생쥐의 난소와 난자에서의 Obox4의 동정과 RNAi를 이용한 기능연구

Characterization and Functional Analysis of Obox4 during Oocyte Maturation by RNA Interference

  • 이현서 (포천중문의과대학교 생명과학전문대학원) ;
  • 이경아 (포천중문의과대학교 생명과학전문대학원)
  • Lee, Hyun-Seo (Graduate School of Lift Science and Biotechnology, Pochon CHA University College of Medicine) ;
  • Lee, Kyung-Ah (Graduate School of Lift Science and Biotechnology, Pochon CHA University College of Medicine)
  • 발행 : 2007.12.31

초록

목 적: 본 연구는 정소에서만 발현한다고 알려져 있는 Obox4에 대한 난소 및 난자에서의 동정과 난자 성숙에 미치는 영향을 알아보고자 수행하였다. 연구방법: RT-PCR을 이용하여 발달 단계별 난소와 정소, 난자에서의 Obox4의 mRNA발현을 확인하였다. 난자 성숙동안에 Obox4의 기능을 알아보기 위해 GV 난자의 세포질에 Obox4의 dsRNA를 미세 주입하는 RNAi 방법을 사용하였다. Obox4 dsRNA를 미세주입한 후, M16 배지에서 16시간 동안 배양하거나, IBMX가 첨가된 M16 배지에서 24시간 동안 배양하면서 난자 성숙율 및 spindle, 염색체의 배치와 형상의 변화를 관찰하였다. Obox4 RNAi후 여러 유전자들의 발현 양의 변화를 RT-PCR을 이용하여 확인하였다. 결 과: Obox4의 mRNA는 난소에서 다른 Obox family들과 비교하여 낮게 발현함을 관찰하였다. Obox4 RNAi를 위해 합성된 dsRNA가 Obox4의 발현만을 특정적으로 감소시켰다. Obox4 RNAi후에 M16배지에서 16시간 배양한 군에서의 난자 성숙률은 대조군의 난자 성숙률과 별다른 차이를 보이지 않았다. 흥미롭게도, IBMX가 첨가된 M16 배지에서 24시간 동안 배양한 군에서는 대조군의 난자들이 GV 상태에 정지되어 있는데 반해, Obox4 RNAi군에서는 IBMX에서 존재함에도 불구하고, MI과 MII로의 난자 성숙이 진행되었다. 또한 Obox4 RNAi 난자의 spindle 구조는 완전히 사라지고 매우 응축되어 있는 염색체를 확인하였다. 결 론: 본 연구에서는, 생쥐의 난소 및 난자에서 Obox4의 발현을 처음으로 밝혔으며, 난자 성숙 동안에 Obox4가 염색체 분리 및 spindle 형성에 관여되어 있는 유전자임을 확인하였다. 또한, CAMP에 의해 조절되는 GV-arrest mechanism에 Obox4가 매우 밀접하게 연관되어 있을 것임을 알게 되었다.

Objective: Previously, we identified differentially expressed genes between GV and MII stage mouse oocytes using ACP technology. When we study one of GV selective genes, Obox family, we found Obox4 mRNA expression in ovaries that has been reported as expressed exclusively in testis. Therefore, this study was conducted for characterization and functional analysis for Obox4. Methods: Expression of Obox4 mRNA was examined in gonads and oocytes by RT-PCR. To determine the role of Obox4 in oocyte maturation, Obox4 dsRNA was microinjected into the cytoplasm of GV oocytes followed by 16 h of incubation in the plain medium or by 24 h of incubation in the medium containing IBMX. After RNAi, phenotypes and maturation rates were observed, change in mRNA expression was evaluated, and chromosomal status was confirmed by orcein staining. Results: Obox4 has minimal expression in the ovary compared to that of the other family members. When oocytes were cultured for 16 h in M16 medium after RNAi, maturation rate was not changed significantly, compared with that of non-injected or buffer-injected control oocytes. Surprisingly, however, when oocytes were cultured for 24 h in M16 containing IBMX, in which oocytes were supposed to arrest at GV stage, Obox4 RNAi oocytes were advanced to MI and MII. Spindle structure was disappeared and the chromosomes were condensed in the oocytes after Obox4 RNAi. Conclusions: This is the first report on the expression of Obox4 in the ovary and oocytes. Results of the study suggest that Obox4 plays a crucial role in spindle formation and chromosome segregation during meiosis in oocytes. In addition, Obox4 may play an important role in cAMP-dependent signal cascades of GV-arrest in mouse oocytes.

