Characteristics of Epigenetic Modifications in Porcine Cloned Embryos During Preimplantation Development

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.