M540 bodies and their impact on flow cytometric analyses of human spermatozoa.

M540 bodies are membrane-surrounded round bodies occurring in semen of sub-fertile men. They appear variable in size and density, virtually devoided of chromatinic material and especially frequent in oligoasthenoteratozoospermic patients. Up to now, data collected by our group suggest that they may be apoptotic bodies that somehow escaped from testicular/epididymal phagocytosis. Indeed, they promptly stain with Merocyanine 540, a probe detecting the changes occurring in the membrane of apoptotic cells. In addition, they exhibit many of the apoptotic markers occurring in testicular apoptosis, including caspases activity, Fas receptor, p53 and DNA fragmentation (the latter detected by TUNEL assay). Due to the similarity in size and density between head sperm and M540 bodies, traditional protocols of sample preparation fail to yield sperm population completely free of M540 bodies, except for swim-up selection. In flow cytometry, it is possible to distinguish sperm from bodies by labelling samples with nuclear probes, because the latter fail to stain M540 bodies. Occurrence of M540 bodies in semen has been revealed only recently, at least in quantitative studies, and ,hence, many flow cytometric studies have not accounted for them. We summarise two studies (one on sperm ubiquitination and another on sperm DNA fragmentation) in which flow cytometric analyses were conducted both including and excluding M540 bodies from the sperm population. We found that in both cases M540 bodies largely affected the results. The study on sperm ubiquitination reveals that the direct correlation between sperm ubiquitination and good semen parameters is unmasked only after exclusion of M540 bodies from the analysis. The study on sperm DNA fragmentation shows that the amount of DNA damage in sub-fertile patients is more dramatic than expected from past investigations that included M540 bodies in the analysis.