Asthenozoospermic Patients Research Articles

Peroxiredoxin I in sperm and reactive oxygen species in seminal plasma in patients with idiopathic asthenozoospermia

To explore the relationship between peroxiredoxin I expression and seminal reactive oxygen species (ROS) in patients with idiopathic asthenozoospermia. Twenty-six infertile male patients were selected from the Reproductive Medical Center, Xiangya Hospital, from September to December in 2012. Fresh semen was collected from an experimental group (26 idiopathic asthenozoospermia patients) and a control group (15 men with fertility history and normal semen). Luminol chemiluminescence method was applied to detect the seminal ROS level. Western blot was used to detect the peroxiredoxin I expression. 1)The seminal ROS level in the experimental group was significantly increased compared with that in the control group (P<0.05), and the seminal ROS level was negatively correlated with mobility of the sperm (r=-0.777, P<0.01). 2) Compared with the control group, the peroxiredoxin I expression was significantly downregulated in the experimental group (P<0.05). The content of sperm peroxiredoxin I in the 2 groups was negatively correlated with the seminal ROS level (r=-0.474, P<0.01). 3) The content of peroxiredoxin I had a positive correlation with human sperm motility(r=0.779, P<0.01). The decline of peroxiredoxin I expression may be one of the crucial factors that leads to idiopathic asthenozoospermia. High level of ROS may be one of the main reasons for sperm vitality decline in patients with idiopathic asthenozoospermia.

Identification of a novel m.9588G &gt; A missense mutation in the mitochondrial COIII gene in asthenozoospermic Tunisian infertile men

Infertility affects 10-15 % of the population, of which, approximately 40 % is due to male etiology consisting primarily of low sperm count (oligozoospermia) and/or abnormal sperm motility (asthenozoospermia). It has been demonstrated that mtDNA base substitutions can greatly influence semen quality. In the present study we performed a systematic sequence analysis of the mitochondrial cytochrome oxidase III (COIII) gene in 31 asthenozoospermic infertile men in comparaison to normozoospermic infertile men (n=33) and fertile men (n=150) from Tunisian population. A novel m.9588G>A mutation was found in the mtDNA sperm's in all asthenozoospermic patients and was absent in the normozoospermic and in fertile men. The m.9588G>A mutation substitutes a highly conserved Glutamate at position 128 to Lysine. In addition, PolyPhen-2 analysis predicted that this variant is "probably damaging".

Relationship between ouabain and asthenozoospermia

A growing number of researches have shown that ouabain can regulate mammalian sperm function and male reproduction by modulating the sperm motility, capacitation and acrosome reaction in vitro. This study further examined the relationship between ouabain and asthenozoospermia. In this study, the rat was intraperitoneally injected with ouabain at different concentrations (low-dose ouabain group: 12.5 μg/kg body weight per day, and high-dose ouabain group: 25 μg/kg body weight per day) for 30 days to establish the asthenozoospermia model. The sperms from 60 males with normal fertility were incubated with ouabain of gradient concentrations (10(-7)-10(-2) mol/L) for 4 h. The sperm motility was evaluated under a microscope. Moreover, the endogenous ouabain (EO) level was determined in seminal plasma of mild or severe asthenozoospermia patients and males with normal fertility by competitive inhibition ELISA. The results showed that the sperm motility was significantly diminished in the rats treated with different concentrations of ouabain. The number of motile sperms (grades a and b) was decreased greatly in a time- and dose-dependent manner in 10(-5)-10(-2) mol/L ouabain groups (P<0.01), while no obvious change in sperm motility was observed in 10(-7)-10(-6)mol/L groups even for 4-h incubation (P>0.05). Furthermore, the EO level was significantly increased in asthenozoospermia patients as compared with that in males with normal fertility (25.27±1.71 μg/L in mild asthenozoospermia patients, 26.52±1.82 μg/L in severe asthenozoospermia patients, 19.31±1.45 μg/L in normal fertility men) (P<0.01). In conclusion, rat asthenozoospermia was successfully established by intraperitoneal injection of ouabain, and 10(-5) mol/L ouabain was sufficient enough to inhibit sperm motility in vitro. Moreover, EO, a normal constituent of seminal plasma, was highly expressed in asthenozoospermia males as compared with normal fertility ones.

