Title page
ACKNOWLEDGEMENT
Contents
ABSTRACT 9
I. INTRODUCTION 12
II. MATERIALS AND METHODS 15
II.1. Animals and total RNA isolation from gonads 15
II.2. Collection of mouse oocytes 15
II.3. Double-stranded RNA preparation and microinjection. 16
II.4. Messenger RNA Isolation 21
II.5. RT-PCR analysis 21
II.6. Spindle visualization by Polscope 22
II.7. Aceto-Orcein staining. 23
II.8. Dual kinase assay 23
III. RESULTS 25
III.1. Expression pattern of Obox family 25
III.2. Specific suppression of Obox4 by Obox4 RNAi 29
III.3. Maturation rates after Obox4 RNAi 31
III.3.1. Cultured in M16 medium for 16 h 31
III.3.2. Cultured in M16 medium supplemented with IBMX for 24 h 31
III.3.3. Oocyte maturation speed 38
III.4. Spindle shape after Obox4 RNAi. 40
III.5. Obox4 is crucial for chromosome segregation during oocyte maturation. 42
III.6. Dual kinase assay 45
IV. Discussion 47
V. Conclusion 52
REFERENCES 53
국문초록 58
Table 1. Sequences of oligonucleotide primers used in this study and their annealing temperature (AT) and expected RT-PCR product sizes. 18
Table 2. Oocyte maturation rate after Obox4 RNAi using 2.0 ㎍/㎕ dsRNA in plain M16 media. Maturation rate was not affected by Obox4 RNAi. 34
Table 3. Oocyte maturation rate after Obox4 RNAi using 2.5 ㎍/㎕ dsRNA in plain M16 media. Maturation rate was arrested at MI stage after Obox4 RNAi. 35
Table 4. Oocyte maturation rate after Obox4 RNAi using 2.0 ㎍/㎕ dsRNA in M16 supplemented medium with IBMX. Resumption of meiosis induced in Obox4 RNAi group despite of the presence of IBMX in the medium. 36
Table 5. Oocyte maturation rate after Obox4 RNAi using 2.5 ㎍/㎕ dsRNA in M16 supplemented medium with IBMX. Resumption of meiosis induced more oocytes were arrested at MI stage in the medium. However, compared to 2.0 ㎍/㎕ dsRNA microinjection in which oocytes were arrested at GV stage. 37
Figure 1. Schematic diagram showing how to design Obox4 primers. For the preparation of Obox4 dsRNA, I used different set of primers to produce dsRNA and to confirm the knock down of the Obox4 mRNA. 19
Figure 2. Diagram for the experimental strategy. GV oocytes were microinjected with 2.0 ㎍/㎕ or 2.5 ㎍/㎕ Obox4 dsRNA, and maturation rate was recorded during culture in plain M16 medium for 16 h or M16 supplemented with 0.2 mM IBMX for 24 h. 20
Figure 3. Differential expression of Obox family mRNA according to the ovarian and testicular developmental stages. Total RNA from the ovaries and testes at different developmental stages were reverse transcribed and amplified by PCR. A, Expression of all 6 Obox family genes according to the ovarian... 27
Figure 4. Specific suppression of Obox4 mRNA expression by Obox4 RNAi. Injection of Obox4 dsRNA into the GV oocytes markedly suppressed Obox4 mRNA expression, but not Obox3 or Obox5, of which coding sequences are highly homolog with the Obox4 coding sequence. The mRNA equibalent to a... 30
Figure 5. Maturation rates of the mouse oocytes after Obox4 RNAi followed by IVM. A, B. Oocytes were cultured for 16 h in the plain M16 medium after Obox4 RNAi; C, D. Oocytes were cultured for 24 h in M16 medium supplemented with 0.2 mM IBMX after Obox4 RNAi. A, C. 2.0 ㎍/㎕ dsRNA... 33
Figure 6. Oocyte maturation after Obox4 RNAi scored at 4h interval. A, B. Oocytes were cultured for 16 h in the plain M16 medium after Obox4 RNAi; C, D. Oocytes were cultured for 24 h in M16 medium supplemented with 0.2 mM IBMX after Obox4 RNAi. A, C. 2.0 ㎍/㎕ dsRNA was microinjected; B, D. 2.5 ㎍... 39
Figure 7. Non-invasive analysis for spindle structure by using Polscope. Spindle structure was observed under the Polscope after Obox4 RNAi (2.5 ㎍/ ㎕ dsRNA) followed by culture in the plain M16 medium for 16 h. Control MI stage oocytes were culture for 8 h (A, E), Control MII stage oocytes were... 41
Figure 8. Chromosomal configuration in oocytes after Obox4 RNAi with 2.0 ㎍/ ㎕ dsRNA followed by IVM. A. Control oocytes showing typical chromosomal configuration during IVM. B. MII oocytes cultured in plain M16 medium for 16 h after Obox4 RNAi. C. Oocytes at each stages cultured in M16 medium... 43
Figure 9. Chromosomal configuration in oocytes after Obox4 RNAi with 2.5 ㎍/ ㎕ dsRNA followed by IVM. A. Control oocytes showing typical chromosomal configuration during IVM. B. MII oocytes cultured in plain M16 medium for 16 h after Obox4 RNAi. (a)-(c), GV; (d)-(f), MI. C. Oocytes at each stages... 44
Figure 10. Dual kinase activity assay for MPF and MAPK. Phosphorylation of the substrates Histone H1 and MBP reflect the kinase activities of MPF and MAPK, respectively. Each lane contained one oocyte at specific stage after Obox4 RNAi followed by IVM. RNAi was conducted with 2.5 ㎍/㎕ Obox4... 46