The Effect of Copper Supplementation on in vitro Maturation of Porcine Cumulus-Oocyte Complexes and Subsequent Developmental Competence after Parthenogenetic Activation

국문 초록 (Abstract)

Copper (Cu) 이온은 강한 산화 환원 활성을 갖고 있어서 세포호흡, 라디칼 해독과정, 철 대사과정에 관여하는 여러 효소들의 보조인자로 작용한다. 이러한 Cu의 중요성에도 불구하고 아직까지 Cu가 돼지 난자의 성숙에 미치는 영향에 대한 연구는 진행된 바가 없다. 본 논문에서는, 돼지 난자의 체외성숙 기간동안 Cu를 첨가하는 것이 난자의 핵성숙과 세포질 내 글루타티온 및 활성산소의 수준, 난구세포의 확장에 미치는 효과와 세포사멸, 발달 능, 그리고 Cu 수송 관련된 유전자들의 발현양의 변화를 알아보았다. 뿐 만 아니라 단성생식 후 배아발달에 미치는 영향 또한 확인하였다.
돼지 난자의 체외성숙 기간 동안 Copper(II) Chloride Dihydrate를 성숙 배지에 첨가하였고 이 때의 농도는 0, 0.7, 1.4, 2.8 μg/mL로 설정하였다. 그 결과, Cu를 첨가한 그룹에서 난자의 metaphase II rate가 증가하였다. 한편, 1.4 μg/mL Cu 그룹에서의 글루타티온 수준이 유의적으로 증가하였고, 활성산소종은 모든 농도의 실험군에서 증가하였다. 게다가 난구세포 확장과 관련한 유전자인 Has2의 mRNA 발현이 모든 농도의 실험군에서 증가하는 것으로 나타났다. 난구세포 확장 지표는 대조군 대비 0.7과 1.4 μg/mL 농도의 실험군에서 유의적으로 높았다. 흥미롭게도 0.7 μg/mL Cu 그룹에서 항산화 효소인 SOD1와 발달능 관련 유전자인 PCNA, Zar1, NPM2의 mRNA 발현이 대조군 대비 유의적으로 증가하였다. 세포 사멸율은 난구세포와 난자에서 모두 Cu가 첨가된 실험군에서 감소하는 것으로 나타났다. 마지막으로 0.7 μg/mL Cu를 IVM 동안 첨가한 그룹의 분할율과 배반 포 형성율이 대조군 대비 유의적으로 증가했음을 확인하였다. 결론적으로, 체외성숙 배지에 적절농도의 Cu를 첨가하는 것이 돼지 난자에 존재하는 SOD1 효소의 활성과 유전자 발현을 증가시킴으로써 산화 스트레스와 세포사멸을 감소시킨다. 이러한 효과는 돼지 난자 성숙과 배아 발달을 향상시킬 수 있다고 생각된다.

다국어 초록 (Multilingual Abstract)

Copper (Cu) ions, having redox activity, act as cofactors of enzymes related to respiration, radical detoxification, and iron metabolism. However, despite the known importance of Cu, there are no previous studies regarding its effect on the maturation of porcine oocytes. In this study, copper (II) chloride dihydrate (CuCl2·2H2O) was supplemented into the in vitro maturation (IVM) media of porcine oocytes, at concentrations 0 (control), 0.7, 1.4, and 2.8 μg/mL. The effects of this on nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, cumulus expansion, and the levels of various genes related to apoptosis, developmental competence, Cu transport, and embryonic development were examined following parthenogenetic activation (PA). It was noted that Cu supplementation significantly increased the number of oocytes at the metaphase II stage. Further, the 1.4 μg/mL Cu group showed significantly higher intracellular GSH levels than those in the control group. However, Cu supplementation increased intracellular ROS levels regardless of their concentration. Additionally, the mRNA levels of Has-2, the cumulus cell expansion-related gene, were higher in all the Cu-treated groups than in the control group. Cumulus cell expansion index was higher in the 0.7 and 1.4 μg/mL Cu groups than in the others. In the 0.7 μg/mL Cu group, the mRNA expression levels of PCNA, Zar1, and NPM2, which are related to developmental competence, were significantly higher compared to those in the control group. Moreover, increased levels of Sod1 transcript correlated with anti-oxidative response were observed in the 0.7 and 1.4 μg/mL Cu groups. The apoptosis rate in Cu-treated cumulus cells and oocytes was decreased compared to that in the corresponding control groups. In the evaluation of subsequent embryonic development after PA, the 0.7 μg/mL Cu group showed significantly improved cleavage and blastocyst formation rate compared to the control group. In conclusion, Cu supplementation at an appropriate concentration in IVM medium reduced oxidative stress and apoptosis in oocytes by increasing SOD1 activity and expression. These effects may improve porcine oocyte maturation and subsequent embryonic development. Our findings are relevant to improving the efficiency of porcine oocyte maturation.

목차 (Table of Contents)

• Ⅰ. INTRODUCTION ····································································· 1
• Ⅱ. MATERIALS AND METHODS ··················································· 4
• 1. Chemicals ······························································································ 4
• 2. Oocytes collection and in vitro maturation ························································ 4
• 3. Assessment of nuclear maturation ·································································· 5
• 4. Estimation of cumulus cell expansion index ····················································· 5
• 5. Measurement of intracellular GSH and ROS levels ·············································· 5
• 6. Gene expression analysis by quantitative real-time polymerase chain reaction (qRT-PCR) 6
• 7. Parthenogenetic activation and in vitro culture··················································· 9
• 8. Embryo evaluation and total cell count ···························································· 9
• 9. Experimental design ·················································································10
• 10. Statistical analysis ···················································································11
• Ⅲ. RESULTS·············································································· 12
• 1. Effect of Cu supplementation during IVM on oocyte nuclear maturation ·····················12
• 2. Effect of Cu supplementation during IVM on intracellular GSH and ROS levels ············14
• 3. Effect of Cu supplementation during IVM on cumulus cell expansion ························16
• 4. Contribution of Cu supplementation during IVM to expression levels of developmental-related genes in cumulus cells and oocytes···························································· 18
• 5. Contribution of Cu supplementation during IVM to expression levels of Cu transport-related genes in cumulus cells and oocytes··································································· 21
• 6. Effect of Cu supplementation during IVM on embryonic development after PA············· 23
• Ⅳ. DISCUSSION ········································································· 26
• Ⅴ. REFERENCES ···························································· 30
• SUMMARY IN KOREAN ······························································ 36