멜라토닌이 생쥐 미성숙 난자의 체외성숙과 난구세포의 세포자연사에 미치는 영향
Effect of melatonin on the maturation of mouse germinal vesicle(GV)-stage oocytes and apoptosis of cumulus cells in vitro
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멜라토닌(N-acetyl-5-methoxytryptamine)은 포유동물의 뇌의 송과선에서 분비되는 호르몬으로 수면과 생체리듬 등을 조절하고 난소기능과 번식에도 영향을 미친다. 또한 강력한 scavenger로서 항산화제의 역할을 한다. 이 연구의 목적은 멜라토닌이 생쥐 난구세포-핵낭(germinal vesicle, GV) 시기 난자 복합체의 체외성숙에 미치는 영향을 알아보는 것이다. 3 주령의 ICR 암컷 생쥐의 난소에서 회수된 난자-난구세포 복합체를 0, 0.1 nM, 10 nM, 1000 nM 의 멜라토닌이 첨가된 배양액에서 18 시간 동안 배양하고, 제1 극체의 방출 여부를 확인하여 성숙율을 확인하였다. 체외성숙 후 TUNEL assay와 면역효소흡착법, 실시간역전사연쇄중합법을 통하여 세포자연사(apoptosis)와 난구세포 내 멜라토닌 수용체(Mtnr1)의 mRNA 발현량을 측정하였다. 체외성숙 배양액에 멜라토닌을 첨가한 결과 핵낭시기 난자의 핵 성숙율이 유의하게 증가하였으며, 멜라토닌을 1000 nM 첨가한 군의 성숙율이 가장 높게 나타났다. 체외성숙된 난자에서는 세포자연사가 나타나지 않았으나 난구세포에서는 관찰되었으며 1000 nM을 첨가하여 배양한 군의 난구세포는 유의하게 낮은 세포자연사를 나타냈다. 그리고 1000 nM의 멜라토닌을 첨가한 군의 난구세포에서 멜라토닌 수용체의 mRNA가 대조군에 비해 낮게 발현되었다. 이상의 결과를 종합하면 체외성숙 배양액에 첨가된 멜라토닌은 난구세포의 세포자연사를 줄여줌으로써 생쥐 미성숙 난자의 체외성숙을 향상시키는 역할을 하는 것으로 사료된다.
Abstract
Melatonin(N-acetyl-5-methoxytryptamine), a major hormone of pineal gland in vertebrates, is known to be associated with regulation of the dynamic physiological functions in general and has some functions on reproduction in the ovarian follicles in particular. And its antioxidant properties as a scavenger are also reported. The aim of this study was to investigate the effect of melatonin on the in vitro maturation of mouse germinal vesicle(GV)-stage oocytes. Oocyte maturation, apoptosis, and mRNA expression of melatonin receptor were analyzed in the cumulus cell-enclosed oocytes(CEOs) cultured with melatonin for 18 h. The CEOs were obtained from 3 wk-old ICR female mice cultured in media with 0, 0.1 nM, 10 nM, or 1000 nM melatonin for 18 h. And then the extrusion of the first polar body was assessed to evaluate the maturation rate. The apoptosis and mRNA expression of melatonin receptor(Mtnr1-a and Mtnr1-b) in cumulus cells of each group were measured by TUNEL assay, ELISA, and real time RT-PCR after in vitro maturation(IVM). The addition of melatonin in the IVM medium significantly improved nuclear maturation of the mouse GV oocytes and the highest maturation rate were obtained from the group treated with 1000 nM melatonin. Apoptosis was not detected in IVM oocytes, but detected in cumulus cells. And cumulus cells treated with 1000 nM melatonin exhibited significantly lower apoptosis. In the group treated with 1000 nM melatonin, the expression of melatonin receptor mRNA was decreased in CEOs. In conclusion, melatonin has a potentially important role for regulating oocyte maturation and reduces the apoptosis of cumulus cells in vitro.
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