Activity In Human Seminal Plasma Research Articles

DNase activity in human seminal plasma and follicular fluid and its inhibition by follicular fluid chelating agents

Research questionWhat is the mechanism by which human follicular fluid inhibits seminal plasma DNase activity? DesignHuman genomic DNA was incubated with human follicular fluid and seminal plasma (reaction mixture) under different experimental conditions; increasing volumes of human follicular fluid; proteinase K digested or heat inactivated human follicular fluid; and the addition of Ca2+ or Mg2+ to the reaction mixture. ResultsIncreasing volume of human follicular fluid resulted in a dose-dependent inhibition of seminal plasma DNase activity. Inhibition was not caused by proteins in the human follicular fluid as digestion with proteinase K or heat inactivation of human follicular fluid failed to abolish its inhibitory effect. Addition of divalent cations resulted in a reversion of the inhibitory effect, providing evidence that human follicular fluid inhibition of seminal plasma DNase activity seems to be mediated by a compound with chelating activity. Furthermore, incubation of genomic DNA with human follicular fluid in the presence of divalent cations served to elicit the existence of DNase activity. ConclusionsHuman follicular fluid seems to contain a molecule or molecules with chelating capacity that inhibits DNase activity of both follicular fluid and seminal plasma. Our findings provide new insight to understanding sperm preservation and the physiology of fertilization biology.

Original Article. Matrix Metalloproteinase Mmp-2 and Mmp-9 Activities in Seminal Plasma

SummaryMatrix metalloproteinases (MMPs) areagroup of proteases containing Zn ions asacofactor, which are involved in degrading ofalarge number of extracellular matrix proteins, and bioactive molecules. They also playamajor role in processes such as cell proliferation, cell migration, differentiation and apoptosis. Very little is known about the expression and function of MMPs in the male reproductive tract. Occurrence of MMP-2 and MMP-9 activity in human seminal plasma has been previously reported but their origin and function are still not fully understood. The aim of this study was to examine the presence of MMP-2 and MMP-9 in normal and abnormal human sperm samples and find if any correlation existed between the levels of expression of MMPs and fertilization potential of the spermatozoa. Human spermsamples were examined for the presence of MMP-2 and MMP-9 by gel zymography and western blot analysis. A DNAfragmentation test was performed. The samples were divided into two groups - samples with normozoospermia and teratozoospermia. The gelatin zymography showed gelatinolytic bands with molecular weight 64 and 72 k Da corresponding to active and inactive form of MMP-2. MMP-9 was not detected. The MMP-2 enzymatic activity appeared to be much higher in samples with compromised sperm morphology as compared to the normozoospermic samples. The mean DNAfragmentation index (DFI) of the group with teratozoospermia was relatively higher (22.16%) and over the upper reference limits, compared to the normozoospermic group, in which it was within the normal range (17.26%).

Mobile phones electromagnetic radiation and NAD+-dependent isocitrate dehydrogenase as a mitochondrial marker in asthenozoospermia

NAD+-dependent Isocitrate Dehydrogenase (NAD+-IDH) could be one of the cell phone radiation targets. Enzyme activity alteration may lead to decline in sperm motility during radio-frequency electromagnetic waves (RF-EMW) exposure. The current case control study aimed to investigate the possible relationship between mitochondrial NAD+-IDH activity in human seminal plasma and sperm motility among asthenozoospermic cellular phone users. A total number of ninety idiopathic infertile males referred from the Department of Dermatology and Andrology, were enrolled in this study. NAD+-IDH activity was measured in human seminal plasma by spectrophotometer. Computer-aided sperm analysis (CASA) following WHO criteria has been used for semen analyses. The results showed that IDH activity was increased in patients with prolonged cell phone daily use ≥4 h/day. Its level, correlated negatively with either the motility ratio percentages (r = −0.46, p < 0.001) or the progressive motility percentages (r = −0.50, p < 0.001) in the study groups. The current study suggests that NAD+-IDH in human seminal plasma could be one of seminal plasma biomarkers reflecting the mitochondrial function of spermatozoa. Alteration of its level could reflect the defective motility of sperms among some cases of cellular phone users.

Editorial Comment

Editorial Comment

Characterization of Nuclease Activity in Human Seminal Plasma and its Relationship to Semen Parameters, Sperm DNA Fragmentation and Male Infertility

Some studies have shown that complementary biomarkers are needed in semen analysis to provide a more accurate diagnosis for couples with infertility problems. To our knowledge no study has been done to determine the relationships among nuclease activity in seminal plasma, semen parameters, sperm DNA fragmentation and male infertility. A total of 94 semen samples were collected according to WHO 2010 semen analysis parameters. Samples were analyzed using the single radial enzyme diffusion method for nuclease activity in seminal plasma, and alkaline and neutral Comet assay for sperm DNA fragmentation. Samples were obtained from 11 fertile donors with proven fertility, 17 patients with normozoospermia in an infertile couple, and 16 patients with asthenozoospermia, 19 with teratozoospermia, 21 with asthenoteratozoospermia and 10 with azoospermia. Nuclease activity analyzed in seminal plasma was higher in patients than in controls. It correlated with sperm motility and morphology, and sperm DNA fragmentation measured by the alkaline Comet assay. No correlation with sperm DNA fragmentation was measured by the neutral Comet assay. ROC curves to determine male infertility revealed 0.658 sensitivity, 0.727 specificity and 0.705 cm(2) AUC for the single radial enzyme diffusion method, 0.918, 1 and 0.994 cm(2) for the alkaline Comet assay, and 0.917, 0.250 and 0.373 cm(2), respectively, for the neutral Comet assay. Nuclease activity in seminal plasma corrected by sperm count is a good variable to predict male infertility. Results indicate that it could be a useful complementary parameter for male infertility diagnosis.