키워드

참고문헌

  1. Yoon SJ, Chung HM, Cha KY, Kim NH, Lee KA. Identification of differential gene expression in germinal vesicle vs. metaphase II mouse oocytes by using annealing control primers. Fertil Steril 2005; 83: 1293-6
  2. Barnes FL, Sirad MA. Oocyte maturation. Semin Reprod Med 2000; 18: 123-31 https://doi.org/10.1055/s-2000-12551
  3. Siard MA. Resumption of meiosis: mechanism involved in meiotic progression and its relation with developmental competence. Theriogenology 2001; 55: 1241-54 https://doi.org/10.1016/S0093-691X(01)00480-0
  4. Rajkovic A, Van C, Van W, Klysik M, Matzuk MM. Obox, a family of homeobox genes preferentially expressed in germ cells. Genomics 2002; 79: 711-7 https://doi.org/10.1006/geno.2002.6759
  5. Thomas FH, Vanderhyden BC. Oocyte-granulosa cell interactions during mouse follicular development: regulation of kit ligand expression and its role in oocyte growth. Reprod Bio Endocrinol 2006; 4: 19 https://doi.org/10.1186/1477-7827-4-19
  6. Cheng WC, Li HMH, Yeh TJ, Li H. Mice lacking the obox6 homeobox gene undergo normal early embryonic development and are fertile. Dev Dyn 2007; 236: 2636-42 https://doi.org/10.1002/dvdy.21261
  7. Fire A. RNA-triggered gene silencing. Trends Genet 1999; 15: 358-63 https://doi.org/10.1016/S0168-9525(99)01818-1
  8. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 2001; 15: 188-200 https://doi.org/10.1101/gad.862301
  9. Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001; 409: 363-6 https://doi.org/10.1038/35053110
  10. Hammond SM, Bernstein E, Beach D, Hannon GJ. An RNAdirected nuclease mediates post-trasncriptional gene silencing in Drosophila cells. Nature 2000 16; 404: 293-6 https://doi.org/10.1038/016293a0
  11. Hutvagner G, Zamore PD. RNAi: nature abhors a double-strand. Curr Opin Genetics Dev 2002; 12: 225-32 https://doi.org/10.1016/S0959-437X(02)00290-3
  12. Wianny F, Zemicka-Goetz M. Specific interference with gene function by double-stranded RNA in early mouse development. Nat Cell Bio 2000; 2: 70-5 https://doi.org/10.1038/35000016
  13. Svoboda P, Stein P, Hayashi H, Schultz RM. Selective reduction of dormant maternal mRNA in mouse oocytes by RNA interference. Dev 2002; 127: 4147-56
  14. Gil J, Esteban M. Induction of apoptosis by the dsRNAdependent protein kinase (PKR): mechanism of action. Apoptosis 2000; 5: 107-14 https://doi.org/10.1023/A:1009664109241
  15. Richard FJ, Tsafriri A, Conti M. Role of phosphodiesterase type 3A in rat oocyte maturation. Biol Reprod 2001; 65: 1444-51 https://doi.org/10.1095/biolreprod65.5.1444
  16. Mongillo M, Zaccolo M. A complex phosphodiesterase system controls -adenoreceptor signaling in cardiomyocytes. Biochem Soc Trans 2006; 34: 510-11 https://doi.org/10.1042/BST0340510
  17. Chen J, Hudson E, Chi MM, Chang AS, Moley KH, Hardie DG, et al. AMPK regulation of mouse oocyte meiotic resumption in vitro. Dev Biol 2006; 291: 227-38 https://doi.org/10.1016/j.ydbio.2005.11.039
  18. Jones S, Sgouros J. The cohesion complex: sequence homologies, interaction networks and shared motifs. Genome Biol 2001; 2: 0009.1-0009.12
  19. Michaelis C, Ciosk R, Kim N. Cohesins: Chromosomal proteins that prevent premature separation of sister chromatids. Cell 1997; 91: 35-45 https://doi.org/10.1016/S0092-8674(01)80007-6
  20. Rajkovic A, Pangas SA, Ballow D, Suzumori N, Matzuk MM. NOBOX deficiency disrupts early folliculogenesis and oocytespecific gene expression. Science 2004; 30: 1157-9