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Author Spotlight: Understanding DNA Damage Response in Mammalian Oocytes and Preimplantation Embryos
Published on: June 23, 2023
DNA double-strand breaks disrupted the spindle assembly in porcine oocytes
HaiYang Wang1, YiBo Luo1, Ming-Hui Zhao1
1Department of Animal Sciences, Chungbuk National University, Naesudong-ro, Seowon-gu, Cheongju-si, Chungcheongbuk-do, Korea.
Abstract:
We used etoposide (25-100 µg/mL) to induce DNA double-strand breaks (DSBs) in porcine oocytes at the germinal vesicle (GV) stage to determine how such damage affects oocyte maturation. We observed that DNA damage did not delay the rate of germinal vesicle breakdown (GVBD), but did inhibit the final stages of maturation, as indicated by the failure to extrude the first polar body. Oocytes with low levels of DSBs failed to effectively activate ataxia telangiectasia-mutated (ATM) kinase, while those with severe DNA DSBs failed to activate checkpoint kinase 1 (CHK1)--the two regulators of the DNA damage response pathway--indicating that porcine oocytes lack an efficient G2/M phase checkpoint. DSBs induced spindle defects and chromosomal misalignments, leading to the arrest of these oocytes at meiotic metaphase I. The activity of maturation-promoting factor also did not increase appropriately in oocytes with DNA DSBs, although its abundance was sufficient to promote GVBD and chromosomal condensation. Following parthenogenetic activation, embryos from etoposide-treated oocytes formed numerous micronuclei. Thus, our results indicate that DNA DSBs do not efficiently activate the ATM/CHK1-dependent DNA-damage checkpoint in porcine oocytes, allowing these DNA-impaired oocytes to enter M phase. Oocytes with DNA damage did, however, arrest at metaphase I in response to spindle defects and chromosomal misalignments, which limited the ability of these oocytes to reach meiotic metaphase II.
Insights
Porcine oocytes with DNA double-strand breaks (DSBs) fail to activate crucial DNA damage checkpoints, leading to maturation arrest and developmental issues in resulting embryos. This highlights a compromised DNA damage response in oocyte maturation.
Area of Science:
- Reproductive Biology
- Cellular Biology
- Genetics
Background:
- Oocyte maturation is critical for successful fertilization and embryonic development.
- The DNA damage response (DDR) pathway is essential for maintaining genomic integrity during cell division.
- Understanding DDR in oocytes is crucial for addressing infertility and developmental abnormalities.
Purpose of the Study:
- To investigate the impact of induced DNA double-strand breaks (DSBs) on porcine oocyte maturation.
- To determine the activation efficiency of key DNA damage response regulators, ATM and CHK1, in response to DSBs.
- To elucidate the consequences of DSBs on meiotic progression and subsequent embryonic development in pigs.
Main Methods:
- Porcine oocytes at the germinal vesicle (GV) stage were treated with etoposide to induce DSBs.
- Oocyte maturation, germinal vesicle breakdown (GVBD), and polar body extrusion were monitored.
- Activation of ataxia telangiectasia-mutated (ATM) kinase and checkpoint kinase 1 (CHK1) was assessed.
- Spindle organization, chromosome alignment, and maturation-promoting factor (MPF) activity were analyzed.
- Parthenogenetic activation was performed on treated oocytes to evaluate embryonic development.
Main Results:
- Induced DSBs did not delay GVBD but inhibited final maturation, evidenced by failed first polar body extrusion.
- Low levels of DSBs impaired ATM activation, while severe DSBs inhibited CHK1 activation, indicating a deficient G2/M checkpoint.
- DSBs led to spindle defects and chromosomal misalignments, causing arrest at meiotic metaphase I.
- MPF activity did not increase appropriately, despite sufficient levels for GVBD and condensation.
- Embryos derived from etoposide-treated oocytes exhibited micronuclei formation after parthenogenetic activation.
Conclusions:
- Porcine oocytes exhibit an inefficient ATM/CHK1-dependent DNA-damage checkpoint, allowing DNA-impaired oocytes to enter M phase.
- Spindle defects and chromosomal misalignments resulting from DSBs cause metaphase I arrest, limiting progression to metaphase II.
- The study reveals significant defects in the DNA damage response during porcine oocyte maturation, impacting developmental potential.
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