Sumo1‐ylation of human spermatozoa and its relationship with semen quality

International Journal of Andrology (Impact Factor: 3.7). 11/2011; 34(6pt1):581 - 593. DOI: 10.1111/j.1365-2605.2010.01118.x


Sumoylation is a post-translational modification involved in the regulation of several cell functions. Recent studies suggest its involvement in spermatogenesis, but occurrence and function of SUMO (small ubiquitin-like modifier) in mature spermatozoa remain unknown. We report the occurrence of several SUMO1-conjugated proteins, in a range of 20–85 kDa, in ejaculated spermatozoa. By cytofluorimetric analysis, we evaluated the percentage of SUMO1-positive spermatozoa in 58 subjects undergoing semen analysis in our laboratory and correlated the obtained values with semen parameters. We found that the percentage of SUMO1-positive spermatozoa was inversely correlated with total (r = −0.35, p < 0.01) and progressive motility (r = −0.29, p < 0.05). Such correlations become stricter when only asthenospermic subjects were included in the analysis (r = −0.58, p = 0.01 for progressive motility, n = 17) and were lost in non-asthenospermic subjects. By immunofluorescence and immunoconfocal fluorescence, we demonstrated that SUMO1 is mainly located in the nucleus and, occasionally, in the midpiece of spermatozoa. Immunoelectron microscopy as well as a long permeabilization protocol demonstrated a massive localization of SUMO-1 in the nucleus. By using a fluorescent probe to distinguish dead/live cells, we show that SUMO1 is mainly present in live spermatozoa. In conclusion, sumoylation of human spermatozoa may be involved in the regulation of motility.

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    • "2008), implying that a PI brighter sperm with damage in its DNA may be motile and/or morphologically normal. Moreover, we have previously shown that, with such a protocol, the great majority of sumoylated PI brighter sperm are alive (Marchiani et al. 2011). The occurrence of a significance correlation between SDF and sumoylation and, most importantly, the demonstration that most SUMO1-ylated sperm, in a given sample, is concomitantly DNA fragmented, indicate that SUMO1, when evaluated using a short-time permeabilisation protocol, might identify a fraction of live DNA-fragmented sperm in PI brighter population. "
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    ABSTRACT: We previously demonstrated that SUMO1 is associated with poor sperm motility when evaluated with a protocol that reveals mostly SUMO1-ylated live sperm. Recently, with another protocol, it has been shown that SUMO is expressed in most sperm and is related to poor morphology and motility, suggesting that sumoylation may have multiple roles depending on its localization and targets. We show here, by confocal microscopy and co-immunoprecipitation, that DRP1, RanGAP1 and Topoisomerase IIα, SUMO1 targets in somatic and/or germ cells, are SUMO1-ylated in mature human spermatozoa. DRP1 co-localizes with SUMO1 in the midpiece whereas RanGAP1 and Topoisomerase IIα in the post-acrosomal region of the head. Both SUMO1 expression and co-localization with the three proteins were significantly higher in morphologically abnormal sperm supporting that sumoylation represents a marker of defective sperm. DRP1 sumoylation at the midpiece level was higher in sperm from asthenospermic men. Since in somatic cells DRP1 sumoylation is associated with mitochondrial alterations, this protein may represent the link between SUMO and poor motility. Since SUMO pathways are involved in responses to DNA damage, another aim of our study was to investigate the relationship between sumoylation and sperm DNA fragmentation (SDF). By flow cytometry, we demonstrated that SUMO1-ylation and SDF are correlated (r=0.4, p<0.02, n=37) and most sumoylated sperm shows DNA damage in co-localization analysis. When SDF was induced by stressful conditions (freezing and thawing and oxidative stress), SUMO1-ylation increased. Following freezing and thawing SUMO1-Topoisomerase IIα co-localization and co-immunoprecipitation increased suggesting an involvement in formation/repair of DNA breakage.
    Reproduction (Cambridge, England) 08/2014; 148(5). DOI:10.1530/REP-14-0173 · 3.17 Impact Factor
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    • "A typical flow cytometric result out of 11. Note that PI staining unveils two different sperm populations indicated as PI br and PI dim (Muratori et al. 2008, Marchiani et al. 2011, Meamar et al. 2012) and that only PI br spermatozoa show 8-OHdG fluorescence. "
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    ABSTRACT: Oxidative stress (OS) is involved in many disoders including male infertility. Human spermatozoa are very sensitive targets of reactive oxygen species (ROS) and most sperm functions are impaired in the case of OS. In addition unbalanced production of ROS is considered one of the most important causes of sperm DNA fragmentation, a semen trait of infertile men. The relationship between oxidative damage and semen quality is partially controversial, probably due to the different methods and/or targets used to reveal the OS. In this study, by fluorescence microscopy and flow cytometry, we compared two methods to reveal 8-hydroxy,2-deoxyguanosine (8-OHdG), the hallmark of oxidative DNA damage: an immunofluorescence method and the commercial OxyDNA kit. We found that although both methods localized the labelling in sperm nuclei they yielded different measures, and only with the immunofluorescence method was the labelling specific for sperm 8-OHdG. The immunofluorescence method, coupled to flow cytometry, was thus selected to analyse the 8-OHdG content in semen samples from 94 subfertile patients and to investigate the relationship with semen quality. We found that the percentages of spermatozoa with 8-OHdG (mean±s.d., 11.4±6.9%) were related to sperm count (Pearson's correlation coefficient (r)=-0.27, P=0.04 (ANOVA and student's t-test)), motility (progressive: r=-0.22, P=0.04; non-progressive: r=0.25, P=0.01), and normal morphology (r=-0.27, P=0.01). In conclusion, we demonstrate that immunofluorescence/flow cytometry is a reliable and specific method to detect 8-OHdG at single-cell level and show that oxidative damage only partially overlaps poor semen quality, suggesting that it could provide additional information on male fertility with respect to routine semen analysis.
    Reproduction 03/2013; 145(3):227-35. DOI:10.1530/REP-12-0404 · 3.17 Impact Factor
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    ABSTRACT: Flow cytometry is now a recognized methodology within animal spermatology, and has moved from being a research tool to become routine in the assessment of animal semen destined to breeding. The availability of ‘bench-top’ flow cytometers and of newer and versatile markers for cell structure and function had allowed the instrumentation to measure more sperm parameters, from viability to reactiveness when exposed to exogenous stimuli, and to increase our capabilities to sort spermatozoa for potential fertilizing capacity, or chromosomal sex. The present review summarizes the state of the art regarding flow cytometry applied to animal andrology, albeit keeping an open comparative intent. It critically evaluates the present and future capabilities of flow cytometry for the diagnostics of potential fertility and for the development of current reproductive technologies such as sperm freezing, sperm selection and sperm sorting. The flow cytometry methods will probably further revolutionize our understanding of the sperm physiology and their functionality, and will undoubtedly extend its application in isolating many uncharacterized features of spermatozoa. However, continuous follow-up of the methods is a necessity owing to technical developments and the complexity of mapping spermatozoa.Keywords: animals; fertility; flow cytometry; semen analysis; spermatozoa; sperm functionality; sperm intactness
    Asian Journal of Andrology 04/2011; 13(3):406-419. DOI:10.1038/aja.2011.15 · 2.60 Impact Factor
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