Chen, Wenchao
(2011)
Manipulation of oocyte maturation to improve porcine somatic cell nuclear transfer.
PhD thesis, University of Nottingham.
Abstract
Porcine somatic cell nuclear transfer (SCNT) offers new opportunities for fundamental science, medicine and agriculture. Ten countries or regions have developed the technology of porcine cloning and at least 42 groups have succeeded in producing piglets. Although successful, the efficiencies and reproducibility of porcine SCNT are extremely variable.
The technique of SCNT involves multiple steps, each of which can affect subsequent development. In particular, the synchrony of maturation, biochemical status of the matured oocytes and methods of parthenogenetic activation are thought to be major factors influencing development. The objectives of these studies were to optimise these steps to produce an efficient and reproducible method of porcine SCNT.
Cycloheximide (CHX) and cyclic AMP (cAMP) have been reported to maintain oocytes at the germinal vesicle (GV) stage and synchronise subsequent maturation. The effectiveness of these two treatments in inducing synchronisation was evaluated. Then nuclear status of oocytes was examined by aceto-orcein staining after release from CHX and cAMP at 0 h, 12 h, 22 h, 28 h, 36 h and 44 h. Data was analysed by chi-square test. At 28 h, 78.89%, 77.78% and 73.33% of control, CHX and cAMP oocytes reached metaphase of the first meiotic division (MI) respectively (p > 0.05). At 36 h, the frequency of oocytes at metaphase of the second meiotic division (MII) of the cAMP group (8.64%) was significantly lower than those of control and CHX groups (74.29% and 47.31 %, respectively; p<0.00 1). At 44 h, there was no difference between control and cAMP groups (91 % and 83.72%, respectively; p>0.05), however, the proportion of MII oocytes in the CHX group (56.57%) was lower (p < 0.05). The results demonstrate that CAMP is more effective than CHX in synchronising porcine oocyte maturation with oocytes reaching MII during a shorter time window.
Parthenogenetic development of porcine oocytes synchronised by CHX and cAMP treatments was compared by the frequency of cleavage at 48 h post onset of activation (hpa) and blastocyst formation at 168 hpa. No significant differences were observed in the frequency of cleavage (96.7 ± 2.1%, 81.4 ± 11.6% and 84.5 ± 5.7%, respectively), development to blastocyst (28.3 ± 11.4%, 27.1 ± 5.7% and 32.8 ± 5.3%, respectively) between control, CHX or cAMP treated oocytes respectively (chi-square test, P>0.05). However, total cell number was significantly higher in CHX group than cAMP group (42.7 ± 4.1 and 31.8 ± 2.0, respectively; t-test, P<0.05). The results demonstrate that synchronisation of porcine oocytes by treatment with either CHX or cAMP does not affect subsequent parthenogenetic development as judged by blastocyst formation, however the total cell numbers after CHX treatment were higher than those after cAMP treatment (P < 0.05). cAMP was selected to synchronise porcine oocytes because maturation to MII was more controlled and occurred over shorter period. The meiotic progression of cAMP treated oocytes was recorded at 38-44 h post onset of maturation (hpm) with telophase of the first meiotic division (TI; 35.6 ± 12.8%) peaking at 38 hpm, hence 36 -38 hpm chosen as a time window for TI enucleation.
The percentage of TI porcine oocytes successfully enucleated was 98.1 ± 1.9%. Caffeine (5,10 or 20 mM) had no significant effects on either maturation promoting factor (MPF) or mitogen-activated protein kinase (MAPK) activities of oocytes of TI and early MII arrested oocytes after 6 hours (t-test, P>0.05). Although MPF and MAPK activities in TI enucleated oocytes at 44 hpm were higher than those at 38hpm reaching maximum level at 44 hpm (two-way ANOVA, P<0.05), 5 mM caffeine did not change either of MPF and MAPK activities in TI enucleated oocytes (two-way ANOVA, P>0.05). Finally, development to blastocyst stage of SCNT embryos using TI enucleated oocytes treated with 5 mM caffeine was recorded. The frequency of blastocyst formation obtained was 8.8 ± 0.7 % with average total cell number 29.7 ± 0.9.
In conclusion, these studies have optimised several steps for porcine SCNT and produced porcine SCNT embryos using a homogenous population of porcine oocytes enucleated at earlier stages (TI stage) and treated with caffeine. These studies, along with further research may aid in the design of more successful methods of porcine somatic cell cloning.
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