Human leucocytes in asthenozoospermic patients: endothelial nitric oxide synthase expression

In a basic study at the Andrology Unit, Department of Clinical and Molecular Sciences, Polytechnic University of Marche, Ancona, Italy, we evaluated the pattern of mRNA endothelial nitric oxide synthase (eNOS) expression in human blood leucocytes isolated from normozoospermic fertile and asthenozoospermic infertile men to elucidate any pathogenic involvement in sperm cell motility. Forty infertile men with idiopathic asthenozoospermia and 45 normozoospermic fertile donors, age-matched, were included. Semen parameters were evaluated, and expression analysis of mRNA was performed in human leucocytes using reverse transcription polymerase chain reaction. Sperm volume, count, motility and morphology were determined, and eNOS expression and Western blotting analyses were performed. A positive correlation was observed between the concentrations of NO and the percentage of immotile spermatozoa. The mRNA of eNOS was more expressed in peripheral blood leucocytes isolated from asthenozoospermic infertile men versus those of fertile normozoospermic men (7.46±0.38 versus 7.06±0.56, P=0.0355). A significant up-regulation of eNOS gene in peripheral blood leucocytes was 1.52-fold higher than that of fertile donors. It is concluded that eNOS expression and activity are enhanced in blood leucocytes in men with idiopathic asthenozoospermia.

S-allyl cysteine ameliorates the quality of sperm and provides protection from age-related sperm dysfunction and oxidative stress in rats.

Reactive oxygen species play a central role in the pathophysiology of the age-related decrease in male fertility. It has been reported that the total protein of DJ-1 was decreased in a proteomic analysis of seminal plasma from asthenozoospermia patients and a DJ-1 protein acts as a sensor of cellular redox homeostasis. Therefore, we evaluated the age-related changes in the ratio of the oxidized/reduced forms of the DJ-1 protein in the epididymis. In addition, the protective effects of S-allyl cysteine (SAC), a potent antioxidant, were evaluated against sperm dysfunction. Male rats aged 15–75 weeks were used to assess age-associated sperm function and oxidative stress. Sperm count increased until 25 weeks, but then decreased at 50 and 75 weeks. The rate of sperm movement at 75 weeks was decreased to approximately 60% of the rate observed at 25 weeks. Expression of DJ-1 decreased, but oxidized-DJ-1 increased with age. In addition, 4-hydroxy-2-nonenal modified proteins in the epididymis increased until 50 weeks of age. The total number and DNA synthetic potential of the sperm increased until 25 weeks, and then decreased. In rats 75 weeks of age, SAC (0.45% diet) attenuated the decrease in the number, motility, and DNA synthesis of sperm and inhibited the oxidized proteins. These results suggest that SAC ameliorates the quality of sperm subjected to age-associated oxidative stress.

Involvement of Interleukin-1β, cyclooxygenase-2, and hypoxia-inducible factor -1α in idiopathic asthenozoospermia

Asthenozoospermia may result from different biochemical and functional defects. In particular, it has been demonstrated that reactive oxygen species (ROS) have a role in its pathogenesis. These reactive molecules may mediate the biological effects of cytokines, acting as intracellular signals. Human sperm cells are able to produce biologically active interleukin-1β (IL-1β), which stimulates cyclooxygenase-2 (COX-2) in various cells and tissues, including human reproductive tissues, even if its effects on male reproductive biology are largely unknown. COX-2 is the inducible isoform of the key enzyme in the biosynthesis of prostaglandins, and it could be also induced by hypoxia-inducible factor -1α (HIF -1α), which regulates many genes important for oxygen homeostasis. It has been recently proposed an involvement of COX-2 in male fertility disorders. In this scenario, our aim was to evaluate sperm parameters, as well as the expression of IL-1β, COX-2, and HIF -1α in spermatozoa isolated from normospermic fertile donors and asthenozoospermic infertile patients, by means of immunohistochemical analysis and enzyme-linked immunosorbent assay (ELISA). Sperm cells obtained from the asthenozoospermic group showed a higher expression of IL-1β, COX-2, and HIF-1α, compared with those from normospermic fertile subjects. Our results can lead us to speculate that the increased expression of these substances may influence the kinetic characteristics of spermatozoa. Nevertheless, further studies are in progress in order to assess whether these bioactive mediators may represent targets for future therapeutic strategies.

