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.

Human Cervical Mucus Can Act in Vitro as a Selective Barrier Against Spermatozoa Carrying Fragmented Dna and Chromatin Structural Abnormalities

“…In this connection, it must be remembered that several studies have demonstrated that a variable but consistent fraction of human spermatozoa exhibits abnormalities in chromatin structure, not always detected using classical parameters of evaluation (12)(13)(14)(15)(16)(17)(18)(33)(34)(35). Moreover the existence of a close relationship between chromatin and DNA structural abnormalities and male infertility has been well pointed out.…”

“…During the last decade, a number of studies have shown that variable but consistent fractions of human spermatozoa not always detectable on the basis of classic morphological parameters, contain serious chromatin structural abnormalities and DNA strand breaks (12)(13)(14)(15)(16)(17)(18). The existence of an association between sperm chromatin malformation and male infertility has been repeatedly observed in humans and structural chromatin abnormalities have been found to exert negative effects both on the percentage of fertilization and on embryonic development (17,(19)(20)(21).…”

“…CMA 3 is a useful tool for rapid screening of subfertility in man, as it seems to allow an indirect visualization of protamine-deficient DNA (14,15,22,23). This molecule has been shown to bind as a Mg 2+ -coordinated dimer at the minor groove of GC-rich DNAs and to induce a conformational perturbation in the double helix resulting in a wider and shallower minor groove at its binding site (24,25).…”

CASA derived human sperm abnormalities: correlation with chromatin packing and DNA fragmentation

“…For staining, 20 μl of CMA3 stain was pipetted onto the slide, covered with glass coverslip (22×22 mm) and incubated at room temperature for 20 min. Slides were rinsed in PBS and mounted with buffered glycerol [17]. Sperm head staining pattern was observed under fluorescent microscope with an oil immersion objective(Zeiss microscope, Axioskope 2 plus; Carl Zeiss, Gottingen, Germany) at 63 x magnification with appropriate filters (460-470 nm).…”

Changes in spermatozoal chromatin packaging and susceptibility to oxidative challenge during aging

“…Our flow cytometry-based chromomycin A3 (CMA3) quantification was adapted from the slide-based method (47). The CMA3 was dissolved in McIlvaine's buffer (17 mL of 0.1 mol/L citric acid mixed with 83 mL of 0.2 mol/L Na 2 HPO 4 and 10 mmol/L MgCl 2 , pH 7.0) to a concentration of 0.25 mg/mL.…”