DNA breakage detection-FISH (DBD-FISH) in human spermatozoa: technical variants evidence different structural...

Area of Science:

• Spermatozoa research
• Genetics
• Molecular biology

Background:

• Sperm DNA integrity is crucial for male fertility and successful reproduction.
• Assessing DNA damage in spermatozoa requires sensitive and specific techniques.
• X-ray irradiation is a known genotoxic agent that can induce DNA damage.

Purpose of the Study:

• To evaluate two variations of in situ DNA breakage detection-FISH (DBD-FISH) for whole-genome DNA breakage detection in human spermatozoa.
• To investigate the effect of X-ray irradiation on sperm DNA integrity using DBD-FISH.
• To establish a dose-response relationship for X-ray-induced DNA damage in sperm chromatin.

Main Methods:

• Human spermatozoa were subjected to X-ray irradiation (80 Gy) or left non-irradiated.
• Two DBD-FISH protocols were applied: 1) alkaline unwinding followed by lysis, and 2) lysis followed by alkaline unwinding/denaturation.
• Fluorescence intensity and signal distribution were analyzed to quantify DNA breakage and chromatin structure.

Main Results:

• The lysis-before-unwinding method revealed two sperm subpopulations with varying degrees of DNA breakage.
• X-ray irradiation significantly increased DNA breakage, indicated by enhanced fluorescence intensity and signal dispersion.
• A linear dose-response was observed, with 30 Gy being the lowest dose to detect X-ray-induced damage.

Conclusions:

• DBD-FISH is effective for in situ detection of DNA breakage in human spermatozoa.
• The technique can differentiate between irradiated and non-irradiated sperm and reveal chromatin structural alterations.
• DBD-FISH provides a sensitive method for assessing DNA damage and establishing dose-response relationships in sperm chromatin.