Metabolomics fingerprinting of the human seminal plasma of asthenozoospermic patients

It is estimated that 20% of couples are infertile, and half of these infertility cases are linked to men. One of conditions that can affect male fertility is asthenozoospermia. We applied Raman spectroscopy to the analysis of the metabolome of the human seminal plasma, and used chemometrics on the patterns of Raman spectra obtained. Significant changes were observed in the metabolome of the human seminal plasma of asthenozoospermic patients.

Association of interleukin-1beta C + 3953T gene polymorphism with human male infertility

Cytokines are involved in the regulation of spermatogenesis likely mediating the crosstalk among Sertoli and germ cells to facilitate germ cell movement across the seminiferous epithelium during cellular events such as germ cell differentiation. Members of the Interleukin-1 (IL-1) family are pleiotropic cytokines that are involved in inflammation, immunoregulation, and other homeostatic functions. Interleukin-1 alpha (IL-1α), IL-1β, and the IL-1 antagonistic molecule (IL-1 Ra) are present in the testis under normal homeostasis and they further increase upon infection/inflammation. In the present study we have examined the association of C + 3953T polymorphism of the human IL-1B gene with human male infertility. The case control study comprised of two groups: 222 infertile patients and 230 fertile healthy control men. Genotyping for SNP C + 3953T IL-1B was carried out by polymerase chain reaction followed by analysis with specific endonucleases (PCR-RFLP). DNA sequencing was used to validate the PCR-RFLP results. The genotype frequencies of the IL-1B Taq C/T polymorphism were compared between infertile men and controls. The frequency was significantly higher in asthenozoospermic patients compared to fertile control men (odds ratio = 10.4, CI: 2.50- 43.96, p = 0.001). The C + 3953T of the IL-1B gene is associated with male infertility risk in the asthenozoospermic patients from an Indian population.

A novel m.6307A&gt;G mutation in the mitochondrialCOXIgene in asthenozoospermic infertile men

Infertility affects 10-15% of the population, of which approximately 40% is due to male etiology and consists primarily of low sperm count (oligozoospermia) and/or abnormal sperm motility (asthenozoospermia). Recently, it has been demonstrated that mtDNA substitutions can influence semen quality. In this study, we performed a sequence analysis of the mitochondrial cytochrome oxidase I (COXI) gene in 31 infertile men suffering from asthenozoospermia in comparison to 33 normozoospermic infertile men and 100 fertile men from the Tunisian population. A novel m.6307A>G mutation was found in sperm mitochondrial DNA (mtDNA). This mutation was found in six asthenozoospermic patients, and was absent in normozoospermic and fertile men. We also detected 21 known substitutions previously reported in the Human Mitochondrial Database. The m.6307A>G mutation substitutes a highly conserved asparagine at position 135 to serine. In addition, PolyPhen-2 analysis predicted that this variant is "probably damaging.

