Oosit Maturasyonu Sürecinde Global DNA Metilasyonunun Değişimi

Year 2024, Volume: 10 Issue: 1, 47 - 52, 01.01.2024

Gözde Şükür Nazlıcan Bozdemir Özgür Çınar

https://doi.org/10.53394/akd.1095184

Abstract

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.

Keywords

Epigenetik, Global DNA metilasyonu, Oogenez, Oosit

Supporting Institution

TÜBİTAK

Project Number

120S175

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

Project Number

120S175

References

• 1. Uysal F, Ozturk S. DNA Methyltransferases in Mammalian Oocytes. Results Probl Cell Differ. 2017;63:211-22.
• 2. Reik W, Walter J. Genomic imprinting: parental influence on the genome. Nat Rev Genet. 2001;2(1):21-32.
• 3. Turek-Plewa J, Jagodzinski PP. The role of mammalian DNA methyltransferases in the regulation of gene expression. Cell Mol Biol Lett. 2005;10(4):631-47.
• 4. Araujo FD, Croteau S, Slack AD, Milutinovic S, Bigey P, Price GB, et al. The DNMT1 target recognition domain resides in the N terminus. J Biol Chem. 2001;276(10):6930-6.
• 5. Posfai J, Bhagwat AS, Posfai G, Roberts RJ. Predictive motifs derived from cytosine methyltransferases. Nucleic Acids Res. 1989;17(7):2421-35.
• 6. Bestor TH. The DNA methyltransferases of mammals. Hum Mol Genet. 2000;9(16):2395-402.
• 7. Chuang LS, Ian HI, Koh TW, Ng HH, Xu G, Li BF. Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1. Science. 1997;277(5334):1996-2000.
• 8. Easwaran HP, Schermelleh L, Leonhardt H, Cardoso MC. Replication-independent chromatin loading of Dnmt1 during G2 and M phases. EMBO Rep. 2004;5(12):1181-6.
• 9. Pan Z, Zhang J, Li Q, Li Y, Shi F, Xie Z, et al. Current advances in epigenetic modification and alteration during mammalian ovarian folliculogenesis. J Genet Genomics. 2012;39(3):111-23.
• 10. Kageyama S, Liu H, Kaneko N, Ooga M, Nagata M, Aoki F. Alterations in epigenetic modifications during oocyte growth in mice. Reproduction. 2007;133(1):85-94.
• 11. Uysal F, Akkoyunlu G, Ozturk S. Dynamic expression of DNA methyltransferases (DNMTs) in oocytes and early embryos. Biochimie. 2015;116:103-13.
• 12. Uysal F, Ozturk S, Akkoyunlu G. DNMT1, DNMT3A and DNMT3B proteins are differently expressed in mouse oocytes and early embryos. J Mol Histol. 2017;48(5-6):417-26.
• 13. Lucifero D, Mann MR, Bartolomei MS, Trasler JM. Gene-specific timing and epigenetic memory in oocyte imprinting. Hum Mol Genet. 2004;13(8):839-49.
• 14. Moore LD, Le T, Fan G. DNA methylation and its basic function. Neuropsychopharmacology. 2013;38(1):23-38.
• 15. Saitou M, Kagiwada S, Kurimoto K. Epigenetic reprogramming in mouse pre-implantation development and primordial germ cells. Development. 2012;139(1):15-31.
• 16. Yang X, Smith SL, Tian XC, Lewin HA, Renard JP, Wakayama T. Nuclear reprogramming of cloned embryos and its implications for therapeutic cloning. Nat Genet. 2007;39(3):295-302.
• 17. Shirane K, Toh H, Kobayashi H, Miura F, Chiba H, Ito T, et al. Mouse oocyte methylomes at base resolution reveal genome-wide accumulation of non-CpG methylation and role of DNA methyltransferases. PLoS Genet. 2013;9(4):e1003439.
• 18. Smallwood SA, Tomizawa S, Krueger F, Ruf N, Carli N, Segonds-Pichon A, et al. Dynamic CpG island methylation landscape in oocytes and preimplantation embryos. Nat Genet. 2011;43(8):811-4.
• 19. Smallwood SA, Kelsey G. De novo DNA methylation: a germ cell perspective. Trends Genet. 2012;28(1):33-42.
• 20. Saadeh H, Schulz R. Protection of CpG islands against de novo DNA methylation during oogenesis is associated with the recognition site of E2f1 and E2f2. Epigenetics Chromatin. 2014;7:26.
• 21. Costello KR, Leung A, Trac C, Lee M, Basam M, Pospisilik JA, et al. Sequence features of retrotransposons allow for epigenetic variability. Elife. 2021;10.
• 22. Sendzikaite G, Kelsey G. The role and mechanisms of DNA methylation in the oocyte. Essays Biochem. 2019;63(6):691-705.
• 23. Yu B, Dong X, Gravina S, Kartal O, Schimmel T, Cohen J, et al. Genome-wide, Single-Cell DNA Methylomics Reveals Increased Non-CpG Methylation during Human Oocyte Maturation. Stem Cell Reports. 2017;9(1):397-407.
• 24. Hwang GH, Hopkins JL, Jordan PW. Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II. J Vis Exp. 2018(132).
• 25. Guo H, Zhu P, Yan L, Li R, Hu B, Lian Y, et al. The DNA methylation landscape of human early embryos. Nature. 2014;511(7511):606-10.
• 26. Yuan P, Guo Q, Guo H, Lian Y, Zhai F, Yan Z, et al. The methylome of a human polar body reflects that of its sibling oocyte and its aberrance may indicate poor embryo development. Hum Reprod. 2021;36(2):318-30.