체외성숙 과정 중 생쥐와 돼지 난자의 malate dehydrogenase (Mor2)의 기능에 대한 비교 분석
Comparative functional analysis of the malate dehydrogenase (Mor2) during in vitro maturation of the mouse and porcine oocytes
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생쥐의 난자는 특별한 첨가물이나 난구세포 없이 체외 배양해도 성숙율이 높은 반면 돼지 난자의 체외성숙율은 매우 낮다. 본 연구는 이러한 차이의 원인을 연구하기 위하여 생쥐와 돼지 난자의 에너지 생성에 관여하는 유전자인 malate dehydrogenase (Mor2)의 기능을 RNAi를 이용하여 비교분석 하였다. 생쥐와 돼지 각각의 Mor2 double-stranded RNA (dsRNA)를 제작하고, 생쥐는 난구세포를 제거한 미성숙 (GV) 난자의 세포질에 mMor2 dsRNA를 미세주입 한 후 16시간 동안 배양하였고, 돼지는 난구세포를 제거하거나 (DO) 혹은 그대로인 상태 (COCs)로 pMor2 dsRNA를 미세주입 한 후 48시간 동안 배양하였다. 사용한 배양액은 M199에 10%의 porcine follicular fluid, pyruvate, p-FSH, EGF, cystein, estradiol-17등이 첨가된 배양액을 사용하였고 배양 후 난자의 형태학적 변화를 관찰하고 Mor2 mRNA양의 변화를 RT-PCR로 확인하여 RNAi 결과 염기서열 특이적으로 Mor2발현이 억제됨을 확인하였다. 돼지 난자의 pMor2 RNAi 결과, DO 난자에서는 약 58%의 난자가 MI에서 성숙이 억제되었으나 COCs에선 84.4%가 MII로 성숙하는 것을 볼 수 있었다. 이는 돼지의 난구세포가 난자성숙에 매우 중요하며, 특히 malate 공급에 관여할 것임을 시사한다. 선행연구 결과 Mor2 RNAi 결과 생쥐는 GV에서, 본 연구에서 돼지는 MI에서 성숙이 정지되었기 때문에 이러한 차이가 배양액의 차이인지 다시 mMor2 RNAi 후 생쥐 난자를 M199 배양액에 16시간 동안 배양한 후 성숙율을 관찰하였더니 MII로의 성숙율은 62%로 대조군과 비교하여 큰 차이가 없었다 (non-injected: 76.8%, buffer-injected: 73.3%). 이는 mMor2 RNAi 결과가 배양액의 조성에 의해 극복될 수 있음을 보여준 결과다. 생쥐와 돼지에서 서로 다른 대사과정을 갖고 있음은 바로 이 두 종에서의 체외성숙율 차이의 원인이 될 수 있을 것이며, 앞으로 두 종에서의 난자와 난구세포간의 상호작용 및 대사경로 등에 관한 심도 깊은 비교분석 연구를 통하여 돼지의 체외성숙율을 증가시킬 수 있는 배양 시스템의 개발이 가능할 것으로 기대된다.
Abstract
Contrast to mouse where its in vitro maturation rates are high without specific supplements or presence of the cumulus cells, there are some species, such as porcine, where its in vitro oocyte maturation rates are still very low. This comparative study was conducted to investigate the role of malate dehydrogenase (Mor2) during oocyte maturation by RNAi in the mouse and porcine. The Mor2 double-stranded RNA (dsRNA) was prepared species-specifically and microinjected into the cytoplasm of denuded germinal vesicle (GV) oocytes. Oocytes were cultured for 48h (porcine) and 16h (mouse) in M199 with 10% porcine follicular fluid, pyruvate, p-FSH, EGF, cystein, and estradiol-17We measured changes in oocyte morphology, maturation rates and mRNA levels after Mor2 RNAi. We confirmed gene sequence-specific knock down of Mor2 mRNA in both species after Mor2 RNAi. In contrast to our previous finding that mMor2 RNAi resulted in GV arrest in the mouse, we found that pMor2 RNAi resulted in MI arrest in denuded porcine oocytes (58%), but developed to MII (84.4%) in COCs. To determine whether this difference between mouse and porcine RNAi is due to differences in culture media, we cultured mouse oocytes in the M199 media for 16 hours after mMor2 RNAi. Mouse oocytes were developed to MII stage (62%) and there was no statistical difference compared to that of non-injected (76.8%) and buffer-injected (73.3%) control groups. Therefore, we concluded that the mouse and porcine oocytes are having different metabolic systems in relation to malate dehydrogenase for oocyte maturation. This could be a basis for differences in maturation rates in vitro in two species. Further scrutinized studies on the metabolic pathways would led us in finding better culture system to improve oocyte maturation rates in vitro, especially in more challenging species like the porcine.
참고문헌 (19)
- "Oocyte maturation", 18, pp.123~131 (2000)
- "Involvement of enzymes of amino acid metabolism and tricarboxylic acid cycle in bovine oocyte maturation in vitro", 126, pp.753~763 (2003)
- "Metabolism of porcine oocytes matured in vivo and in vitro", 64(Suppl 1), pp.138~ (2001)
- "Metabolism of radiolabeled glucose by mouse oocytes and oocyte-cumulus cell complexes", 60, pp.1446~1452 (1999)
- "Intercommunication between mammalian oocytes and companion somatic cells. Bioessays 13:569-574." (1991)
- "Mammalian oocyte growth and development in vitro", 44, pp.260~273 (1996)
- "Relationship between the developmental programs controlling nuclear and cytoplasmic maturation of mouse oocytes. Dev Biol 164", pp.19~ (1994)
- "Enhanced glycolysis after maturation of bovine oocytes in vitro is associated with increase developmental competence", 53, pp.19~26 (1999)
- "A comparative analysis of metabolism and viability in porcine oocytes during in vitro maturation.", 98, pp.72~96 (2007)
- "Mitochondrial malate-aspartate shuttle regulates mouse embryo nutrient consumption", 280, pp.18361~18367 (2005)
- "Developmental capacity, energy metabolism and ultrastructure of mature oocytes from prepubertal and adult sheep", 8, pp.1029~1037 (1996)
- "The relationships between the ATP content of preimplantation mouse embryos and their development in vitro during culture. J Reprod Fertil 35" (1973)
- "Changes in the metabolism of glucose glutamine and glycine during maturation of cattle oocytes in vitro. J Reprod Fertil 100" (1994)
- "Resumption of meiosis: mechanism involved in meiotic progression and its relation with developmental competence", 55, pp.1241~1254 (2001)
- "Oocyte metabolism predicts the development of cat embryos to blastocyst in vitro.", 56, pp.163~171 (2000)
- "ATP content of human oocytes and developmental potential and outcome after in vitro fertilization and embryo transfer", 10, pp.415~424 (1995)
- "Cumulus-oocyte complex interactions during oocyte maturation.", 236, pp.251~291 (2004)
- "Differentially expressed mRNA profiles between immature GV and Mature MII mouse oocytes.", 8, pp.35~42 (2004)
- "Role of cytosolic malate dehydrogenase in the oocyte maturation and embryo development.", 85, pp.193~203 (2006)