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.

No abstract

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 ...

The increasing interest in stress/heat shock proteins (Hsps) as markers of exposure to environmental stress or disease requires an easily applicable method for Hsp determination in peripheral blood cells. Of these cells, monocytes preferentially express Hsps upon stress. An appropriate fixation/permeabilization procedure was developed, combined with immunofluorescence staining and flow cytometry for the detection of the inducible, cytosolic, 72 kDa Hsp (Hsp70) in human monocytes. Higher relative fluorescence intensity was observed in cells exposed to heat shock (HS), reflecting a higher expression of Hsp70 in these cells as compared with cells kept at 37 degrees C. The heat-inducible increased Hsp70 expression was temperature- and time-dependent. Expression of Hsp70 was not uniform within the monocyte population, indicating the presence of subpopulations expressing variable levels of Hsp70 in response to HS. Simultaneous measurements of intracellular Hsp70 and membrane CD14 expression revealed that the higher Hsp70 inducibility coincided with the higher CD14 expression. Comparisons performed with biometabolic labelling, Western blotting, immunofluorescence and immunoperoxidase microscopic analysis, showed a high concordance between these different methods; however, cytometry was more sensitive for Hsp70 detection than Western blotting. Flow cytometric detection of intracellular Hsp70 is a rapid, easy and quantitative method, particularly suited for the determination of protein levels in individual cells from an heterogeneous population such as peripheral mononuclear blood cells, and applicable to cohort studies.

In this study we investigated the relationship between the presence of bound protamine on mouse and human sperm DNA and the level of chromomycin A3 (CMA3) and 4'6-diamidino-2-phenylindole (DAPI) fluorescence. This was accomplished by performing a competition assay between salmon protamine and fluorochromes on decondensed spermatozoa that had their nuclear proteins extracted and were fixed on slides. Various concentrations (0, 0.005, 0.0225, 0.05, 0.225, 0.5 and 5 mg/ml) of salmon protamine were added to either the CMA3 or DAPI staining solutions. Fluorescence emission measurements of stained sperm nuclei were then performed using a microfluorometer. When the treated decondensed sperm heads were stained with either CMA3 or DAPI all spermatozoa were found to fluoresce intensely. The addition of protamines to the spermatozoa led to an elimination of CMA3 fluorescence, while the intensity of DAPI staining was decreased to approximately 50% at the highest concentrations of protamine. The addition of increasing amounts of salmon protamine also induced the sperm nuclei to regain their initial condensed appearance. This study shows that protamine retains a strong affinity for sperm DNA in situ and that CMA3 fluorescence is a strong indicator of the protamination state of spermatozoa.