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much better in synchronising porcine oocyte maturation if judged by the effects on cumulus expansion after long-time culture, the effectiveness of synchronisation and the percentage of MII oocytes obtained at 44 h. Treatment with cAMP also produced a more homogenous (less aged) population of oocytes as compared to unsynchronised controls (Figure 3.4). In conclusion, a reliable porcine IVM system was developed. Also, a less time-consuming, environmentally favorable and economical protocol for oocyte nuclear maturation assessment was introduced. Finally, cAMP method is more effective in synchronising porcine oocyte maturation. This is the first detailed study reporting synchronisation of meiosis of porcine oocytes by cAMP. 70
CHAPTER 4 Parthenogenetic development of porcine oocytes synchronised by CHX or cAMP and maturation timing of cAMP treated oocytes for TI enucleation 4.1 INTRODUCTION The term parthenogenesis was first employed by Richard Owen referring to reproduction without the immediate influence of a male (Owen, R., 1849). Subsequently parthenogenesis was redefined as "the production of an embryo, with or without eventual development into an adult, from a female gamete in the absence of any contribution from a male gamete" (Kaufman, 1979). Oocytes can be parthenogenetically activated physically and chemically. There are a lot of factors influencing the results of parthenogenetic activation, including oocyte age, activation media, duration and strength of electric stimulation and post-treatments (cytochalasin B or D, CHX and 6-DMAP, etc. ). Oocytes activated by different methods may not have equal competence for development. In addition, various results have been evaluated to determine the success of parthenogenetic activation such as the frequency of pronucleus formation, cleavage or blastocyst and the total cell number at blastocyst stage. Porcine parthenotes have been obtained using a range of activation stimuli including ethanol (Didion et al., 1990), electroporation (Hagen et al., 1991), calcium ionophores (A23187; Funahashi et al., 1994), electroporation plus CHX (Nussbaum and Prather, 1995), ethanol plus CHX (Petr et al., 1996), staurosporine (Wang et al., 1997b), thimerosal (Machäty et al., 1997), electroporation plus butyrolactone I (Dinnyes et al., 71
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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
Page 35 and 36: A Nuclear NEBD PCC reformation DNA
Page 37 and 38: 1.2.3.2 MPF, MAPK, NEBD and PCC in
Page 39 and 40: Little is known about the mechanism
Page 41 and 42: development of porcine embryos prod
Page 43 and 44: short-wavelength, UV excitable fluo
Page 45 and 46: The procedure is to expel the PBI a
Page 47 and 48: The majority of successful porcine
Page 49 and 50: Until 1997, the established dogma i
Page 51 and 52: deacetylases (HDACs). Trichostatin
Page 53 and 54: Figure 1.5 presents the general exp
Page 55 and 56: CHAPTER 2 General materials and met
Page 57 and 58: 2.3 Oocyte staining and assessment
Page 59 and 60: holding pipette enucleation pipette
Page 61 and 62: ! 00-0 !".. ww "'') ^ýýe ;ý ý
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: oocyte (Chen et al., 1990). In the
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 and 112: Figure 5.2 MPF and MAPK activities
Page 113 and 114: A 4500000 N 4000000 3500000 Z 30000
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
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Christmann, L., Jung, T. and Moor,
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and human ovarian oocytes. Nature,
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Funahashi, H., Cantley, T. C. and D
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Hartwell, L. H. (1973) Three additi
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Jelinkovä, L., Kubelka, M., Motlik
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Kubelka, M., Anger, M., Pavlok, A.,
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Le Bourhis, D., Beaujean, N., Ruffi
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Liu, L., Oldenbourg, R., Trimarchi,
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Minshull, J., Blow, J. J. and Hunt,
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Oback, B. and Wells, D. N. Cloning
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A. E., Garst, A. S., Moore, M., Dem
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Rossomando, A. J., Payne, D. M., We
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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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00 Cl) h 'Cb a) Ü^W wv F NC a b d
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In In ý. ö O V %) ö ö N _ ö O
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aý yy U a 2 w I-N O N 'T N 1-1 NO
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. 10 CA aý v 2 4 1-1 1-1 o Np cc o
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