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oocytes peaked at 38 hpm, so we decided to select and enucleate TI oocytes at 36 -38 hpm and then transferred these enucleated oocytes back into maturation medium for completion of cytoplasmic maturation and further study. In the third experiment, the efficiency of enucleation at TI was determined and found to be 98.1 ± 1.9%. This number was close to that (97.8%) obtained by TI enucleation in ovine cloning (Lee and Campbell, 2006). However, TI arrested oocytes progressed to MII stage very fast (about 30 min) after removal of the cumulus cells. The rapid progression from TI to MII stage in denuded porcine oocytes was different from that observed in in vitro matured ovine oocytes arrested at TI stage, which progressed more slowly. One possibility was that repression of GVBD by cAMP allows the oocyte to synchronise proteins required for maturation. Thus maturation may occur more rapidly. In addition, laser-assisted microdissection of the zona pellucida has been used in SCNT to facilitate enucleation since a noncontact infrared diode laser system was designed (Rink et al., 1994). In this experiment, laser-assisted microdissection was found to be more time-consuming than using sharp pipette because size of the hole is related to the laser exposure time and it took more time to make holes in zona pellucida using laser. For each oocyte, it would take at least 2-3 sec to make a hole by laser but sharp pipette helped make a hole directly. In conclusion, a reliable parthenogenetic activation system was developed and cAMP method was selected to synchronise oocytes based on the expansion of the cumulus cells of the oocytes, the effectiveness in synchronising porcine oocyte maturation and parthenogenetic development. Secondly, cAMP treatment produce MII stage oocytes during a shorter time window (36 - 44 hpm) and 36 -38 hpm was chosen to do TI enucleation. Finally, the percentage of enucleated porcine oocytes was 98.1 ± 1.9%. 84
CHAPTER 5 Effect of caffeine treatment on MPF and MAPK activities in TI enucleated porcine oocytes and on parthenogenetic development and the development of SCNT embryos reconstructed using TI enucleated oocytes treated with caffeine 5.1 INTRODUCTION Meiosis (originally maiosis) was the term coined in 1905 by Farmer and Moore. It refers to a typical type of cell division that occurs in the production of germ cells (eggs and sperm). Meiosis consists of two nuclear divisions but only one round of DNA replication. Meiotic maturation of mammalian oocytes is regulated by the cytoplasmic protein kinases, MPF and MAPK which may also have effects on reprogramming following SCNT. MPF consists of p34odoz (CDK1, a catalytic subunit) and cyclin B (a regulatory subunit). It is a family of serine/threonine protein kinases and promotes all the visible changes associated with meiosis and mitosis. MAPK is a family of serine/threonine protein kinases. MAPK signaling cascade plays an important role in both mitosis and meiosis (Chapter 1). For nuclear reprogramming of donor cell nuclei following SCNT, cell cycle coordination of karyoplast and cytoplast could be essential (Chapter 1). MPF in cytoplasts (oocytes) has been demonstrated to induce NEBD, PCC and dispersion of nucleoli in the transferred nucleus which may be beneficial for nuclear reprogramming (Collas, et al., 1992). 85
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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
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 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: In addition to the listed benefits
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
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
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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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In In ý. ö O V %) ö ö N _ ö O
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