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Hatfield, United Kingdom

Chang S.-P.,Genes and Development Group | Chang S.-P.,Molecular Pathology Research and Development Laboratory | Mullins J.J.,University of Edinburgh | Morley S.D.,Queens Medical Research Institute | West J.D.,Genes and Development Group
Organogenesis | Year: 2011

Mice showing mosaic expression of an appropriate marker gene that is activated during development provide simple tools for investigating cell lineages. We used the mosaic β-galactosidase staining patterns in adrenal cortices of 21OH/LacZ transgenic mice to study both organogenesis and maintenance of the adult tissue. Randomly orientated mosaic patterns present in embryonic day 14.5 (E14.5) adrenals changed progressively during the perinatal period from discrete spots, via patches and radial arrays, to radial stripes, which first emerged between postnatal days 0 and 7 (P0 and P7). The mosaic radial stripe pattern was fully established by P21 and remained unchanged throughout the adult period (8-52 weeks). The mouse adrenal gland grew continuously between E14.5 and P21, including the period during which stripes emerge. Ki67-positive, proliferative cells in the adrenal cortex were mainly localized to the outer cell layers between E18.5 and P3. By P10, cell proliferation had increased, and the proliferative region had expanded but was still mainly confined to the outer cortex. Correlation of changes in mosaic patterns in 21OH/LacZ adrenal cortices with the locations of adrenocortical cell proliferation suggest that the radial stripes arise by edge-biased growth during the perinatal period, even if they are maintained by stem cells in adults. The stability of the adult stripe pattern suggests that stem cell function is unchanged between 8 and 52 weeks. © 2011 Landes Bioscience. Source

Salavati M.,University of Bedfordshire | Salavati M.,Genes and Development Group | Ghafari F.,Coventry University | Zhang T.,Bournemouth University | Fouladi-Nashta A.A.,Genes and Development Group
Theriogenology | Year: 2013

Phosphodiesterase (PDE) inhibitors have been utilized for invitro maturation (IVM) of oocytes to manipulate the meiotic resumption and progression. Premature chromatin condensation and DNA replication of the oocytes, immediately after the decrease in the cAMP level, are the difficulties in canine IVM. Caffeine, a nonselective competitive PDE inhibitor, due to its structural similarity to adenosine molecule maintains the cAMP level by occupying PDE enzymes such as PDE-3A inside the oocyte and PDE-4 and PDE-5 in the cumulus cells. In this study, the effects of 12-hour caffeine pretreatment in a biphasic IVM protocol were assessed on maturation rates of canine oocytes. Sixty hours of culture after a 12-hour of 10 mM caffeine pretreatment resulted in 16.9% ± 2.4 of the oocytes reaching metaphase II stage (MII) and 25.9% ± 5.2 degeneration rate compared with the control group with 2.2% ± 2.2 MII and 37.6% ± 4.3 degeneration rates (P < 0.05). Caffeine pretreatment induced higher mitogen-activated protein kinases (MAPK1 and MAPK3) phosphorylation and maturation-promoting factor activity at 12 hours and activated MAPK1 and maturation-promoting factor at 48 hours after culture in cumulus-oocyte complexes (COCs) compared with the control group (P < 0.05). Fresh canine COCs were also analyzed before IVM using brilliant cresyl blue (BCB) staining. Oocytes showed difference in meiotic resumption (MI-MII) (BCB+ = 16.11% ± 5.5, BCB- = 9.86% ± 5.0; P < 0.05) after 60 hours of culture following 12-hour caffeine pretreatment. The BCB+ canine oocytes had higher MII rate than the BCB- group under caffeine pretreatment (10.2% ± 2.9 vs. 1.1% ± 1.1, respectively; P < 0.05). Results indicated that 12-hour caffeine pretreatment of canine COCs improves the MII maturation rates at 72 hours and BCB+ oocytes have higher competency invitro for nuclear maturation. © 2013 Elsevier Inc. Source

Salavati M.,University of Bedfordshire | Salavati M.,Genes and Development Group | Ghafari F.,Coventry University | Zhang T.,Bournemouth University | Fouladi-Nashta A.A.,Genes and Development Group
Reproduction | Year: 2012

Canine oocytes require an extended period of culture (72 h) in vitro for nuclear maturation to the metaphase II stage, which also results in high degeneration. Canine cumulus oocyte complexes were isolated by slicing from ovaries collected after ovariohysterectomy and cultured in serum-free synthetic oviductal fluid incubated at low (5%) or high (20%) oxygen levels. Changes in oocyte nuclear maturation rates, H2O2 levels within the oocytes and mRNAs of reactive oxygen species inhibitory genes superoxide dismutase 1 and 2 (SOD1 and 2), glutathione reductase (GSR), glutathione peroxidase (GPX1), and catalase (CAT) were quantified. Higher meiotic resumption from germinal vesicle breakdown up to MII was observed in low O2 (41.8±13.1%) compared to high O2 (15.8±8.2%) (P=0.014) after 52 h of culture (n=112). Extension of the culture period up to 84 h at low O2 (n=457 oocytes) produced the highest meiotic resumption at 72 h (64.1±6.0%; P=0.008), compared with 52 h. Oocytes (n=110) cultured in high O2 contained higher levels of peroxidase measured using the 20, 70-dichlorodihydrofluorescein diacetate fluorescence assay after 72 h of culture compared with low O2 (P=0.004). High O2-cultured oocytes also showed higher amounts of SOD1, SOD2, GSR, GPX1, and CAT mRNA. Vitamin E in high oxygen level was able to decrease degeneration (P=0.008) but had no improving effect on percentage of oocytes in MII. These results for the first time showed that low oxygen gas composition improves nuclear maturation rates and alleviates the oxidative stress for canine oocytes during in vitro maturation.© 2012 Society for Reproduction and Fertility. Source

Marei W.F.A.,Genes and Development Group | Marei W.F.A.,Cairo University | Salavati M.,Genes and Development Group | Fouladi-Nashta A.A.,Genes and Development Group
Molecular Human Reproduction | Year: 2013

Biological functions of hyaluronan (HA) depend on its molecular size. Small size HAs are known to regulate cell proliferation; however, the expression of HA synthases (HASs) and hyaluronidase-2 (HYAL2) and their role during early embryo development have not been previously identified. In this paper, we have shown by immunostaining that HA is produced by bovine in vitro-produced embryos at all stages of early development to blastocyst. Addition of HA-synthesis inhibitor (4-methylumbelliferone; 4MU) to in vitro embryo culture inhibited blastocyst formation. HASs HAS2 and HAS3mRNAwere expressed at all stages of embryo development; however, relativemRNAexpression of HAS2 was significantly reduced as the embryos develop to the blastocyst stage. HAS1 was detected during 2- and 4-cell stages but was barely detectable in subsequent stages. HYAL2 mRNA expression was detected in oviducts at the early luteal phase but was only detected in the embryos at morula and blastocyst stages (Day 6 and 7 post-fertilization). Addition of HYAL2 to embryo culture media at Day 2 post-fertilization increased phosphorylated mitogen-activated protein kinases (MAPK1 and 3) in the embryos and improved development to the blastocyst stage and increased embryocell numbers. Addition of an anti-CD44 antibody or anMAPKinhibitor (U0126) abrogated the positive effects ofHYAL2on blastocyst rates. In conclusion, we demonstrate that the expression of different HAS genes and HYAL2 in bovine embryos varies according to the stage of development and that the supplementation ofHYAL2 in vitro mimics oviductal conditions and is shown to improve the blastocyst rate and embryo quality, an effect which requires CD44 activity and MAPK signalling. © The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. Source

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