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Figure 3.1 Classification of meiotic stages of porcine oocytes by nuclear morphology (black arrow: the nucleus of the oocyte) GVI, characterised by a distinct nuclear envelope, a nucleolus and chromatin in the form of a ring or a horseshoe; GVII, characterised by a nucleolus and a few orcein-positive zones (chromocenters) localised on the nuclear membrane; GVIII, characterised by a nuclear membrane, chromatin distributed in separate clumps and filamentous bivalents; GVIV, characterised by less distinct nuclear membrane, no nucleolus and the chromatin as an irregular network, or else the individual filamentous bivalents; ProMi, characterised by less conspicuous nuclear area of a single lump or smaller discrete fragments; MI, characterised by chromosomes formed and arranged in an orderly sequence on the equator of a bipolar spindle; Al, characterised by the chromosomes migrating towards the poles of an elongated spindle; TI, characterised by the chromosomes reaching the polar regions of the spindle; Mli, characterised by the first polar body abstracted from the egg, and the chromosomes arranged on the equator of the spindle. Scale bar = 50 Nm. 58
3.3 RESULTS 3.3.1 Conventional maturation of porcine oocytes in modified NCSU-23 medium When gilt oocytes were cultured in modified NCSU-23 medium, a large proportion of MII oocytes (91.0 ± 1.4%) were observed at 44 h. At this time, the cumulus cells of the oocytes were highly expanded and could be easily removed from the oocytes by gentle pipetting (Figure 3.2). 3.3.2 Assessment of oocyte nuclear maturation A simple aceto orcein staining protocol was developed, which was to fix oocytes in 1.5 ml tubes, stain oocytes in recycled four-well dishes and then mount the stained oocytes onto slides to observe nuclear status. Nuclear morphology of meiotic stages was classified as GV (GVI-IV), prometaphase I, metaphase I (MI), anaphase I (Al), telophase I (TI), and metaphase II (MII) with details shown in Figure 3.1. 3.3.3 Comparison of CHX and cAMP for synchronisation of porcine oocyte maturation Morphologically, control and cAMP treated oocytes were similar at all times (Figure 3.2). After 36 h, rapid expansion of the cumulus cells was visible in both groups. In contrast, CHX treated oocytes were morphologically distinct particularly during latter stages (post 36 h) when little expansion of the cumulus cells was observed. At 44 h, the cumulus cells of control and cAMP treated oocytes could be almost entirely removed by gently mechanical pipetting. However, hyaluronidase was required for removal of cumulus cells from CHX treated oocytes at this time. Meiotic progression of oocytes cultured in the presence of 5 µg/ml CHX for 12 h or I mM cAMP for 22 h and then in the control medium with conventional IVM as control, as is shown in Figure 3.3 and Figure 3.4 (for detailed data, see data in the attached 59
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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
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
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: I 57
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
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
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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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