Page 59 - PRECONGRESS COURSE 01

TUNEL assay. TUNEL-positive nuclei (with double-strand nuclear DNA fragmentation) of spermatozoa as represented by the intense (A) and dull (B) Texas red fluorescence in the nuclear region. The healthy nuclei (without DNA fragmentation) are stained blue with DAPI (C) used as counterstain.
Angelopoulou et al., Reprod Biol Endocrinol, 5, 36, 2007
Sperm Chromatin Structure Assay – SCSA
0 100
Evenson et al., 1980
Denatured ss DNA: red fluorescence
100
200 cells /minute
5000 cells analysed/sample
Detection of DNA damage: methods
Single‐cell gel electrophoresis (Comet assay)
TUNEL
Acridine Orange Technique (AOT)
Sperm Chromatin Structure Assay (SCSA) In situ nick translation
Sperm Chromatin Dispersion Test (SCD)
Acid Aniline blue
Chromomycin A3 – CMA3 .......
 Evaluates DNA integrity (single & double‐ strand breaks)
 Evaluates DNA fragmentation (single & double‐strand breaks)
 Distinguish between single & double‐ stranded
 Acid DNA denaturation
 Single‐strand DNA breaks
 Examines susceptibility of sperm DNA to acid denaturation
 Stains lysine residues from persisting histones
 Indirect visualization of nicked denatured DNA
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PRECONGRESS COURSE 01 I GENEVA, SWITZERLAND – 2 JULY 2017 59
Green Fluorescence