돼지 체외성숙란 및 수정란에서 미토콘드리아 특이적 Rhod-2 발현 양상 확인
Confirmation of Mitochondrial Specific Rhod-2 Expression Patterns in Matured and Fertilized Porcine Oocytes In Vitro
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초록
미토콘드리아는 세포질 칼슘 항상성 및 ATP생산에 중요한 역할을 하는 세포 소기관으로 이러한 미토콘드리아의 기능은 성숙과 수정 그리고 배 발달에 매우 중요한 역할을 한다. 미토콘드리아 칼슘 축적은 기능장애를 일으킨다. 그러나 돼지 체외성숙란 및 수정란에서 미토콘드리아 칼슘 변화의 관련성에 관한 연구는 보고된 적이 없다. 본 연구의 목적은 미토콘드리아 칼슘 지시자로 알려진 Rhod-2 염색을 이용하여 성숙란 및 수정란에서 미토콘드리아 칼슘 축적의 변화를 확인하였다. 형태학적 모습의 기준을 통해 난구세포의 세포층과 세포질의 균질도를 바탕으로 G1과 G2로 나누어서 체외성숙을 진행하였다. 이후 두 그룹에서 핵 성숙율을 비교하였을 때, G2가 G1에 비해 낮게 나타났다(p<0.001). 돼지 체외성숙란 및 수정란에서 평균적인 Rhod-2 spot의 수는 G1보다 G2에서 더 많이 나타났다(6시간째 체외수정란: p<0.05). 다음으로 Rhod-2 spot수에 따른 난모세포의 비율을 확인하기 위해 Rhod-2 spot의 수를 4개의 군(n<10, 10≤n<20, 20≤n<30, 그리고 30≤n)으로 나누어 해당 난모세포의 비율을 확인하였다. 체외성숙란 및 체외수정란 모두 G1이 G2에 비해 10개 미만(n<10)인 Rhod-2 spot의 수를 가지는 난모세포가 많았으며, 체외수정란에서는 유의적으로 높았다(p<0.05). 마지막으로 체외성숙란 및 수정란에서 Rhod-2 intensity값을 측정하여 두 그룹을 비교하였을 때, G2가 G1에 비해 유의적으로 높은 것을 확인 할 수 있었다(성숙란; p<0.001 그리고 수정란; p<0.05). 본 연구의 결과를 토대로 돼지에서 미성숙 난포란의 형태학적인 품질은 체외성숙 및 체외수정 과정 동안 미토콘드리아 내 칼슘 축적과 관련이 있음을 확인하였다.
Abstract
Mitochondria plays an important role in regulating cytoplasmic calcium (Ca2+) homeostasis for ATP production during early embryonic development in mammals. Accumulation of mitochondrial Ca2+ induces mitochondrial dysfunctions. However, its relevance with change of mitochondrial Ca2+ in matured and fertilized oocytes has not been reported. The aim of this study was to identify the changes of mitochondrial Ca2+ level by using Rhod-2 staining analysis which is known as mitochondrial Ca2+ indicator in matured and fertilized oocytes in vitro. Here, we divided into two groups (Group 1: G1, compact cumulus cells and homogeneous cytoplasm and Group 2: G2, non-compact cumulus cells and heterogeneous cytoplasm) according to the morphological difference of COCs. Meiotic maturation rate decreased in G2 compared to G1 (p<0.001). Average number of Rhod-2 spots in matured and fertilized oocytes was higher in G2 than in G1 (6 h after IVF: p<0.05). We also investigated the detailed expression pattern of Rhod-2 spots dividing it into four parts (n<10, 10≤n< 20, 20≤n<30, and 30≤n). The ratio of fertilized oocytes with less than 10 Rhod-2 spots was higher in G1 (p<0.05). In addition, the Rhod-2 intensity in matured and fertilized oocytes in G2 were all higher than that in G1 (matured oocytes; p<0.001 and zygotes; p<0.05). Based on these results, we confirmed that morphological difference of immature oocytes was correlated with mitochondrial Ca2+ accumulation during porcine oocyte maturation and fertilization in vitro.
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