-210 Nottingham - Nottingham eTheses - The University of Nottingham

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varie 1 mmature ooc CHXIcAMP IVM e TI Enucleation lEnucleated ooc I Cell transfer ou Iet Fusion and activation lActivated cou let In vitro embryo culture torulae or blastocyst Figure 1.5 Flow chart of the experimental design The black text refers to the conventional nuclear transfer procedures. The red text highlights sections of the procedure which were modified during these studies for manipulation of the porcine oocyte maturation. CHX and cAMP were compared to synchronise porcine oocyte maturation. Oocytes were to be enucleated at TI stage and treated with caffeine before cell transfer. Couplets are made from enucleated oocytes treated with caffeine and donor cells by cell transfer and fusion. Followed by fusion and activation, embryo culture, the developmental potential to blastocyst stage is to be determined. 38
CHAPTER 2 General materials and methods All chemical reagents used were purchased from Sigma-Aldrich (UK) unless otherwise stated. Detailed experimental design and protocols are given in the following chapters. Methods of IVM of porcine oocytes (using modified NCSU-23 medium), synchronisation of porcine oocyte maturation (using 1 mM cAMP), oocyte staining and assessment of nuclear maturation (using a modified aceto-orcein staining protocol), selection of TI and MII oocytes (using 0.05 sucrose), TI enucleation of porcine oocytes, caffeine treatment of TI enucleated porcine oocytes and NT using TI enucleated porcine oocytes treated with caffeine were developed in these studies. 2.1 In vitro maturation of porcine oocytes Ovaries were collected at a slaughterhouse and maintained at 25-30°C in a thermos flask filled with Dulbecco's Phosphate Buffered Saline (DPBS) during transport to the laboratory. On arrival, they were washed three times with DPBS at 39°C, and then held at 39°C during aspiration. Translucent antral follicles from 3 to 8 mm in diameter were aspirated using an 18-gauge needle attached to a 10 ml disposable syringe and the follicular fluid was placed into 50 ml conical centrifuge tubes. The aspirated follicular fluid was allowed to settle for 10 min at 39°C and then 75% of the supernatant was discarded and the remaining fluid was diluted with three times its volume of modified TL-HEPES-PVA medium containing 114 mM NaCl, 3.2 mM KC1,2 mM NaHCO3,0.34 mM NaH2PO4.2H20 (Fisions), 10 mM sodium lactate, 0.5 mM MgC12.6H20,2 mM CaCI2.2H20,10 mM HEPES, 0.19 mM sodium pyruvate, 25 µg/ml gentamicin, 65 µg/ml penicillin GS 5 mM D-Glucose and 0.1 g/l polyvinylalcohol (PVA) and transferred into a 90 mm Petri dish (Nunc). Oocytes surrounded by at least 3 layers of cumulus cells were selected at 39°C under a 39
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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 and 52: deacetylases (HDACs). Trichostatin
Page 53: Figure 1.5 presents the general exp
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
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e ýo; +ý \.. 4.0 ob "i "s NW Ir
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5.3.4 Effects of caffeine treatment
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Figure 5.6 Effects of 5 mM caffeine
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5.4 DISCUSSION In the first experim
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In the final experiment, developmen
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CHAPTER 6 General discussion 7.1 Ma
Page 127 and 128:
cumulus cells could be observed spe
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parthenogenetic development excludi
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demonstrated treatment of the TI en
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REFERENCES Abeydeera, L. R., Wang,
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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
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-
Page 169 and 170:
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