Numerous studies have shown the presence of DNA strand breaks in human ejaculated spermatozoa. The nature of this nuclear anomaly and its relationship to patient etiology is however poorly understood. The aim of this study was to investigate the relationship between nuclear DNA damage, assessed using the TUNEL assay and a number of key apoptotic markers, including Fas, Bcl-x, and p53, in ejaculated human spermatozoa from men with normal and abnormal semen parameters. We also determined the nature of the DNA damage by examining the percentage of ejaculated spermatozoa exhibiting DNA damage using the comet assay and by challenging sperm chromatin to attack by micrococcal nuclease S7 and DNase I. We show that TUNEL positivity and apoptotic markers do not always exist in unison; however, semen samples that had a low sperm concentration and poor morphology were more likely to show high levels of TUNEL positivity and Fas and p53 expression. In addition, the DNA damage in ejaculated human sperm is represented by both single- and double-stranded DNA breaks, and access to the DNA is restricted by the compacted nature of ejaculated spermatozoa. This DNA protection is poorer in men with abnormal semen parameters. We propose that the presence of DNA damage is not directly linked to an apoptotic process occurring in spermatozoa and arises due to problems in the nuclear remodeling process. Subsequently, the presence of apoptotic proteins in ejaculated spermatozoa may be linked to defects in cytoplasmic remodeling during the later stages of spermatogenesis.
The formation of mature spermatozoa is a unique process involving a series of meioses and mitoses, changes in cytoplasmic architecture, replacement of somatic cell-like histones with transition proteins and the final addition of protamines, leading to a highly packaged chromatin (Kumaroo et al., 1975; Goldberg et al., 1977; Poccia, 1986). Mature mammalian spermatozoa contain high percentages of protamines, for example, human and mouse sperm nuclei contain more than 85% and 95% protamines in their nucleoprotein component, respectively (Gatewood et al., 1987; Bellvé et al., 1988; Debarle et al., 1995). In mice, protamines allow the mature sperm nuclei to adopt a volume 40 times less than that of normal somatic nuclei (Ward and Coffey, 1991).In many mammals, spermatogenesis leads to the production of spermatozoa that appear highly homogeneous in form and function. However, in humans, it is apparent that there are large differences between the form and function of spermatozoa among males and within the ejaculate of an individual. Classically, analyses of the differences in spermatozoa among men have been measured by examining sperm concentration, motility and morphology. Although this analysis gives a broad clinical insight, it does not explain why and where differences originate.For a number of years, many laboratories have concentrated on analysing differences in sperm populations by examining chromatin structure. These studies have shown that the major factor affecting chromatin packaging in ejaculated human spermatozoa appears to be linked to faulty or incomplete protamine deposition during spermiogenesis. In numerous studies, spermatozoa from infertile men were found to exhibit sperm chromatin anomalies related to the deposition of protamines (Balhorn, 1982; Foresta et al., 1992; Belokopytova et al., 1993; de Yebra et al., 1993). These anomalies range from altered ratios of protamine 1 and 2 (Balhorn et al., 1988; Belokopytova et al., 1993) to the complete absence of protamine (de Yebra et al., 1993).During the 1990s, several groups have analysed the sperm nucleus further by examining the integrity of the DNA in mature human spermatozoa. This review summarizes the accumulated knowledge concerning DNA damage in mature human spermatozoa and how this may be related to male infertility. Furthermore, we will speculate on how and why DNA damage may originate in certain males and how it influences the genetic project of a mature spermatozoon. DNA packaging in mammalian spermatozoaThe chromatin contained in the nuclei of mature mammalian spermatozoa is an extremely compact and stable structure. Sperm DNA must be organized in a specific manner (Fig. 1), which differs substantially from that of somatic cells, to achieve this unique condensed state (Poccia, 1986; Ward and Coffey, 1991). This DNA organization not only permits transfer of the very tightly packaged genetic information to the egg, but also ensures that the DNA is delivered in such a physical and chemical form that the developing embryo can access the ...
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.
During spermiogenesis, mammalian chromatin undergoes replacement of nuclear histones by protamines, resulting in a DNA that is highly condensed in the mature sperm. We have previously demonstrated that a percentage of human spermatozoa exhibit 1) positivity to the guanine-cylosine-specific chromomycin A3 (CMA3) fluorochrome and 2) the presence of endogenous nicks in their DNA. In situ protamination of mature human sperm limits the percentage of sperm positive to CMA3 and exhibiting endogenous nicks. In this study, we report further investigations that aim to clarify the relationship existing between levels of CMA3 stainability and the presence of endogenous nicks in the DNA of mature human spermatozoa. Human spermatozoa from 25 different samples showed values of sensitivity to the CMA3 fluorochrome ranging from 13% to 75%. The same samples showed a percentage of sensitivity to endogenous nick translation ranging from 1% to 38%. A strong correlation (r = 0.86) was evident between these two parameters. Prior staining of sperm with the CMA3 fluorochrome drastically reduced sensitivity to nick translation. In contrast, previously nick-translated sperm stained with CMA3 showed very little difference from samples that had not been pretreated. The presence of nicked sperm in the ejaculate may indicate anomalies during spermiogenesis and be an indicator of male infertility.
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