Lucia Mazzi

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.

Resistin, interleukin-6, tumor necrosis factor-alpha, and human semen parameters in the presence of leukocytospermia, smoking habit, and varicocele

OBJECTIVE To explore the relationships between resistin, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) and semen parameters, sperm apoptosis, and necrosis in infertile patients and in control subjects with unknown reproductive potential with/without smoking habits, leukocytospermia, and varicocele. DESIGN Prospective study. SETTING Sperm laboratory. PATIENT(S) A total of 110 selected men. INTERVENTION(S) Family history, clinical/physical examination, ELISA determination (resistin, IL-6, TNF-α), semen analysis, annexin V/propidium iodide assay. MAIN OUTCOME MEASURE(S) Relationships among resistin, IL-6, and TNF-α and semen parameters in the presence of smoking habits, varicocele, leukocytospermia, and in infertile subjects. RESULT(S) Resistin level was higher in semen than in serum. Resistin semen levels showed negative correlations with sperm motility and positive correlations with apoptotic, necrotic sperm and TNF-α and IL-6 levels. Resistin, TNF-α, and IL-6 levels were higher in smokers compared with nonsmokers and in cases with leukocytospermia, in which an increase in necrotic sperm and a decrease in the number of sperm with normal morphology and motility were observed. Cytokine levels were significantly higher in infertile patients compared with control subjects with unknown reproductive potential. A total of 74.5% of infertile patients showed leukocytospermia. CONCLUSION(S) Semen resistin correlated with IL-6, TNF-α, and sperm quality; in cases of leukocytospermia and smoking habits, resistin concentrations were increased, suggesting that resistin may play a regulatory role in inflammation of the male reproductive system.

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.

Immunolocalisation of ghrelin and obestatin in human testis, seminal vesicles, prostate and spermatozoa

The role of ghrelin and obestatin in male reproduction has not completely been clarified. We explored ghrelin and obestatin localisation in the male reproductive system. Polyclonal antibodies anti‐ghrelin and anti‐obestatin were used to detect the expression of these hormones in human testis, prostate and seminal vesicles by immunocytochemistry, while in ejaculated and swim up selected spermatozoa by immunofluorescence. Sertoli cells were positive for both peptides and Leydig cells for ghrelin; germ cells were negative for both hormones. Mild signals for ghrelin and obestatin were observed in rete testis; efferent ductules were the most immune reactive region for both peptides. Epididymis was moderately positive for ghrelin; vas deferens and seminal vesicles showed intense obestatin and moderate ghrelin labelling; prostate tissue expressed obestatin alone. Ejaculated and selected spermatozoa were positive for both peptides in different head and tail regions. This study confirms ghrelin localisation in Leydig and Sertoli cells; the finding that ghrelin is expressed in rete testis, epididymis, vas deferens and seminal vesicles is novel, as well as the localisation of obestatin in almost all tracts of the male reproductive system. This research could offer insights for stimulating other studies, particularly on the role of obestatin in sperm physiology, which is still obscure.

Effect of Quercetin-loaded liposomes on induced oxidative stress in human spermatozoa

A strategy to circumvent the poor polyphenols bioavailability is to load these compounds into liposomes. We evaluated the in vitro effects of quercetin (Q) and Q-loaded liposomes (QLL, 30, 50, 100μM) on motility, viability and chromatin integrity of swim-up selected human sperm. Antioxidant power was assayed against tert-butylhydroperoxide induced lipid peroxidation (LPO) using C11-BODIPY581/591 fluorescent probe and transmission electron microscopy. QLL showed decreased toxicity for sperm motility and viability and increased DNA damage compared to Q. The percentage of sperm with fluorescence, marker of LPO, was decreased in samples incubated with Q vs QLL (P<0.001). The ultrastructure of acrosomes and membranes was preserved with Q 30/100μM, whereas QLL did not prevent membrane injury. Q alone appeared more effective than Q incorporated into liposomes; however liposomes could be considered as carriers that may convey different compounds inside sperm; they may therefore represent a field of research rich of many applications.

Effect of chocolate and Propolfenol on rabbit spermatogenesis and sperm quality following bacterial lipopolysaccharide treatment.

Abstract The aims of the study were to evaluate the effects of chocolate and propolis-enriched diets on rabbit spermatogenesis, sperm motility, and ultrastructure following bacterial lipopolysaccharide (LPS) treatment. Thirty-two New Zealand White rabbits were divided into four groups. The LPS-Propolfenol® group received propolis (500 mg/kg/day) in their diet for 15 days, while the LPS-chocolate group was fed 70% cacao chocolate (1 g/1 kg/day) for the same period. Following the diet treatments, rabbits in the LPS-Propolfenol® and LPS-chocolate groups, and an LPS group received a single intraperitoneal dose of 50μg/kg LPS, and the control group received only saline. Kinematic sperm traits were evaluated with a computer assisted sperm analyzer (CASA) system, and ultrastructural characteristics were examined by transmission electron microscopy (TEM). Testicular and epididymal tissues were observed by light microscopy and TEM and multiplex real time reverse transcriptase-polymerase chain reaction (RT-PCR) assay was used to detect and quantify toll-like receptor-4 (TLR-4) gene expression. The values of the analyzed semen parameters of rabbits treated with LPS-Propolfenol® and LPS-chocolate did not show any variations compared with the control group, but they were lower in rabbits treated only with LPS. Alterations observed in the testicular tissue of LPS treated-rabbits were not detected in specimens from the LPS-chocolate and LPS-Propolfenol® groups, which showed normal spermatogenesis. The TLR-4 mRNA expression was similar in controls, in LPS treated, and in LPS-chocolate groups, but it was significantly (p < 0.01) decreased in LPS-Propolfenol® rabbits. In conclusion, a chocolate and propolis-enriched diet showed a protective effect on the spermatogenetic process of buck rabbits following LPS treatment.

