (n = 33), and control (n = 37) groups were incubated in tyrodes medium containing 10 µM Cell Tracker blue for 30 min and transferred under fluorescence microscope, with digital images analysed by image J software. To evaluate the embryonic development, several oocytes from IVM-B10 (n = 145), IVM-B100 (n = 137), and control (n = 143) groups were subjected to parthenogenetic activation by applying 1 min of exposure to 2.5 µM ionomycin followed by 2 mM 6-DMAP treatment for 3 h. After stimulation, oocytes were cultured in CR1aa medium for 7 days under the conditions stated previously. Four replications were performed. The metaphase-II rate, cleavage, and blastocyst rates were compared by x2 analysis. The GSH content was analysed by one-way ANOVA. A P-value of less than 0.05 was considered significant. The results showed that metaphase-II rate was higher in the IVM-B100 group (88.7%), as compared with the control group (77.3%), but not significant as compared with that in the IVM-B10 group (84.5%). No difference was also found between the IVM-B10 group and control group in terms of the metaphase-II rate. Oocytes in the IVM-B10 group revealed a higher (96.8%) GSH content than both of the IVM-B100 (86.9%) and control (86.3%) groups. There was, however, no difference in the GSH content between the IVM-B100 group and control group. The proportion of cleaved embryos was not different between the groups; however, the blastocyst rate was higher in both the IVM-B10 (37.9%) and IVM-B100 (39.3%) groups compared with the control group (22.4%). Collectively, the results of this study showed that supplementation of IVM media with BDNF promoted nuclear maturation, increased GSH content, and stimulated in vitro embryonic development in ovine.
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Pryor, J. H., J. F. Hasler, L. Strøbech, B. Avery, N. Hashem, S. Menges, C. R. Long, G. Shewfelt, and C. R. Looney. "86 IMPROVED BOVINE EMBRYO PRODUCTION USING NOVEL IN VITRO CULTURE SYSTEMS." Reproduction, Fertility and Development 28, no. 2 (2016): 172. http://dx.doi.org/10.1071/rdv28n2ab86.
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Development and testing of new embryo production components is important to improve the outcome following in vitro production of bovine embryos. The objective of this study was to compare media used in two bovine embryo production systems (control and EmbryoTrans Biotech: ETB). In Exp. 1, abattoir-derived cumulus-oocyte complexes were randomly assigned and in vitro matured (IVM) in either control [Medium 199 with Earles salts (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 1% penicillin/streptomycin (Invitrogen), 0.2 mM sodium pyruvate, 2 mM L-glutamine (Sigma Chemical Co., St. Louis, MO, USA), and 5.0 µg mL–1 of Folltropin®-V (Vetoquinol, Pullman, WA, USA)] or ETB BO-IVM medium for 21 to 24 h. IVF was conducted in 500 µL of pre-equilibrated modified Tyrode-lactate medium for control (Pryor et al. 2011 Theriogenology 75, 24–33) or ETB BO-IVF in Nunclon® 4-well multi-dishes (VWR Scientific, Pittsburgh, PA, USA). Seventeen hours post-insemination, presumptive zygotes were cleaned of cumulus cells and cultured in either Bovine Evolve (Zenith Biotech, Guilford, CT, USA) supplemented with 4 mg mL–1 of Probumin BSA (EMD Millipore, Norcross, GA, USA), under oil (Irvine Scientific, Santa Ana, CA, USA) or ETB BO-IVC medium under BO-oil for 7 days (8 days post-IVF). All cultures were performed at 38.5°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 using BT37 incubators (Planer Plc, Sunbury, UK). For Exp. 2, all conditions were maintained except a modified ETB BO-IVCA medium was used. On Day 8 of IVC, grade 1 and 2 blastocysts (BL) through hatching blastocysts (HBL) were counted and used to calculate total viable rates. In Exp. 2, these embryos were fixed in cold methanol, washed in PBS/0.1% Tween 20, mounted in 10 μg mL–1 Hoechst 33342/glycerol, and viewed under UV light to count cells (n = 49 and 107 for control and ETB, respectively). Each experiment was replicated 3 times with a total of 425 oocytes in Exp. 1 and 430 in Exp. 2, divided equally between treatments. Percentage data were transformed using arcsine square root function before analysis and means compared using a paired Student’s t-test. For Exp. 1, there were no differences in rates of cleavage or viable embryos between control and ETB systems (81.3% and 42.9% v. 80.5% and 48.4%, respectively). In Exp. 2, ETB was superior to control for percent viable, HBL, and combined HBL/expanded BL (51.9, 23.9, 45.8% v. 29.2, 5.8, 20.5, respectively; P < 0.05). Differences between mean cell counts for viable embryos were significant (control = 127.0 ± 6.7 s.e.m. and ETB = 162.7 ± 5.7; P < 0.0001). Embryo viability decreased in control media between Exp. 1 and 2 (42.9 v. 29.2%; P < 0.05). Seasonal differences may have contributed via heat stress with temperatures ranging from 23.8°C for Exp. 1 to 33.8°C for Exp. 2. Interestingly, embryo development in the ETB media did not decrease under the same conditions. In conclusion, ETB media produced more high-quality embryos than control under varying conditions experienced by commercial IVF companies.
