S-Space College of Veterinary Medicine (수의과대학) Dept. of Veterinary Medicine (수의학과) Theses (Ph.D. / Sc.D._수의학과)
Application of sonic hedgehog signaling-induced in vitro oocyte maturation to klotho-knockout pig production
Sonic hedgehog signaling이 촉진된 체외난자성숙체계의 확립 및 klotho 유전자 제거 돼지 생산을 위한 이의 적용
- 수의과대학 수의학과
- Issue Date
- 서울대학교 대학원
- 학위논문 (박사)-- 서울대학교 대학원 : 수의과대학 수의학과, 2018. 2. 이병천.
- Pigs have been widely used as animal models in biomedical research, because of the similarity of their anatomy and physiology to those of humans. In particular, genetically modified pigs could be used as models for studying human disease and for xenotransplantation by eliminating specific genes. Several techniques for in vitro production of preimplantation porcine embryos have made it possible to produce animals for specific purposes. Among them, somatic cell nuclear transfer (SCNT) is one of the most efficient methods for producing genetically modified pigs. However, the developmental competence of porcine oocytes matured in vitro, the most important requirement for SCNT, still remains low. Although numerous studies have been performed to improve in vitro maturation (IVM) of porcine oocytes using specific compounds, little consideration has been given to the understanding of physiology of oocyte maturation and underlying mechanism by which they affect porcine cumulus oocyte complexes (COCs).
In growing ovarian follicles, communication among the oocyte, granulosa cell and theca cell compartments regulates each other’s proliferation and differentiation via signaling pathways such as sonic hedgehog signaling (Shh), which are essential for follicle development. In addition, targets (Ptch, Smo and Gli1) of active Shh signaling were found in the granulosa and cumulus cell layers of porcine ovary. For these reasons, Shh is considered an important signaling that could affect cumulus expansion and oocyte maturation. Therefore, in this study, the applicability of Shh signaling-induced in vitro oocyte maturation to klotho-knockout pig production was investigated.
Firstly, the relationship between the beneficial effects of resveratrol or melatonin on porcine IVM and Shh signaling was assessed. To clarify the underlying mechanism by which resveratrol or melatonin directly acts on porcine COCs, cumulus expansion, oocyte nuclear maturation, subsequent embryo development and expression of Shh signaling related genes and proteins were evaluated. Either a resveratrol or melatonin-induced increase in cumulus expansion, expression of Shh signaling genes, and proteins in cumulus cells and subsequent embryo development was prevented by a Shh signaling inhibitor (cyclopamine). Therefore, it was demonstrated that Shh signaling mediates resveratrol or melatonin to improve cumulus expansion, developmental competence of porcine oocytes and subsequent preimplantation embryo development. Nextly, possible synergistic effects of the combination of resveratrol and melatonin on porcine IVM was investigated. The combination of resveratrol and melatonin has synergistic effects on oocyte nuclear maturation and total cell numbers of parthenogenetic blastocysts and it finally improved cloning efficiency.
Secondly, this improved IVM system was applied to production of klotho knockout pigs. The klotho gene is considered to be one of the aging-suppressor genes that controls life span. As limited information is available on the functions of the klotho gene in large animals such as pigs, klotho monoallelic knockout fetal fibroblast cell lines were established by recovery of fetuses cloned via SCNT using non-selected porcine fibroblasts transfected with Cas9-sgRNA ribonucleoproteins, targeting exon 3 of the porcine klotho locus. Using these klotho-knockout cell lines as nuclear donors, klotho-knockout cloned embryos were generated and transferred to eleven recipients to produce klotho-knockout pigs. Seven from eleven recipients (63.6%) became pregnant. Nextly, a klotho monoallelic knockout cell line inducibly expressing a human Klotho was established by transfection of tetracycline (Tet)-inducible vector (pB-Tet-hKlotho-F2A-mCherry) and cloned embryos derived from this cell line were transferred to three recipients. All recipients (100.0%) became pregnant.
In conclusion, Shh signaling mediated the beneficial effects of resveratrol or melatonin on porcine IVM. Furthermore, the combination of resveratrol and melatonin during IVM had synergistic effects on porcine IVM and finally improved cloning efficiency. In addition to this, klotho monoallelic fetal fibroblast cell lines with or without inducibly expressing a human klotho gene were firstly established in the pig. Using oocytes matured in vitro with the combination of resveratrol and melatonin and klotho-knockout cell lines as nuclear donors, cloned embryos were generated via SCNT and transferred to recipients to produce klotho-knockout pigs. Successful implantation was confirmed through ultrasound imaging 28 days after transfer. Shh signaling-induced IVM system is demonstrated to be applicable in generating genetically modified pigs which are potential animal models for studying human disease.