Cytoskeleton Aberrations in Alkaptonuric Chondrocytes

Alkaptonuria (AKU) is an ultra‐rare autosomal genetic disorder caused by a defect in the activity of the enzyme homogentisate 1,2‐dioxygenase (HGD) that leads to the accumulation of homogentisic acid (HGA) and its oxidized product, benzoquinone acetic acid (BQA), in the connective tissues causing a pigmentation called “ochronosis.” The consequent progressive formation of ochronotic aggregates generate a severe condition of oxidative stress and inflammation in all the affected areas. Experimental evidences have also proved the presence of serum amyloid A (SAA) in several AKU tissues and it allowed classifying AKU as a secondary amyloidosis. Although AKU is a multisystemic disease, the most affected system is the osteoarticular one and articular cartilage is the most damaged tissue. In this work, we have analyzed for the first time the cytoskeleton of AKU chondrocytes by means of immunofluorescence staining. We have shown the presence of SAA within AKU chondrocytes and finally we have demonstrated the co‐localization of SAA with three cytoskeletal proteins: actin, vimentin, and β‐tubulin. Furthermore, in order to observe the ultrastructural features of AKU chondrocytes we have performed TEM analysis, focusing on the Golgi apparatus structure and, to demonstrate that pigmented areas in AKU cartilage are correspondent to areas of oxidation, 4‐HNE presence has been evaluated by means of immunofluorescence. J. Cell. Physiol. 232: 1728–1738, 2017.

CagA-Positive Helicobacter pylori Infection and Reduced Sperm Motility, Vitality, and Normal Morphology

Helicobacter pylori (HP) infection, particularly when caused by strains expressing CagA, may be considered a concomitant cause of male and female reduced fertility. This study explored, in 87 HP-infected males, the relationship between infection by CagA-positive HP strains and sperm parameters. HP infection and CagA status were determined by ELISA and Western blotting; semen analysis was performed following WHO guidelines. The amino acid sequence of human enzymes involved in glycolysis and oxidative metabolism were “blasted” with peptides expressed by HP J99. Thirty-seven patients (42.5%) were seropositive for CagA. Sperm motility (18% versus 32%; P < 0.01), sperm vitality (35% versus 48%; P < 0.01) and the percentage of sperm with normal forms (18% versus 22%; P < 0.05) in the CagA-positive group were significantly reduced versus those in the CagA-negative group. All the considered enzymes showed partial linear homology with HP peptides, but four enzymes aligned with four different segments of the same cag island protein. We hypothesize a relationship between infection by strains expressing CagA and decreased sperm quality. Potentially increased systemic levels of inflammatory cytokines that occur in infection by CagA-positive strains and autoimmune phenomena that involve molecular mimicry could explain the pathogenetic mechanism of alterations observed.

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 effects in vitro of TNF-α and its antagonist 'etanercept' on ejaculated human sperm.

Tumour necrosis factor (TNF)-α is primarily involved in the regulation of cell proliferation and apoptosis; in addition it possesses pro-inflammatory properties. Anti-TNF-α strategies involve either administration of anti-TNF-α antibody or soluble TNF receptor to mop up circulating TNF-α. Etanercept, a recombinant human TNF-α receptor, was found to be effective in the treatment of rheumatoid arthritis. The impact of TNF-α inhibitors on human fertility is of notable interest. This in vitro study investigated the effect of different concentrations of TNF-α and etanercept used alone or in combination on sperm viability, motility, mitochondrial function, percentage of apoptosis and chromatin integrity in swim-up selected human spermatozoa. A negative effect of TNF-α (300 and 500ng mL-1) and etanercept (from 800µg mL-1 to 2000µg mL-1) individually on sperm viability, motility, mitochondrial function, percentage of apoptotic spermatozoa and sperm DNA integrity was demonstrated. However, at concentrations of 100 and 200µg mL-1, etanercept can block, in a significant way, the toxic effects of TNF-α (500ng mL-1) on studied sperm characteristics. Our results confirm that TNF-α has a detrimental effect on sperm function and suggest, for the first time, that etanercept may counteract the in vitro toxic action of TNF-α. This data appears to be quite promising, although further studies, both in vivo and in vitro, are needed to understand the exact mechanism of action of TNF-α and TNF-α antagonists on sperm function.