Oosit Maturasyonu Sürecinde Global DNA Metilasyonunun Değişimi

Öz

Amaç: Bu çalışmada, global DNA metilasyonunun Germinal Vezikül (GV) aşamasından Metafaz II (MII) aşamasına kadar olan oosit maturasyonu sürecinde değişim gösterip göstermediğinin ortaya konulması amaçlanmıştır. Yöntem: Bu çalışmada, 4 haftalık Balb/C farelerinin GV ve in vivo MII oosit evreleri arasındaki global DNA metilasyonu farkı immünofloresan yöntemi kullanılarak incelendi. Bu amaçla GV ve MII aşamasındaki oositlerde 5-metil sitozin (5mC) işaretlemesi sonrası Zeiss LSM-880 Airyscan konfokal mikroskopta alınan optik kesitlerinden elde edilen görüntülerden Image-J yazılımı kullanılarak hesaplanan sinyal yoğunlukları değerlendirildi. Bulgular: Global DNA metilasyonu, 5-metil sitozin (5mC) işaretlemesi sonrası değerlendirildiğinde, GV aşamasındaki oositlerde, çekirdek bölgesinde gözlemlenirken, MII aşamasındaki oositlerde metafaz plağına uygun lokasyonda, olduğu izlendi. Global DNA metilasyonunun göreceli sinyal yoğunluğu değerlendirildiğinde; MII aşamasında GV aşamasına göre 3,2 katlık istatistiki olarak anlamlı bir azalma olduğu saptandı. Bu azalışın birinci mayoz sonrası DNA miktarındaki azalmaya bağlı olup olmadığı için yapılan hesaplamalar da bunun sadece DNA miktarındaki azalmadan kaynaklanmadığını gösterdi. Sonuç: Oositlerde, GV aşamasına kıyasla MII evresinde global DNA metilasyon seviyesinin üç kattan daha fazla azalmış olması, fertilizasyon öncesi oositteki DNA metilasyonunun çeşitli mekanizmalarla kontrol edildiğini ve bunun fertilizasyon dinamiğinde önemli olabileceğini göstermiştir.

Anahtar Kelimeler

• Epigenetik
• Global DNA metilasyonu
• Oogenez
• Oosit

Alteration of Global DNA Methylation in the Oocyte Maturation Process

Abstract

Objective: In this study, it was aimed to reveal whether alteration of global DNA methylation occurs or not during the oocyte maturation from Germinal Vesicle (GV) to Metaphase II (MII). Method: The difference in global DNA methylation between the GV and in vivo MII oocyte stages of 4-week-old Balb/C mice was examined by using the immunofluorescence method. For this purpose, oocytes at GV and MII stages were labeled with anti-5-methyl cytosine (5mC) antibody and images from optical sections taken with Zeiss LSM-880 Airyscan confocal microscope were obtained. Signal intensities of the images were digitalized and calculated by using Image-J software. Result: When the global DNA methylation was evaluated after 5-methyl cytosine (5mC) labeling, 5mC signals were observed in nucleus of GV stage oocytes whereas, at metaphase plate of MII stage. Relative signal intensities of global DNA methylation were evaluated, and statistically significant (p<0.0001) 3.2-fold decrease was found at MII stage compared to GV. Calculations aiming to reveal whether this significant decrease depended on the reduction in the amount of DNA after meiosis I or not, showed that the decrease in global DNA methylation may not be caused by meiosis itself. Conclusion: The result that the global DNA methylation level was decreased more than three times at the MII stage compared to the GV stage oocytes suggests that DNA methylation is controlled by various mechanisms, and this may have vital role fertilization dynamics.

Keywords

• Epigenetic
• Global DNA methylation
• Oogenesis
• Oocyte

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