-210 Nottingham - Nottingham eTheses - The University of Nottingham

More documents

Recommendations

Info

and its derivative and CHX were only inhibitors shown to improve development of porcine oocytes to blastocyst stage after IVF or have been used for porcine SCNT (Funahashi et al., 1997b; Day, 2000a; Ye et al., 2005; Betthauser et al., 2000; Yin et al., 2002). Caffeine was shown to be beneficial for reprogramming in porcine SCNT embryos using oocytes enucleated at MII stage (Kawahara et al., 2005; Iwamoto et al., 2005; Kwon et al., 2008) and ovine SCNT embryos using Al/TI enucleated oocytes (Lee and Campbell, 2006). It was hypothesized that a homogenous population of porcine oocytes enucleated at earlier stages (TI stage) and treated with caffeine could be beneficial for the establishment of a porcine SCNT. In this study, I manipulated porcine IVM and for the first time, used TI enucleation (Lee and Campbell, 2006) on porcine oocytes to establish the developmental potential and develop reproducible techniques of porcine SCNT. To achieve this, the basic strategy was to use an inhibitor to synchronise and optimise porcine oocyte IVM and then obtain oocytes maximally at TI stage and porcine NT embryos reconstructed using caffeine treated TI oocytes as recipient cells. Moreover, the use of an inhibitor would allow control of maturation to produce a homogenous population of porcine oocytes. Edwards (1965) first reported that porcine oocytes could mature in vitro. This was followed by various porcine IVM systems studied. The length for maturation of porcine oocytes varies from labs to labs because of the difference among media, culture methods, selection of oocytes and culture conditions. Day et al. (2000b) discussed about the methods that resulted in higher frequencies of male pronuclear formation or blastocysts. In the experiments described in Chapter 3, a reproducible oocyte IVM system using modified NCSU-23 medium were firstly introduced. The recipe of the modified NCSU-23 was different from media used by other reports and the percentage of MII oocytes reached over 90% at 44 hpm. This result was comparable to that (93%) reported by Wang et al. (1997a). Also, expansion of 110
cumulus cells could be observed specially after 36 hpm. Nagai et al. (2006) pointed out the importance of cumulus cells for IVM of porcine oocytes and the relationship among oocytes, cumulus cells and culture conditions. It was suggested that cumulus cells are involved in the nuclear and cytoplasmic maturation required for the developmental competence after fertilisation such as male pronucleus formation and further development to the blastocyst stage. In this experiment, the expansion of the cumulus cells indicated a good nuclear and cytoplasmic maturation of the oocytes. This gave a reliable basis for in vitro production of porcine embryos. A less time-consuming, environmentally favorable and economical assessment protocol for oocyte nuclear maturation was reported by fixing oocytes in 1.5 ml tubes, using recycled four-well dishes for orcein staining and then mounting the stained oocytes on slides to observe nuclear status. This aceto-orcein staining method was developed by modification of conventional oocyte fixation and staining method (Spalding et al., 1955; Hunter and Polge, 1966; Motlik and Fulka, 1976) and benefited a lot for assessment of the nuclear status. The observations on the morphological changes in the nucleus of the porcine oocytes were quite clear by this modified aceto-orcein staining system. Based on this system, the assessment of the nuclear status of the meiosis in oocytes in the following experiments was performed. Next, CHX and cAMP were selected as candidates for synchronisation to optimise porcine IVM respectively. By using a chemical inhibitor, porcine oocytes in vitro were synchronised in order that most of the oocytes could simultaneously reach maturation in a narrower window than normal maturation and this would improve early embryonic development of porcine embryos. Both CHX and cAMP could synchronise the porcine oocytes and the meiotic inhibition was reversible, as was consistent with Ye et al. (2005) and Betthauser et at. (2000). CHX treatment inhibited cumulus expansion which is an indicator for the nuclear and cytoplasmic maturation of the oocytes (Chen et at., 1990). The resumption of meiotic maturation in oocytes 111
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: CHAPTER 6 General discussion 7.1 Ma
Page 129 and 130: parthenogenetic development excludi
Page 131 and 132: demonstrated treatment of the TI en
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?