CHROMOMYCIN A(3)-STAINING AS AN INDICATOR OF PROTAMINE DEFICIENCY ANDFERTILIZATION

Mature mammalian spermatozoa have a compact and stable nuclear structu re conferred by protamines instead of histones, which are present in a ll other cellular types. Chromomycin A(3) (CMA(3)) is a useful tool fo r the detection of protamine deficiency in sperm chromatin. The purpos e of this study was to correlate the percentage of spermatozoa stainin g positively for CMA(3) with sperm parameters and in-vitro fertilizati on of human oocytes. Spermatozoa were collected from 56 fertile and 18 infertile men, and washed twice in PBS, fixed in two changes of metha nol : acetic acid (3 : 1 v : v) spread on rinsed slides treated with A PES and dried. Twenty-four of the semen samples were subjected to both Percoll and swim-up, and were stained subsequently with CMA(3). CMA(3 )-stained spermatozoa were expressed as a percentage in a count of 200 spermatozoa. A substantial variation in the percentage of CMA(3)-stai ned cells was observed in ejaculated human spermatozoa, varying betwee n 8% and 77%. A strong negative correlation (r = -0.64, p < 0.001) was found between sperm count and the percentage of CMA(3)-stained sperma tozoa. No correlation was found between CMA(3)-stained spermatozoa and their motility, while excessive sperm morphological abnormalities wer e related positively to CMA(3)-staining. Spermatozoa in samples exhibi ting low (8-62%) CMA(3)-staining had significantly higher fertilizing rates in vitro than did samples exhibiting high (49-77%) CMA(3)-staini ng. The mean percentage of CMA(3)-stained spermatozoa after swim-up or Percoll preparation (26% vs 31%) did not differ significantly These r esults demonstrate a close relationship between CMA(3)-staining, ferti lization and sperm count, and suggest potential application of this ma rker for the prediction of sperm quality and fertilizing capacity.