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5.3.2 Effects of caffeine treatment on MPF and MAPK activities of TI and early MII oocytes At 38 hpm, denuded oocytes arrested at TI or early MII stage were cultured in modified NCSU-23 with different concentrations of caffeine (0,5,10 and 20 mM) for 6h and then MPF and MAPK activities at 44 hpm were assayed (Figure 5.3). No statistically differences in either kinase activities were observed between 0,5,10 and 20 mM caffeine treatments (T test, P>0.05). 96
A 4500000 N 4000000 3500000 Z 3000000 u 2500000 2000000 1500000 MPF MAPK 1000000 Y 500000 0 0 SmM 10 mm 20mM MII by normal IVM B 12345 Histone H1 32 kD Figure 5.3 Effects of different caffeine concentrations on MPF and MAPK activities in TI and early MII porcine oocytes A) Levels of MPF and MAPK activities. TI and early MII oocytes were cultured in different concentrations (0,5,10,20 mM) of caffeine for 6 h. B) A representative autoradiograph of histone H1 (MPF) and MBP (MAPK) assay. Lane 1: denuded intact MII oocytes without caffeine treatment at 44 hpm; Lane 2: 20 mM caffeine treatment; Lane 3: 10 mM caffeine treatment; Lane 4: 5 mM caffeine treatment; Lane 5: 0 mM caffeine treatment. Ten oocytes were analysed for each sample per gel and three replicates were performed. Bars represent mean ± SEM. 97
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The University of -210 Nottingham M
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treated oocytes was recorded at 38
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TABLE OF CONTENTS ABSTRACT ........
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2.5.1.2 Preparation of enucleation/
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CHAPTER 6 .........................
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LIST OF FIGURES Figure 1.1 Process
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LIST OF ABBREVIATIONS A23187 AC AI
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MgSO4.7H20 MAPK MAPKK MAPKKK MBP mg
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CHAPTER 1 LITERATURE REVIEW The aim
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The production of multiple offsprin
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cytoplasm which contained the nucle
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pluripotency, as demonstrated by th
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gene-targeting via somatic cell clo
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conventional breeding and genetic m
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Oocyte maturation is generally cont
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ooýcýýommý MPF Time Figure 1.3
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not obtain clones by SCNT using bra
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A Nuclear NEBD PCC reformation DNA
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1.2.3.2 MPF, MAPK, NEBD and PCC in
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Little is known about the mechanism
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development of porcine embryos prod
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short-wavelength, UV excitable fluo
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The procedure is to expel the PBI a
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The majority of successful porcine
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Until 1997, the established dogma i
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deacetylases (HDACs). Trichostatin
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Figure 1.5 presents the general exp
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CHAPTER 2 General materials and met
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2.3 Oocyte staining and assessment
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holding pipette enucleation pipette
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Page 63 and 64: ,rjý. 7!: rY Is All Pi D r. Figure
Page 65 and 66: were washed three times in embryo c
Page 67 and 68: Three replicates of batches of oocy
Page 69 and 70: fertilisation, the occurrence of po
Page 71 and 72: 3.2 MATERIALS AND METHODS 3.2.1 Exp
Page 73 and 74: I 57
Page 75 and 76: 3.3 RESULTS 3.3.1 Conventional matu
Page 77 and 78: ýIC M 00 N N N N 0 öa oÜd U 0 0
Page 79 and 80: Meiotic stage in CAMP treated oocyt
Page 81 and 82: 3.4 DISCUSSION The development of N
Page 83 and 84: 44 h. It was comparable to that (93
Page 85 and 86: oocyte (Chen et al., 1990). In the
Page 87 and 88: CHAPTER 4 Parthenogenetic developme
Page 89 and 90: mechanical and chemical procedures.
Page 91 and 92: COCs were collected and oocytes wer
Page 93 and 94: f. " ý1403: 0.71 14 - 'Ir I A Figu
Page 95 and 96: 100% 90% 80% 70% d 60% 50% 0 40% 30
Page 97 and 98: 4.4 DISCUSSION For successful porci
Page 99 and 100: In addition to the listed benefits
Page 101 and 102: CHAPTER 5 Effect of caffeine treatm
Page 103 and 104: in vitro substrates. In pigs, Naito
Page 105 and 106: 5.2 MATERIALS AND METHODS 5.2.1 Exp
Page 107 and 108: 5.2.2 In vitro maturation of porcin
Page 109 and 110: Blastocysts at day 7 were stained a
Page 111: Figure 5.2 MPF and MAPK activities
Page 115 and 116: e ýo; +ý \.. 4.0 ob "i "s NW Ir
Page 117 and 118: 5.3.4 Effects of caffeine treatment
Page 119 and 120: Figure 5.6 Effects of 5 mM caffeine
Page 121 and 122: 5.4 DISCUSSION In the first experim
Page 123 and 124: In the final experiment, developmen
Page 125 and 126: CHAPTER 6 General discussion 7.1 Ma
Page 127 and 128: cumulus cells could be observed spe
Page 129 and 130: parthenogenetic development excludi
Page 131 and 132: demonstrated treatment of the TI en
Page 133 and 134: REFERENCES Abeydeera, L. R., Wang,
Page 135 and 136: Bromhall, J. D. (1975) Nuclear trna
Page 137 and 138: Christmann, L., Jung, T. and Moor,
Page 139 and 140: and human ovarian oocytes. Nature,
Page 141 and 142: Funahashi, H., Cantley, T. C. and D
Page 143 and 144: Hartwell, L. H. (1973) Three additi
Page 145 and 146: Jelinkovä, L., Kubelka, M., Motlik
Page 147 and 148: Kubelka, M., Anger, M., Pavlok, A.,
Page 149 and 150: Le Bourhis, D., Beaujean, N., Ruffi
Page 151 and 152: Liu, L., Oldenbourg, R., Trimarchi,
Page 153 and 154: Minshull, J., Blow, J. J. and Hunt,
Page 155 and 156: Oback, B. and Wells, D. N. Cloning
Page 157 and 158: A. E., Garst, A. S., Moore, M., Dem
Page 159 and 160: Rossomando, A. J., Payne, D. M., We
Page 161 and 162: Spemann, H. Embryonic development a
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Development. 55(2), 121-127. Törne
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Production of the first cloned came
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oocytes. Reproduction, 125(5), 645-
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