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LIST OF PUBLICATIONS Selected publications from thesis: Chen, W. C., Zhu, J., Fisher, P. and Campbell, K. H. S. (2008) A comparison of effects of cycloheximide and cAMP on synchronisation of porcine oocyte maturation. Society for Reproduction and Fertility annual conference, abstract, 34. (oral presentation) Chen, W. C., Zhu, J., Fisher, P., Amarnath, D. and Campbell, K. H. S. (2009) A comparison of parthenogenetic development of porcine oocytes synchronised during maturation by cycloheximide or cAMP. Reproduction, Fertility and Development, 21(1), 215-216. (poster presentation) Campbell, K. H. S., Fisher, P., Chen, W. C., Choi, I., Kelly, R. D. W., Lee, J-H. and Zhu, J (2007). Somatic cell nuclear transfer: Past, Present and future perspectives. Theriogenology, 68 Supplement 1, S214-S231. Chen, W. C., Zhu, J., Fisher, P., Amarnath, D. and Campbell, K. H. S. A comparison of effects of cycloheximide and 3', 5'- cyclic AMP on synchronisation of porcine oocyte in vitro maturation and parthenogenetic development. (To be submitted to Zygote) Moawad, A. R. *, Choi, I. *, Chen, W. C. *, Zhu, J., and Campbell, K. H. S. Ovine oocytes vitrified at germinal vesicle stage as cytoplast recipients for somatic cell nuclear transfer (SCNT). (equal contribution, to be submitted to Cloning and Stem Cells) ix
LIST OF FIGURES Figure 1.1 Process of nuclear transfer Figure 1.2 Main reproductive hormones as to oocytes Figure 1.3 Changes in maturation promoting factor (MPF) and cytostatic factor (CSF) activities during meiotic divisions of oocytes and mitotic divisions of early zygotes Figure 1.4 Effects of nuclear transfer of karyoplasts at defined cell cycle stages into cytoplasts with either high (group A) or low (group B) MPF activity upon DNA synthesis during the first cell cycle and potential effects upon the ploidy of the reconstructed embryo Figure 1.5 Flow chart of the experimental design Figure 2.1 Diagram of micromanipulation instruments and manipultion set up Figure 2.2 Enucleation of porcine oocytes at telophase of first meiotic division (TI) Figure 2.3 Production of oocyte-somatic cell couplets by cell transfer Figure 3.1 Classification of meiotic stages of porcine oocytes by nuclear morphology Figure 3.2 Morphology of control, CHX and cAMP treated COCs during maturation Figure 3.3 Distribution of meiotic progression (mean ± SEM) in control, CHX and cAMP treatments during maturation Figure 3.4 The percentages of MII oocytes (mean ± SEM) in control, CHX and cAMP treatments during maturation Figure 4.1 Morphology of porcine oocytes and parthenotes in control or synchronised Figure 4.2 groups Meiotic changes of oocytes (mean ± SEM) in control and cAMP treatments at different time points Figure 5.1 Timeline of the treatment of porcine oocytes with caffeine during somatic cell nuclear transfer Figure 5.2 MPF and MAPK activities in porcine oocytes detected with an in vitro double-kinase assay by phosphorylation of histone HI and MBP, respectively Figure 5.3 Effects of different caffeine concentrations on MPF and MAPK activities in TI and early MII porcine oocytes Figure 5.4 Morphology of parthenotes from oocytes treated wit different concentrations of caffeine Figure 5.5 Total cell number counting of blastocyst stage embryos from oocytes treated Figure 5.6 Figure 5.7 with different concentrations of caffeine by DAPI staining Effects of 5 mM caffeine on MPF and MAPK activities in TI enucleated porcine oocytes Hatching porcine SCNT blastocyst x
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: CHAPTER 6 .........................
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 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
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were washed three times in embryo c
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Three replicates of batches of oocy
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fertilisation, the occurrence of po
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3.2 MATERIALS AND METHODS 3.2.1 Exp
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I 57
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3.3 RESULTS 3.3.1 Conventional matu
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ýIC M 00 N N N N 0 öa oÜd U 0 0
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Meiotic stage in CAMP treated oocyt
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3.4 DISCUSSION The development of N
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44 h. It was comparable to that (93
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oocyte (Chen et al., 1990). In the
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CHAPTER 4 Parthenogenetic developme
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mechanical and chemical procedures.
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COCs were collected and oocytes wer
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f. " ý1403: 0.71 14 - 'Ir I A Figu
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100% 90% 80% 70% d 60% 50% 0 40% 30
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4.4 DISCUSSION For successful porci
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In addition to the listed benefits
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CHAPTER 5 Effect of caffeine treatm
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in vitro substrates. In pigs, Naito
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5.2 MATERIALS AND METHODS 5.2.1 Exp
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5.2.2 In vitro maturation of porcin
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Blastocysts at day 7 were stained a
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Figure 5.2 MPF and MAPK activities
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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
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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
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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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