-210 Nottingham - Nottingham eTheses - The University of Nottingham

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Several approaches have been proposed to obtain a more homogenous population of mature MII porcine oocytes. These include (1) treatment of gilts with gonadotrophin (eCG) 72 h before oocyte collection (Funahashi et al., 1996), (2) preincubation of oocytes for 12 h without gonadotrophins (Funahashi et al., 1997a) and (3) synchronisation of oocyte maturation by inhibiting GVBD using reversible inhibitors (Funahashi et al., 1997b). In studies reported in this thesis, an inhibitor to synchronise porcine oocyte maturation was chosen from 2 inhibitors, which were tested to determine the degree of synchrony and subsequent development. 1.6 Objectives of the research and experimental design Previously discussed, TI enucleation facilitates SCNT by removal of a smaller volume of oocyte cytoplasm and a much higher percentage of enucleated oocytes (Lee and Campbell, 2006). A homogenous population of porcine oocytes chemically synchronised led to improved developmental competence of porcine oocytes (Funahashi et al., 1997b; Betthauser et al., 2000; Ye et al., 2005). Also, caffeine was shown to enhance reprogramming in porcine SCNT embryos using oocytes enucleated at MII stage (Kawahara et al., 2005; Iwamoto et al., 2005; Kwon et al., 2008). It can be hypothesized that a homogenous population of porcine oocytes enucleated at earlier stages (TI stage) and treated with caffeine could be advantageous for the establishment of a porcine SCNT system and possibly beneficial for the reprogramming of porcine NT embryos. As mentioned at the beginning of this chapter, the objectives of these studies were to establish reliable and reproducible techniques for porcine SCNT, in particular by manipulation of oocyte maturation to produce a homogenous population to use as cytoplast recipients. 36
Figure 1.5 presents the general experimental design and the red text highlights sections of the procedures which were modified during these studies. A reliable porcine IVM system was to be established. The effects of CHX and cAMP on synchronisation of porcine oocyte maturation were compared in order to produce a homogenous population of oocytes to use as cytoplast recipients. The effects of treatment with CHX and cAMP on maintenance of GV arrest and synchrony of subsequent maturation were determined by a modified aceto-orcein staining method for assessment of nuclear maturation (Chapter 3). A parthenogenetic activation system of porcine oocytes was introduced. The parthenogenetic developmental potential of oocytes synchronised by CHX and cAMP was evaluated. Oocytes were to be enucleated at TI stage and treated with caffeine before cell transfer. The timing for TI enucleation was also determined. The efficiency of TI enucleation in porcine oocytes was investigated (Chapter 4). An MPF assay was developed and the effects of caffeine on porcine TI enucleated oocytes were studied and the optimal concentration defined based on survival development and MPF and MAPK activities. SCNT was then carried out using synchronised TI enucleated oocytes as cytoplast recipients. Cytoplast-karyoplast couplets (reconstructed embryos) were made from enucleated oocytes treated with caffeine and donor cells by cell transfer. Finally, the development of SCNT embryos using TI enucleated oocytes treated with caffeine was finally determined (Chapter 5). 37
Page 1 and 2: The University of -210 Nottingham M
Page 3 and 4: treated oocytes was recorded at 38
Page 5 and 6: TABLE OF CONTENTS ABSTRACT ........
Page 7 and 8: 2.5.1.2 Preparation of enucleation/
Page 9 and 10: CHAPTER 6 .........................
Page 11 and 12: LIST OF FIGURES Figure 1.1 Process
Page 13 and 14: LIST OF ABBREVIATIONS A23187 AC AI
Page 15 and 16: MgSO4.7H20 MAPK MAPKK MAPKKK MBP mg
Page 17 and 18: CHAPTER 1 LITERATURE REVIEW The aim
Page 19 and 20: The production of multiple offsprin
Page 21 and 22: cytoplasm which contained the nucle
Page 23 and 24: pluripotency, as demonstrated by th
Page 25 and 26: gene-targeting via somatic cell clo
Page 27 and 28: conventional breeding and genetic m
Page 29 and 30: Oocyte maturation is generally cont
Page 31 and 32: ooýcýýommý MPF Time Figure 1.3
Page 33 and 34: 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: deacetylases (HDACs). Trichostatin
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 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
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Figure 5.6 Effects of 5 mM caffeine
Page 121 and 122:
5.4 DISCUSSION In the first experim
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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,
Page 141 and 142:
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
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
Page 163 and 164:
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-
Page 169 and 170:
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In In ý. ö O V %) ö ö N _ ö O
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