Analysis of endothelial nitric oxide synthase (eNOS) G894T polymorphism and semen parameters in a Chinese Han population

Previous studies have shown that endothelial nitric oxide synthase (eNOS) gene may be involved in abnormal semen parameters. However, the relationship between eNOS G894T polymorphism and semen parameters remains controversial. The purpose of this study was to investigate the association of eNOS G894T polymorphism and semen parameters. The genotype frequency of eNOS G894T was determined in 270 idiopathic asthenozoospermia patients and 248 ethnically matched healthy volunteers using iPLEX genotyping assays on a MassARRAY(®) (Sequenom, San Diego, CA, USA) platform. The statistical analysis performed with Fisher's exact test showed no significant difference in frequencies of genotypes between both groups. The logistic regression showed that genotypes GT, TT and allele T were nonassociated with increased risk of asthenozoospermia in the patient group with ≤5% or >5% sperm with normal forms. The dependence on genotypes of semen parameters was further investigated in both patients and control group. There was no significant difference as compared to control group (P > 0.05). Our study indicated that eNOS gene G894T polymorphism may not have an adverse effect on semen parameters in a Chinese Han population.

Genetic variation in the Nrf2 promoter associates with defective spermatogenesis in humans

Defective spermatogenesis, which severely impairs male fertility, can be caused by excessive reactive oxygen species (ROS). Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates transcription of genes encoding enzymes important for protection against ROS. In human seminal plasma and spermatozoa, superoxide dismutase isoenzymes (SOD) and glutathione S-transferases (GST) are key antioxidant enzymes. We hypothesized that decreased function of the Nrf2-antioxidant response element (ARE) pathway might predispose individuals to male infertility. In this study, we identified three functional single nucleotide polymorphisms (SNPs) in the Nrf2 promoter regions of 196 idiopathic asthenozoospermic patients, 140 idiopathic oligoasthenozoospermic patients, and 295 controls. We found that two of the Nrf2 SNPs (-617 G > T and -653 T > C) were associated with oligoasthenozoospermia (p = 0.001) and individuals with 617 TT and 653 TT genotypes had higher risk of oligoasthenozoospermia (p = 0.006 and p = 0.002). Four haplotypes of Nrf2 promoters were identified, and two of them (GCC and TCT) had different frequencies in oligoasthenozoospermic patients than in controls (p = 0.019 and p = 0.011). In vitro reporter assay indicated that oligoasthenozoospermia associated genotypes of Nrf2 had significantly decreased transcriptional capabilities. The GCC and TCT haplotypes both showed lower Nrf2 mRNA expression in spermatozoa than GCT. TCT also showed decreased levels of antioxidant gene GSTM1 and SOD2 mRNA. Analysis of total seminal SOD activity elucidated that oligoasthenozoospermic patients had less SOD activity than controls. This study is the first to demonstrate a strong association between functional polymorphisms in Nrf2 promoters with defective spermatogenesis in humans.

Differential RNAs in the sperm cells of asthenozoospermic patients

Alterations in RNAs present in sperm have been identified using microarrays in teratozoospermic patients and in other types of infertile patients. However, so far, there have been no reports on using microarrays to determine the RNA content of sperm from asthenozoospermic patients. We started the present project with the goal of characterizing the RNA abundance in the sperm cells of asthenozoospermic patients when compared with controls. To reach this objective, we initially selected four normal fertile donors and four asthenozoospermic infertile patients. Equal amounts of RNA were extracted from the sperm samples, subjected to different quality controls and hybridized to the Affymetrix U133 Plus version 2 arrays. Several transcripts were identified that were present in different abundance in patients compared with controls. Subsequently, we validated the differential expression of three of the detected transcripts (ANXA2, BRD2 and OAZ3), using real-time PCR in a larger set of samples. A positive correlation between the expression of these transcripts and progressive motility was observed. The sperm cells of asthenozoospermic patients contain an altered amount of some RNAs as detected using microarray analysis and subsequently validated using real-time PCR. These results open up the possibility to investigate the implication of these genes in the pathogenic mechanisms in asthenozoospermia and to consider their potential utility as infertility biomarkers.

