Gaia Terzuoli

In vitro effect of gold and silver nanoparticles on human spermatozoa

The cytotoxicity of Au/Ag nanoparticles (NPs) on human spermatozoa was investigated in vitro. Semen from donors were incubated (37 °C, 60′–120′) with 30, 60, 125, 250 and 500 μM Au/Ag‐NPs. Sperm motility was evaluated following WHO guidelines; sperm viability was assessed with eosin Y test. Au‐NPs were characterised and localised with field emission gun‐based scanning transmission electron microscope/energy dispersive spectroscopy and transmission electron microscopy. Both tested NPs exerted a significant dose‐dependent effect on motility and viability of human spermatozoa (P < 0.001). Ag‐NPs seem to show a slightly elevated toxicity although not significant (P > 0.05). Au‐NPs were localised in spermatozoa, whereas Ag‐NPs were undetectable. In conclusion, Au‐NPs and Ag‐NPs do not appear to be harmful for human spermatozoa up to high concentrations (250–500 μM) that are probably difficult to reach in vivo. It is mandatory to explore the genotoxic effect of NPs in germ cells.

Effect of quercetin, rutin, naringenin and epicatechin on lipid peroxidation induced in human sperm.

Quercetin, rutin, naringenin, epicatechin are flavonoids with diverse properties, including antioxidant potential. We evaluated, in vitro, the cytotoxicity of these flavonoids (20, 30, 50, 100, 200, 400 μM) in swim-up selected human sperm. Antioxidant activity was tested against tert-butylhydroperoxide induced lipid peroxidation using a C11-BODIPY(581/591) probe and transmission electron microscopy. A significant concentration-dependent effect on sperm viability (P<0.001) and motility (P<0.001) was observed. Lipid peroxidation was decreased in samples treated with 30 μM quercetin (P<0.01) and 30 μM rutin (P<0.05) versus samples incubated with tert-butylhydroperoxide alone. Naringenin (50-100 μM) showed a low protective effect and epicatechin (200 μM) was not efficacious. Transmission electron microscopy analysis confirmed the protective action of rutin and in particular quercetin on damages induced by lipid peroxidation. These results underlined the antioxidant properties of quercetin and rutin. A possible role of these compounds in the supplementation of media used during semen handling warrants attention and further studies.

Effects of gold and silver nanoparticles in cultured human osteoarthritic chondrocytes.

The aim of our study was to evaluate the effects of gold (Au) and silver (Ag) nanoparticles (NPs) at different concentrations on cultured human osteoarthritic chondrocytes. Cell viability and inducible nitric oxide synthase expression were evaluated by light microscopy. Using transmission electron microscopy (TEM) and field emission gun‐based scanning transmission electron microscopy/energy dispersive spectroscopy (FEG‐STEM/EDS) allowed us to localize NPs. Gene expression of matrix metalloproteinases 1, 3 and 13 and A disintegrin and metalloproteinase with thrombospondin motifs ‐4 and ‐5 were carried out by real‐time polymerase chain reaction. A cell viability test indicated a significant dose‐dependent cytotoxic effect of both NPs. At concentrations of 160 and 250 μM NP light microscopy showed chondrocytes with signs of apoptosis and an increased presence of inducible nitric oxide synthase. Au‐NPs were characterized by FEG‐STEM/EDS and TEM analysis localized NPs in cytoplasm and in endocytotic vesicles. On the contrary, the Ag‐NPs were undetectable by FEG‐STEM/EDS and TEM. Increased gene expression, particularly in matrix metalloproteinase‐3, was observed for both NPs (160 μM), but at a concentration of 250 μM the expression of the evaluated genes became lower. Our in vitro studies, although preliminary, suggest that engineered Au and Ag‐NPs appear to be harmful for human osteoarthritic chondrocytes in high concentrations (160–250 μM). Copyright

Immunolocalization of aquaporin 7 in human sperm and its relationship with semen parameters

Aquaporins (AQPs) are a family of 13 small hydrophobic trans-membrane proteins expressed in numerous tissues and cells. Some AQPs work as strict water channels, others are permeable to a range of substances, including glycerol. In the male reproductive system their localization in testis, efferent ducts, epididymis, and spermatozoa has been described. We studied the distribution of AQP7 in ejaculated human sperm and the relationship between AQP7 labeling and sperm characteristics. Semen samples from 33 men were examined by light and transmission electron microscopy (TEM). TEM data were quantified using a mathematical formula that calculates a fertility index (FI) and the percentages of sperm apoptosis, immaturity, and necrosis. Immunocytochemistry with a polyclonal antibody anti-AQP7 was performed on the sperm samples. Normal sperm were labeled in the pericentriolar area, midpiece, equatorial segment, and weakly in the tail (grade 1). Abnormal sperm showed a diffuse low intensity of fluorescence evident in the cytoplasmic residues, coiled tails, in the entire head, and acrosome (grade 2). A high number of motile sperm obtained by swim up were labeled in a dotted manner in the mitochondria. A significant positive correlation was found between the spermatozoa with AQP7 grade 1 labeling and the percentage of normal form (P < 0.008), progressive motility and FI (P < 0.005); a negative correlation was noted with the percentages of cytoplasmic residues (P < 0.010) and immaturity (P < 0.006) and coiled tails (P < 0.012). The link between AQP7 distribution and sperm morphology and the particular dotted labeling in swim up selected motile sperm are novel and deserve additional studies.

