-210 Nottingham - Nottingham eTheses - The University of Nottingham

More documents

Recommendations

Info

protocol was a further development based on previous protocol (Ye et al., 2003). The images obtained were close to those in Wehrend and Meinecke (2001) and Kubelka et al. (2002) with 2 clear bands representing MPF and MAPK activities individually. The MPF and MAPK double kinase assay allows the simultaneous measurement of MPF and MAPK activities, minimising the inevitable errors by individual assays. This method has also been successfully applied in assessment of ovine oocyte MPF/MAPK activities recently (data not shown). Next, which caffeine concentration was better to treat enucleated oocytes was determined and toxicity experiments for caffeine treatment were introduced. Caffeine was shown to have no significant effects on either MPF or MAPK activities of porcine oocytes selected at TI and early MII stage after 6 hours incubation with caffeine. Parthenogenetic developmental potential of TI and early MII oocytes treated by different concentration of caffeine was compared and 5 mM caffeine was chosen for future experiments (Chapter 5). Interestingly, by selection of cAMP treated oocytes at relatively earlier maturation stage (TI and early MII), the frequency of blastocyst formation can reach about 50%, which was different from the number (32.8 ± 5.3%) using cAMP treated MII oocytes, indicating selection of oocytes at earlier stage could be beneficial for parthenotes. Also, 5 mM caffeine did not effectively raise either kinase activity in TI enucleated oocytes peaking at 44 hpm during the prolonged culture. Based on this, cell transfer at 42 hpm and activation at 44 hpm were decided. Finally, the development of SCNT embryos using TI enucleated oocytes treated with 5 mM caffeine was examined. Caffeine has been individually reported to result in improved remodeling of the donor genome in porcine and ovine SCNT embryos (Kawahara et al., 2005; Iwamoto et al., 2005; Kwon et al., 2008; Lee and Campbell, 2006). In particular, Lee and Campbell (2006) presented a detailed investigation into the effects of caffeine on ovine oocytes and SCNT produced embryos. They 114
demonstrated treatment of the TI enucleated ovine oocytes with 10 mM caffeine for 6 h led to the elevation of the activities of MPF and MAPK and a rise in the occurrence of NEBD in the donor nucleus and total cell numbers of the resultant blastocyst stage embryos. These observations formed the basis of the studies presented here. Using TI oocytes with actively raised MPF as cytoplast recipients, SCNT embryos were obtained with the frequency of blastocyst formation 8.8 f 0.7 % and average total cell number of 29.7 ± 0.9. These results suggested for the first time the development of porcine SCNT embryos by manipulating oocyte maturation, which used TI enucleated oocytes treated with 5 mM caffeine. However, every coin has two sides. Although TI enucleation seemed to be much easier and more efficient than MII enucleation in porcine oocytes, the number of TI oocytes obtained at 36 - 38 hpm reached 15 -20 maximally among about 150 oocytes because most of the TI arrested oocytes went to early MII very quickly after removal of cumulus cells, as was a disadvantage for TI enucleation. Further research needs to be done to see whether the treatment involved in the studies result in any benefits after the surrogate mothers give birth to the newborn. 7.2 Future experiments First, it is interesting to see whether this cloning strategy of porcine NT embryos reconstructed using caffeine treated TI oocytes or both TI and MII enucleated at 36-38 hpm as cytoplast recipients will bring any benefits to cloning efficiency after those embryos are transferred into the surrogate. The efficiency of porcine cloning is also dependent on the production of cloned offspring. Also, this work was the first step to try to improve the porcine cloning efficiency in based on the previous accumulation (Ye et al., 2005; Lee and Campbell, 2006; Choi and Campbell, 2010). Other methods could be introduced to improve the porcine cloning efficiency such as trying other oocyte synchronising methods. Inhibitor 115
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 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
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
Page 125 and 126: CHAPTER 6 General discussion 7.1 Ma
Page 127 and 128: cumulus cells could be observed spe
Page 129: parthenogenetic development excludi
Page 133 and 134: REFERENCES Abeydeera, L. R., Wang,
Page 135 and 136: Bromhall, J. D. (1975) Nuclear trna
Page 137 and 138: Christmann, L., Jung, T. and Moor,
Page 139 and 140: and human ovarian oocytes. Nature,
Page 141 and 142: Funahashi, H., Cantley, T. C. and D
Page 143 and 144: Hartwell, L. H. (1973) Three additi
Page 145 and 146: 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
Page 165 and 166: Production of the first cloned came
Page 167 and 168: oocytes. Reproduction, 125(5), 645-
Page 169 and 170: 00 Cl) h 'Cb a) Ü^W wv F NC a b d
Page 171 and 172: In In ý. ö O V %) ö ö N _ ö O
Page 173 and 174: - u2 y vý'ý OOU 1-1 00 ^ OO NN ö
Page 175 and 176: aý yy U a 2 w I-N O N 'T N 1-1 NO
Page 177: . 10 CA aý v 2 4 1-1 1-1 o Np cc o
show all

-210 Nottingham - Nottingham eTheses - The University of Nottingham

Create successful ePaper yourself

Delete template?

Save as template?