Nitric oxide synthase and tyrosine nitration in idiopathic asthenozoospermia: an immunohistochemical study

To characterize the expression pattern of constitutive and inducible nitric oxide synthase (NOS) in spermatozoa isolated from normospermic fertile donors and asthenozoospermic infertile patients, by means of immunohistochemistry. Also to evaluate the immunohistochemical expression of citrulline, a marker of NOS activity, and nitrotyrosine, which indicates the formation of peroxynitrite, which may affect sperm functionality through its cytotoxic action. Prospective study. Academic male infertility center. Twenty-nine infertile patients affected by idiopathic asthenozoospermia and 26 age-matched normospermic fertile donors. Sperm parameters were evaluated, and immunohistochemical analysis was performed in isolated spermatozoa. Semen analyses, to ascertain volume, sperm count, motility, and morphology. Immunohistochemical expression of NOS isoforms, citrulline, and nitrotyrosine. Constitutive NOS expression was greater in spermatozoa isolated from normospermic fertile donors. In contrast, the immunohistochemical expression of inducible NOS and nitrotyrosine was higher in asthenozoospermic samples. Our data concerning citrulline indicated enhanced NOS activity in sperm from idiopathic asthenozoospermic patients. Our results support the hypothesis that increased NOS activity and an excess of tyrosine nitration may affect the functional characteristics of spermatozoa in idiopathic asthenozoospermia.

D-Aspartate, a Key Element for the Improvement of Sperm Quality

Introduction: D-Aspartate is an endogenous amino acid involved in LH and testosterone release in humans. In this study we investigate the impact of nutritional supplementation of sodium D-aspartate on the improvement of sperm quality in sub-fertile patients and the rate of pregnancies that occurred with their partners. Materials and Methods: A group of 30 patients affected by oligo-asthenozoospermia and a group of 30 patients affected by asthenozoospermia were treated with a daily dose of sodium D-aspartate for 90 days. After which, the change in spermatozoa concentration and their motility and the pregnancies that occurred with their partners were recorded. Results: We found that the supplementation of D-aspartate significantly increased the concentration and the motility of spermatozoa. In oligo- asthenozoospermic patients the increase of sperm concentration was found to be 2.0-fold, P Conclusions:Treatment of sub-fertile patients with sodium D-aspartate improved the number and the motility of the spermatozoa and consequently improved the rate of pregnancies of their partners.

Altered Profile of Seminal Plasma MicroRNAs in the Molecular Diagnosis of Male Infertility

Although microRNAs (miRNAs) play essential roles in spermatogenesis, little is known about seminal plasma miRNAs in infertile men. We investigated the profile of seminal plasma miRNAs in infertile men to identify miRNAs that are altered in infertility; we then evaluated their diagnostic value. Seminal plasma samples were obtained from 289 infertile men and 168 age-matched fertile control individuals. The stability of the miRNAs was first assessed by time-course and freeze-thaw cycle analyses. The Solexa sequencing technology was used for an initial screen of the miRNAs in samples pooled from 45 patients with nonobstructive azoospermia, 58 patients with asthenozoospermia, and 100 fertile controls. A stem-loop quantitative reverse-transcription PCR (RT-qPCR) assay was conducted in the training and verification sets to confirm the concentrations of the altered miRNAs in 73 patients with nonobstructive azoospermia, 79 patients with asthenozoospermia, 34 patients with oligospermia, and 68 fertile controls. The miRNAs in seminal plasma were stable. The Solexa sequencing analysis demonstrated 19 markedly altered miRNAs in the patient groups, compared with the control group. RT-qPCR analysis identified 7 miRNAs (miR-34c-5p, miR-122, miR-146b-5p, miR-181a, miR-374b, miR-509-5p, and miR-513a-5p) as markedly decreased in azoospermia but increased in asthenozoospermia. The area under the ROC curve for these miRNAs ranged from 0.733 to 0.921, markedly higher than for routine biochemical parameters (0.510-0.622). Moreover, the concentrations of some selected miRNAs were also increased in the semen sperm of the asthenozoospermia patients. The measurement of miRNAs in seminal plasma provides a novel, noninvasive approach for diagnosing male infertility.