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Pérez, Cristina, Nuria Garcia, David San Segundo, Marcos Lopez Hoyos, Margarita Sanchez Beato, Nerea Martinez, Jose Pedro Vaqué, and Miguel Angel A. Piris. "Novel Approaches For Targeted Therapy In Cutaneous T-Cell Lymphomas." Blood 122, no. 21 (November 15, 2013): 3834. http://dx.doi.org/10.1182/blood.v122.21.3834.3834.
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Abstract Introduction Development of targeted therapy for Cutaneous T-Cell Lymphoma (CTCL) patients still requires actionable mutated genes and deregulated pathways to be identified and explored. Although it has been proposed that neoplastic CTCL cells feature increased TCR downstream signaling and also a degree of phenotypic plasticity, the mechanistic nature of the pathogenesis of this disease remains essentially unveiled. Our laboratory has recently studied the mutational status of a number of human CTCL lesions, and detected TCR/PLCG1 and JAK/STAT signaling pathways frequently mutated. Taking advantage of these findings, we have explored the biological effects that targeted inhibition, using specific inhibitors of the two afore mentioned signaling pathways, exerts in proliferation, survival and phenotype in a panel of human CTCL cell lines. Our results suggest that TCR/PLCG1 and JAK/STAT pathways can control proliferation, survival and phenotype of CTCL cells and hence can serve as potential targets for mono or combinatorial therapy in CTCL patients. Material and Methods Cutaneous T-cell lymphoma cell lines used were HH (MF) and MJ (MF) obtained from ATCC (Rockville, MD, US ) and My-La (MF) and HUT-78 (SS) obtained from ECACC (Salisbury, UK).Cell proliferation analyses were performed using CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA) with the appropriate amount of inhibitor. Apoptosis were evaluated by flow cytometry using FlowCellect Annexin Red Kit (EMD Millipore Corporation, Billerica, USA). All data were detected on a FACS Calibur flow cytometer (BD) and analyzed using CellQuest Pro software (BD). Treg/Th17 phenotype was determined by flow cytometry. Cells were stained with monoclonal antibodies: anti-CD4-allophycocyanin (APC)-Cy7 (clone SK3, Becton Dickinson, San Diego, CA). Monoclonal antibodies to detect intracellular antigens were: anti-Foxp3-PE (clone PCH101, eBiosciences, San Diego, CA), anti-RORgt-APC (clone AFKJS-9, eBioscience, San Diego, CA), and IgG2a isotype controls conjugated with PE and APC. The cells were acquired by flow cytometer (FACS Canto-II, Becton Dickinson). Results To test both the effect that inhibition of PLCG1 downstream signaling and the effect that inhibition of JAK downstream signaling provokes in CTCL cell lines, they were incubated with increasing concentrations of specific CaN and JAK inhibitors like FK506 and INCB018424 respectively or DMSO (vehicle) for 0, 24 and 48 h. The results show that increasing concentrations of FK506 affected the proliferation and survival of CTCL cells in a concentration dependent manner. But in the case of INCB018424 inhibitor mainly affects CTCL cell proliferation versus survival. The combined use of FK506 and INCB018424 produced a significant greater inhibition of CTCL cell proliferation than each inhibitor alone except of HUT-78, where the combined treatment was not significant (Figure 1). Because of neoplastic T-cells can acquire Treg and/or Th17 phenotypes at different stages of the disease (Tiemessen, M.M. et al. 2006) and that there is a CTCL phenotypic plasticity(Abraham, Zhang et al. 2011) (Eisenstein and Williams 2009) (Hoechst, Gamrekelashvili et al. 2011), we have decided to study these phenotypes in the four CTCL cell lines. Strikingly we found that most cells in all CTCL cell lines were double positive for the expression of Treg and Th17 markers (Figure 2). The expression of the Treg and Th17 lineage markers (FoxP3 and RORγt respectively) after treatment with FK506 and INCB018424 were tested. FK506 provoked an unbalance in the expression of the Treg and Th17 markers in all CTCL cell lines. (Figure 3). Conclusions These results are the first evidences for the use of a combinatorial targeted therapy developed on the basis of a mutational analysis in CTCL, which in the future may serve as a lead to explore into detail about a potential utilization of this therapeutical approach in the clinic. Surprisingly, our results evidence a novel hybrid Treg/Th17 phenotype in our panel of CTCL cell lines, that we could alter by interfering specifically with TCR/PLCG1 and/or JAK/STAT downstream signaling using FK506 and/or INCB018424 respectively. Although these are still primary results, it will be important to explore how to take clinical advantage of these phenotypic characteristics in patients. Disclosures: No relevant conflicts of interest to declare.
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"Masterplast controls Hoechst masterbatch business in UK." Additives for Polymers 1996, no. 3 (March 1996): 8. http://dx.doi.org/10.1016/s0306-3747(96)90356-5.
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