Light, polarizing, and transmission electron microscopy: Three methods for the evaluation of sperm quality

Semen from 33 patients were evaluated by light microscopy (LM) obtaining sperm concentration, percent motility, percentage of sperm with normal morphology (PAP staining), and percentage of dead sperm (Eosin Y stained). The samples were observed by polarizing microscopy (PM), that evaluates sperm morphology and the viability by birefringence of organelles, and it provides a PM index (percentage of birefringent, viable, motile sperm) and a percentage of dead, non-birefringent sperm. Sperm were processed for transmission electron microscopy (TEM) and TEM data were elaborated with a mathematical formula able to provide a fertility index (FI, number of sperm free of structural defects) and percentages of sperm immaturity and necrosis (dead sperm). To test the reliability of these techniques, the values of normal acrosome, nucleus, midpiece, and tail and the presence of cytoplasmic residues obtained with the three methods were compared. With the exception of cytoplasmic residues (P = 0.40), significant differences in the evaluation of each organelle were observed and TEM analysis resulted as the most stringent screening. In addition, relationships among relevant sperm variables were investigated. Motility showed positive correlations with the percentage of normal tail, midpiece, and PM index (P < 0.01), but it exhibited negative correlations with indices of sperm death (non-birefringent sperm: P < 0.05; percentage of eosin Y stained sperm: P < 0.05; necrosis: P < 0.01), which were positively correlated with each other (P < 0.01). Positive correlations were found between indices expressing normal sperm morphology: FI with PM index (P < 0.01) and with the percentage of normal sperm (PAP staining) (P < 0.01), which in turn were correlated with the PM index (P < 0.001). Sperm immaturity showed positive correlations (P < 0.01) with the presence of cytoplasmic residues detected with the three methods. In conclusion, LM, PM, and TEM are reliable techniques in evaluating sperm quality. PM appears to offer several advantages ‘midway’ between LM and TEM and it should be considered in sperm analysis.

The effect of radio electric asymmetric conveyer treatment on sperm parameters of subfertile stallions: A pilot study

The Radio Electric Asymmetric Conveyer (REAC) has been mostly applied to treat symptoms related to psychological stress. In the study, we demonstrated the effect of REAC-Veterinary Neuro Psycho Physical Optimization (VNPPO) treatment protocol on sperm parameters of subfertile (n=11) and fertile (n=4) stallions. Subfertile stallions showed a reduced sperm concentration, progressive motility and normal morphology compared to fertile stallions. An increase in progressive sperm motility and quality of sperm morphology was found in subfertile stallions after the REAC-VNPPO treatment. The positive effect of the REAC-VNPPO treatment was visible in a reduced number of reacted or absent acrosomes, nuclei with marginated chromatin and presence of cytoplasmic residues. Thus, we suggest that the REAC-VNPPO treatment for stallions with idiopathic subfertility may enhance the reproductive performance of stallions.

In Vitro Effects of Tire Debris Organic Extracts on Human Spermatozoa

Background & objectives: Environmental pollutants have detrimental effects on the reproductive function of human being and of animals. This study was aimed at evaluating the effects of tire debris, a fraction of particulate matter derived from tire wear, on motility and morphology of human spermatozoa. Methods: spermatozoa from ten healthy men were incubated for 4 hours with 10, 50 and 75 μg/ml of tire debris organic extract (TDOE). Sperm motility and morphology were evaluated according to WHO guidelines; the presence of apoptosis and necrosis was assessed by the AnnexinV/Propidium iodide assay. Ultrastructure of sperm organelles was observed by transmission electron microscopy. Results: incubation with 10 � g/ml of TDOE did not affect sperm quality, except for the value of apoptosis (median: 8% vs. 6%; P<0.01). Sperm motility and sperm morphology percentages (P<0.01)decreased when sperm were treated with 50 and 75 μg/ml TDOE and the percentage of apoptotic and necrotic sperm (P<0.01) increased concomitantly. At the same concentrations TEM analysis showed an increased percentage of reacted and swollen acrosomes, disrupted chromatin and altered axonemes. Interpretation & conclusions: the organic part of tire debris is potentially harmful to spermatozoa, particularly at a concentration of 50-75 μg/ml, inhibiting motility and compromising morphological integrity. Nevertheless, these results cannot yet be extrapolated to the in vivo outcome due to the lack of studies focused on this issue.