Protein tyrosine phosphatase non-receptor type 14 is a novel sperm-motility biomarker

To understand the molecular basis of sperm-motility and to identify related novel motility biomarkers. Two-dimensional electrophoresis (2DE) followed by Reverse-phase-nano-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (RP-nano-HPLC-ESI-MS/MS) were applied to establish the human sperm proteome. Then the sperm proteome of moderate-motile human sperm fraction and that of good-motile human sperm fraction from pooled spermatozoa of forty normozoospermic donors (Group 1 subjects) were compared to identify the dysregulated proteins. Among these down-regulated proteins, Protein tyrosine phosphatase non-receptor type 14 (PTPN14) was chosen to reconfirm by Western blotting and semi-quantitative reverse transcription polymerase chain reaction. For clinical application, Western blotting and real-time reverse transcription polymerase chain reaction was performed to compare the expression level of PTPN14 in (Group 2 subjects) nine normozoospermic controls and thirty-three asthenozoospermic patients (including 21 mild asthenozoospermic cases and 12 severe cases). Finally, bioinformatic tools prediction and immunofluorescence assay were performed to elucidate the potential localization of PTPN14. The expression levels of three proteins were observed to be lower in the moderate-motile sperm fraction than in good-motile sperm of group 1 subjects. Among three proteins with persistent down-regulation in the moderate-motile sperm, we reconfirmed that the expression level of PTPN14 was significantly lower in both mRNA and protein levels from the moderate-motile sperm fraction. Further, down-regulation of PTPN14 was found at the translational and transcriptional level in the asthenozoospermic men. Finally, Bioinformatic tools prediction and immunofluorescence assay showed that PTPN14 maybe predominantly localized at the mitochondria in the midpiece of human ejaculated sperm. Proteomics tools were applied to identify three possible sperm motility-related proteins. Among these proteins, PTPN14 was highly likely a novel sperm-motility biomarker and a potential mitochondrial protein.

Down-regulation of DJ-1 protein in the ejaculated spermatozoa from Chinese asthenozoospermia patients

To determine whether the expression of DJ-1 protein, whose levels in spermatozoa have been reported to be highly correlated with male infertility caused by toxicants, is changed in spermatozoa of Chinese asthenozoospermia patients. DJ-1 measurement by Western blotting, quantitive ELISA, and isoelectric-focusing electrophoresis (IFE) combined with immunoblotting. Academic medical center and research laboratories. Asthenozoospermia patients (n = 113), including mild asthenozoospermia patients (n = 70) and moderate asthenozoospermia patients (n = 43), and age-matched control subjects (n = 58). None. DJ-1 in spermatozoa was determined by Western blotting and ELISA, the isoelectric point (pI) of DJ-1 by IFE combined with immunoblotting, and sperm superoxide dismutase (SOD) activity by an assay kit. The sperm DJ-1 concentration in moderate asthenozoospermia patients was lower than those in mild asthenozoospermia patients and control subjects. DJ-1 with a more acidic pI was increased in asthenozoospermia patients. Sperm SOD activity was decreased in asthenozoospermia patients. DJ-1 levels are reduced in moderate asthenozoospermia patients. DJ-1 concentration is positively correlated with sperm motility and sperm SOD activity indicated by partial correlation analysis.

Mutations in exons 5, 7 and 8 of the human voltage-dependent anion channel type 3 (VDAC3) gene in sperm with low motility

Summary Voltage-dependent anion channels (VDACs1-3) are pore-forming proteins located in the outer mitochondrial membrane of various tissues and in human sperm flagella. VDACs are involved in metabolite fluxes between mitochondria and other cell compartments and play a role in the motility of mammalian spermatozoa. VDAC3-deficient male mice lacking exons 5–8 were reported to be healthy but infertile (Sampson et al., 2001). We analysed mutations in these exons of the human VDAC3 (hVDAC3) gene in spermatozoa of 32 asthenozoospermic patients. Previously, we reported on nucleotide substitution mutations in exon 6 (Asmarinah et al., 2005); now, we continue with the examination of exons 5, 7 and 8. Amplification of the exon fragments of the hVDAC3 gene was carried out by polymerase chain reaction (PCR) using specific primers for each exon followed by sequencing. We found various types of mutations in the examined exons of the hVDAC3 gene, such as deletion and nucleotide substitution in spermatozoa from seven of the 30 asthenozoospermic sperm samples (and unconfirmed insertions in two other ones) but none in spermatozoa from normozoospermic controls. Our preliminary data, although of small numbers investigated, suggest that genetic mutations in the hVDAC3 gene could be an explanation for the aetiology of infertility in these asthenozoospermic patients.

