| dc.description.abstract | Somatic cell nuclear transfer (SCNT) in mammals is an assisted reproductive technique
used to produce an animal from a single cell nucleus using an enucleated oocyte as a recipient.
This technique holds great potential to be applied in various biotechnological aspects including
pharmaceutical technologies, stem cell therapy, biomedical applications, increasing productivity
in animal production, preserving endangered species and applying research on gametes and
embryos. However, the success rate of clones was still very low due to the quality of the recipient
oocytes and the reprogramming of the donor cell nucleus.
In Chapter 1, we introduced about our two main objectives in this study. Our first main
objective is to improve the quality of the recipient oocytes. To achieve this target, we established
an optimal system for in vitro maturation (IVM), activation procedure and in vitro culture (IVC)
of porcine oocytes using parthenogenetic model and subsequently apply the system to generate the
recipient oocytes for SCNT and to culture cloned embryos. Next, our second objective is to
reprogram the donor nucleus using HDACi (Scriptaid). Our final examination was on the
characteristics of epigenetic modifications of cloned embryos during preimplantation
development.
In Chapter 2, we investigated effects of modulating agents during in vitro maturation on
the quality of porcine mature oocytes and parthenogenetic diploid embryos. Our results showed
that the combination of 1 mM dbcAMP and 0.01 IU/ml FSH supplemented to the maturation
medium could increase the quality of mature oocytes and improve the preimplantation2
development of parthenogenetic diploid embryos, which produced a higher rate of the embryos
developed to the blastocyst stage and higher number of good quality blastocysts.
Moreover, oocytes are committed to deterioration in quality as they aged due to a long
duration manipulation which leads to the reduced success rate of SCNT. We found that 5 mM
caffeine during Metaphase I- Metaphase II (MI-MII) transition could efficiently improve the
preimplantation development of embryos derived from aging oocytes (48 h, 36.95% blastocysts)
as well as those derived from fresh oocytes (42 h, 38.73% blastocysts, p>0.05).
On the other hand, due to the limited number of fully-grown oocytes on the ovary, the
research on the in vitro maturation and development of immature oocytes could be important in
order to supply a large quantity of mature oocytes to produce embryos for subsequent
experiments. Therefore, we investigated effects of L-ascorbic acid supplementation during IVM
on porcine parthenogenetic diploid oocytes derived from both small antral (2-3 mm) and large
antral (4-6 mm) follicles with L-ascorbic acid supplement during pre-maturation and maturation,
respectively. Moreover, according to the author’s knowledge, no comprehensive work was
dedicated to introduce hCG at the end of pre-IVM to shorten the culture time. Together with the
new culture system, our study suggested that L-ascorbic acid supplementation at 100 μg/ml
sharply enhanced the developmental potential of porcine oocytes to the blastocyst stage and the
quality of blastocysts in both oocytes collected from small and large antral follicles. Moreover,
the L-ascorbic acid-treated embryos showed a significantly higher number of blastomeres and
increased levels of acetylation of H3K9 and dimethylation of H3K4 in blastocyst derived from
both small and large antral follicle.
Our results also showed that FBS supplementation at 5% during the embryo development
significantly increased the number of parthenogenetic diploid embryos reaching late-to-hatched
blastocysts. Especially, FBS supplementation at Day 4 significantly improved the quality of both
parthenogenetic diploid and cloned blastocysts compared to the control.
In Chapter 3, the effects of Scriptaid on preimplantation development of cloned porcine
embryos and the epigenetic modifications were examined. In this study, first, porcine cloned3
embryos were treated with Scriptaid for 6 hours at different concentrations (0, 100, 200, 400 and
800 nM) after activation. Second, we examined the effect of Scriptaid treatment at 42-48h and at
48-54 h post-activation (hpa) on preimplantation development. Last, we investigate the effects of
two-step Scriptaid treatment in which the first step from the time of activation and the second step
from 42 h (pact) on the preimplantation development of porcine cloned embryos. The results of
the first experiment showed that Scriptaid at 200 nM reached the highest number of cloned
blastocysts. The results of the second experiment showed that, cloned embryos treated with
Scriptaid after activation showed the highest number of embryos that developed to the blastocyst
stage (0 hpa, 17.84%). Interestingly, Scriptaid treatment at the early 4-cell stage embryo (42 hpa)
has a comparable percentage of blastocyst compared to the 0 hpa group (p<0.05) and has the
blastocyst rate (14.18%) higher compared to those treated at late 4-cell stage (48 hpa, 7.15%). In
the last experiment, the two-step Scriptaid treatment showed the significantly highest rate of
blastocyst (35.02%) and the highest rate of late/hatched blastocyst (18.65%) among the four
treatments (p<0.05). In addition, it had significantly increased in the total cell number of porcine
cloned blastocysts (68.20, p<0.05). Nevertheless, two-step Scriptaid treatment had increased the
level of histone acetylation of H3K9 and significantly decreased the level of H3K9-Me compared
to the control group, suggesting an open space for gene expression in porcine cloned embryos.
Moreover, a decrease in H3K9-Me could help to enhance the level of gene expression. Our results
indicated that the two-step Scriptaid treatment had improved the correction of aberrant gene
expressions, showing the highest level of OCT4 expression (68.18, p<0.05) among all treatments.
In Chapter 4, the main results of this study were summarized. Based on parthenogenetic
diploid embryos as an efficient model, we had established the culture system to provide high
quality recipient oocytes and the optimal culture system to develop cloned embryos. Importantly,
the two-step Scriptaid treatment had rescued the aberrant modifications of histone in cloned
embryos during preimplantation development. | en_US |
