Effect of Deoxyribonucleic Acid Protamination on Fluorochrome Staining and in Situ Nick-Translation of Murine and Human Mature Spermatozoa1

Abstract

A major event in enhancing sperm chromatin stability is the replacement of the histones by protamines during spermiogenesis. In this study, we present results indicating that chromomycin A3 (CMA3) can be used to show protamine deficiency in sperm chromatin. Fixed chromatin of mature mouse spermatozoa showed high fluorescence after treatment with ethidium bromide (EB), but was completely unstained after treatment with CMA3. The same chromatin was found to be highly resistant to in situ nick-translation. In contrast, a substantial fraction of human spermatozoa were positive for CMA3. The accessibility of CMA3 to the DNA of human sperm was eliminated if the slides were previously treated with protamine in situ. This treatment did not affect the accessibility of EB to the chromatin. Individual human sperm samples revealed a substantial frequency of spermatozoa with endogenous nicks, which was found to be the same as the frequency of spermatozoa responding positively to CMA3 staining. Treatment of preparations with protamines prevented the identification of the endogenous nicks. These data as a whole suggest that CMA3 could represent a useful tool for the detection of protamine deficiency in sperm chromatin. Furthermore, confirmation of experiments relating sensitivity to nick translation and positivity to CMA3 may allow an indirect in situ visualization of nicked and partially denatured DNA, which could correlate with certain forms of male factor infertility.