Nitric oxide and human sperm motility : an immunohistochemical study

Defective sperm function is a common cause of human male infertility. Human spermatozoa are very susceptible to oxidative stress, probably because they contain high levels of polyunsaturated fatty acids and are exposed to ROS produced by contaminating neutrophils or by the spermatozoa themselves. In addition, due to the cytoplasm scarcity, they lack an adequate reserve of antioxidant enzymes. It has been previously shown that human spermatozoa produce nitric oxide (NO) as well as superoxide anion and consequently peroxynitrite, which can rapidly react with proteins, lipids, and nucleic acids. NO is a short-lived free radical produced by three isoforms of NO synthase (NOS): endothelial (eNOS), neuronal (nNOS), and inducible (iNOS), which are responsible for the conversion of L-arginine to L-citrulline and NO. NO plays an important role in sperm physiology, although at high concentrations it could lead to cytotoxicity accompanied by decreased sperm motility. In recent studies it has been evidenced the presence of a significant negative linear correlation between NO concentration and sperm motility, as well as between peroxynitrite production and the kinetic features of spermatozoa. It has been previously demonstrated that the spermatozoa of fertile men express both eNOS and nNOS, which appear to be involved in sperm motility, metabolism, capacitation and acrosome reaction. However, the specific role of NOS isoforms in gametes is not well understood, even if iNOS seems to negatively affect the sperm function through the production of large amounts of NO, and consequently the nitration of protein tyrosine by peroxynitrite. The aim of the present study was to assess the presence and localization of NOS isoforms in human spermatozoa isolated from controls and asthenozoospermic infertile patients, by means of immunohistochemical techniques, in order to characterize the pattern of NOS expression and to evaluate the correlation with sperm function. In addition we studied the immunoexpression of citrulline, to achieve the microscopic visualization of NOS catalytic activity, and nitrotyrosine, since the protein tyrosine nitration represents a shift from the physiological role of NO toward pathogenetic pathways. The main results of our study are that the immunohistochemical expression of constitutive NOS isoforms is lower in the asthenozoospermic group, while the immunoexpression of iNOS and nitrotyrosine is higher in comparison with normozoospermic fertile subjects, thus suggesting a possible pathogenetic role of NO in the reduction of sperm motility.

Association of Seminal Plasma Motility Inhibitors/Semenogelins with Sperm in Asthenozoospermia-Infertile Men

Seminal plasma motility inhibitors (SPMIs) are proteinase-resistant fragments of semenogelin I and II (Sgs), which are the major proteins of semen coagulum. SPMIs inhibit the motility of spermatozoa, and Sgs are thought to be natural regulators of human sperm function. The mechanism underlying sperm motility regulation and its association with defective motility in infertile men remain unclear. The purpose of this study was to investigate the association between SPMIs and spermatozoa in infertile men with asthenozoospermia. Fifty-four semen samples from 37 asthenozoospermic patients and 17 samples from 9 normal healthy subjects were analyzed. Spermatozoa, washed by Percoll density gradients, were immunostained with anti-SPMI antibody and subjected to flow cytometric analysis. The proportion of spermatozoa labeled with the antibody and the average intensity of fluorescence labeling per spermatozoa were analyzed in relation to the parameters used for semen analysis. A significant negative correlation was found between sperm motility and the proportion (R = –0.68) and intensity (R = –0.38) of labeling. These results suggest that SPMIs remain on the sperm surface after liquefaction. This might account for some disorders of sperm motility observed in infertile men with asthenozoospermia.