Extracting and analyzing micronuclei from mouse two-cell embryos fertilized with freeze-dried spermatozoa

Area of Science:

• Reproductive biology
• Cytogenetics
• Developmental biology

Background:

• Abnormal chromosome segregation (ACS) in early embryos is a primary cause of miscarriages.
• Understanding the mechanisms behind ACS is crucial for improving pregnancy outcomes.
• Current limitations in analyzing segregated chromosomes hinder research into ACS causes.

Purpose of the Study:

• To develop a method for extracting micronuclei from mouse embryo blastomeres for detailed analysis.
• To investigate the composition and characteristics of micronuclei in embryos exhibiting ACS.
• To elucidate the underlying mechanisms contributing to abnormal chromosome segregation.

Main Methods:

• Micronuclei were extracted from mouse embryo blastomeres using micromanipulation after cytoskeleton depolymerization (cytochalasin B and colcemid).
• DNA sequencing was performed on extracted micronuclei to analyze chromosome content.
• Cytoskeletal interactions (DNA cross-links, tubulin binding) and chromosomal regions (major satellite regions) of micronuclei were examined.

Main Results:

• Micronuclei were successfully extracted from blastomeres of ACS embryos.
• Extracted micronuclei typically contained single, randomly selected chromosomes or chromosome fragments, not whole chromosomes.
• Specific associations of micronuclei with DNA cross-links, tubulin, and major satellite regions were observed.

Conclusions:

• The developed method enables detailed analysis of micronuclei, offering insights into ACS mechanisms.
• Findings suggest that ACS in embryos involves the mis-segregation of random chromosome portions.
• This research facilitates a deeper understanding of the causes of embryonic aneuploidy and miscarriage.