-210 Nottingham - Nottingham eTheses - The University of Nottingham

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1.3.1 Preparing the cytoplasts In general, oocytes arrested at MII are used as recipient cytoplasts and they can be obtained either in vivo or in vitro. Oocyte in vitro maturation (IVM) is also related to various technical factors. 1.3.1.1 In vivo and in vitro matured oocytes MII oocytes can be matured in vivo and collected following ovulation by surgical or non-surgical methods dependent on species. In contrast, IVM of slaughterhouse derived oocytes provides a cheap and plentiful supply. Oocytes should be stripped of their cumulus cells prior to enucleation, either by mechanical pipetting or using hyaluronidase B. In vivo porcine ovulation occurs at 38 - 42 h after the pre-ovulatory luteinising hormone (LH) peak when oocytes mature and have reached MII. IVM procedures are based on this and typically require a minimum of 36 h to 48 h or 50 h (Coy and Romar, 2002). 1.3.1.2 In vitro maturation system Technical factors, which influence the successful outcome of IVM, include ovary and oocyte delivery (transport temperature and time, etc. ), IVM medium composition including the additives (hormones, growth factors, serum and follicular fluid, etc. ) and culture system (the culture temperature, humidity and gas phase, etc. ) as well as pH and osmolarity. Various media have been used for porcine IVM including NCSU-23, NCSU-37, TCM-199, Waymouth medium, Whitten's medium, and Tyrode's lactate pyruvate medium (TLP), etc (Coy and Romar, 2002). NCSU-23, NCSU-37 and TCM-199 are more widely used for IVM in porcine cloning. NCSU-23 medium gives improved results as compared to TCM-199 medium and modified Whitten's medium for the 24
development of porcine embryos produced by in vitro fertilisation (Wang et al., 1997a). Modifications of in vitro porcine oocyte maturation techniques to increase quality have included: (1) addition of cysteine, glutathione, cysteamine or EGF, (2) co-culture with somatic cells, (3) reduced hormone exposure during IVM, (4) supplementation of maturation media with porcine follicular fluid (pFF), (5) adjustment of the NaCl concentration and (6) synchronisation of maturation by maintaining oocytes at the germinal vesicle stage. These methods result in higher frequencies of male pronuclear formation and blastocyst development possibly by improving nuclear and cytoplasmic maturation of the porcine oocytes (Day et al., 2000a; Appendix 2). A preferred medium for maturation would be one which does not require co-culture with somatic cells or their secretions (Day et al., 2000b). 1.3.2 Preparing the karyoplasts Donor cells are called karyoplasts. Their preparation involves cell cycle and culture system, technically. 1.3.2.1 Cell cycle of the donor cells Donor cells induced to become quiescent (GO) by serum starvation (Campbell et al., 1996b) as well as nuclei in GI, S or G2 and M phases have been successfully applied to clone pigs (Polejaeva et al., 2000; Lai et al., 2002b). 1.3.2.2 Donor cell culture system Dulbecco's modified Eagle medium (DMEM) with 10%, 15% or 20% FCS/FBS is widely used for porcine donor cell culture. They are prepared by primary cell culture, passaging from frozen cells, serum starvation or contact inhibition, and. It is important to use as early passage as possible. 25
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: Little is known about the mechanism
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 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
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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
Page 99 and 100:
In addition to the listed benefits
Page 101 and 102:
CHAPTER 5 Effect of caffeine treatm
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in vitro substrates. In pigs, Naito
Page 105 and 106:
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
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-
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