Title Page
ACKNOWLEDGEMENT
Contents
ABSTRACT 10
I. INTRODUCTION 12
II. MATERIALS AND METHODS 16
II. 1. Collection of porcine immature oocytes and in vitro maturation 16
II. 2. Sperm preparation and in vitro fertilization 17
II. 3. In vitro culture of porcine embryos and counting the cell number ofblastocysts 17
II. 4. Cell death detection using TUNEL and ELISA assay in blastocystsand cumulus cells 18
II. 5. Glutathione assay 20
II. 6. Visualization of normal spindle in oocytes matured in vitro by thePolScope 21
II. 7. Analysis of gene expression in oocytes using reverse transcriptasepolymerasechain reaction 21
II. 8. Statistical Analysis 22
III. RESULTS 23
III. 1. Effect of folic acid on the oocytes maturation and subsequentembryonic development of porcine 23
III. 2. Intracellular GSH contents and gene expression in oocytesmatured in media with different concentration of folic acid 31
III. 3. Effect of folic acid on anti-apoptosis of cumulus cell after IVM andsubsequently developed blastocysts 33
III. 4. Existence of spindle during IVM 37
IV. DISCUSSION 40
V. CONCLUSION 44
REFERENCE 45
국문초록 53
Fig. 1 The folic acid pathway is comprised of folate cycle, methionine cycle, and glutathione metabolism. 15
Fig. 2A Effect of folic acid on in vitro maturation of porcine oocytes using rough range. Data are shown as mean ± SEM for five replications. 25
Fig. 2B Effect of folic acid on cleavage and embryonic development of in vitro matured porcine oocytes using rough range. 26
Fig. 3A Effect of folic acid on in vitro maturation of porcine oocytes using narrow range. Data are shown as mean ± SEM for six replications. 27
Fig. 3B Effect of folic acid on cleavage and embryonic development of in vitro matured porcine oocytes using narrow range. 28
Fig. 4A Effects of folic acid on the number of total cells and inner cell mass of in vitro produced blastocysts. 29
Fig. 4B Differentially stained inner cell mass (ICM, blue) and trophectoderm (TE, pink) in in vitro produced porcine blastocysts depending on the folic acid concentration. 30
Fig. 5 Glutathione concentration in porcine oocytes matured in in vitro maturation (IVM) media with different concentration of the folic acid (A), and relative mRNA expression of Smarca 2 depending on the folic acid concentration. mRNA levels of Smarca 2 were measuredby RT-PCR. 32
Fig. 6 Quantification of apoptosis in cumulus cells (CCs) from in vitro matured cumulus-oocyte complexes (COCs) depending on the folic acid concentration. The extent of apoptosis in the cumulus cells was measured by ELISA. 34
Fig. 7 The number of total cells (A) and apoptotic cells (B) in in vitro produced porcine blastycysts depending on the folic acid concentration. 35
Fig. 8 Hoechst 33342 (total cells, blue) staining and TUNEL (apoptotic cells, red) in in vitro produced porcine blastycysts depending on the folic acid concentrate. 36
Fig. 9 The rate of intact spindle in in vitro matured metaphase II oocytes depending on the folic acid concentration. Spindle image was observed by non-invasive PolScope. 38
Fig. 10 Spindle morphology in porcine metaphase II oocytes depending on the folic acid concentration by non-invasive PolScope. 39