An Improved Neutral a-Glucosidase Assay for Assessment of Epididymal Function-Validation and Comparison to the WHO Method.

Neutral α-glucosidase (NAG) activity in human seminal plasma is an important indicator for epididymis functionality. In the present study, the classic World Health Organization (WHO) method has been adapted to enhance assay robustness. Changes include modified enzyme reaction buffer composition and usage of an alternative enzyme inhibitor for background correction (glucose instead of castanospermine). Both methods have been tested in parallel on 144 semen samples, obtained from 94 patients/donors and 50 vasectomized men (negative control), respectively. Passing-Bablok regression analysis demonstrated equal assay performance. In terms of assay validation, analytical specificity, detection limit, measuring range, precision, and cut-off values have been calculated. These data confirm that the adapted method is a reliable, improved tool for NAG analysis in human semen.

Malate dehydrogenase activity in human seminal plasma and spermatozoa homogenates

Purpose: Malate Dehydrogenase is an important enzyme of the Krebs cycle, most cells require this enzyme for their metabolic activity. We evaluated the Malate Dehydrogenase (NAD/NADP) activity in human seminal plasma and sperm homogenates in normozoospermic, fertile and infertile males. Also glucose and fructose concentrations were determined in the seminal plasma samples. Material and Methods: Malate Dehydrogenase (NAD/NADP) activity in human seminal plasma and sperm homogenates of normozoospermic and infertile males was determined by spectrophotometric method. Semen analysis was considered according to the WHO Criteria. Results: Malat Dehydrogenase-NAD value in seminal plasma (the mean ± SD, mU/ml) of asthenoteratospermic (40.0±25.7) and azospermic (38.0±43.6) groups were significantly lower than normozoospermic, (93.9±52.1) males. Malat Dehydrogenase-NAD value in sperm homogenates (the mean ± SD, mU/ 20x106 sperm) of teratospermic group (136.8±61.8) was significantly higher compared to the normozoospermic (87.3±26.5) males. Glucose concentration (mg/dl) in asthenoteratospermic (4.0±1.4) and azospermic (15.4±6.4) groups were significantly higher than fertile (2.0±2.1) males. Also fructose concentration (mg/dl) in asthenoteratospermic (706.6±143.3) and azospermic (338.1±228.2) groups were significantly high compared to the normozoospermic (184.7±124.8) group. Conclusion: Sperm may be some part of the source of Malat Dehydrogenase activity in semen. Malat Dehydrogenase activity in seminal plasma has an important role on energy metabolism of sperm. Intermediate substrates of Krebs cycle might have been produced under the control of Malat Dehydrogenase and these substrates may be important for sperm motility and male infertility.Key words:

UP-2.033: Antibacterial Activity of Human Seminal Plasma and Low Molecular-Mass Antibacterial Peptides from Human Seminal Plasma

UP-2.033: Antibacterial Activity of Human Seminal Plasma and Low Molecular-Mass Antibacterial Peptides from Human Seminal Plasma

Cationic polypeptides contribute to the anti‐HIV‐1 activity of human seminal plasma

Mucosal surfaces of the reproductive tract as well as their secretions have important roles in preventing sexual transmission of HIV-1. In the current study, the majority of the intrinsic anti-HIV-1 activity of human seminal plasma (SP) was determined to reside in the cationic polypeptide fraction. Antiviral assays utilizing luciferase reporter cells and lymphocytic cells revealed the ability of whole SP to prevent HIV-1 infection, even when SP was diluted 3200-fold. Subsequent fractionation by continuous flow acid-urea (AU)-PAGE and antiviral testing revealed that cationic polypeptides within SP were responsible for the majority of anti-HIV-1 activity. A proteomic approach was utilized to resolve and identify 52 individual cationic polypeptides that contribute to the aggregate anti-HIV-1 activity of SP. One peptide fragment of semenogelin I, termed SG-1, was purified from SP by a multistep chromatographic approach, protein sequenced, and determined to exhibit anti-HIV-1 activity against HIV-1. Anti-HIV-1 activity was transient, as whole SP incubated for prolonged time intervals exhibited a proportional decrease in anti-HIV-1 activity that was directly attributed to the degradation of semenogelin I peptides. Collectively, these results indicate that the cationic polypeptide fraction of SP is active against HIV-1, and that semenogelin-derived peptides contribute to the intrinsic anti-HIV-1 activity of SP.

Melatonin and aromatase stimulating activity of human seminal plasma

Melatonin concentrations and aromatase stimulating activity were determined in human seminal plasma and correlated with sperm density and motility. Aromatase stimulating activity was determined with an in vitro rat granulosa cell system and melatonin by radioimmunoassay. Compared to normal semen, aromatase stimulating activity was lower in azoospermic individuals, while melatonin was higher in oligospermic and azoospermic samples. Aromatase stimulating activity correlated positively with sperm concentrations and a negative correlation was found between melatonin and sperm progression. These findings suggest that low sperm production is associated with low aromatase stimulating bioactivity in seminal plasma; and melatonin may have an effect upon both sperm production and motility.

Prostatic origin of pyruvate kinase activity in human seminal plasma.

Pyruvate kinase activity is found in normozoospermic, oligozoospermic, azoospermic and vasectomised human seminal plasma. The split ejaculate technique was used to determine the origin of this enzyme. Sperm number, acid phosphatase activity and fructose concentrations were the control parameters to ensure proper collection. Evidence is presented that the prostate is the major source of pyruvate kinase in human seminal plasma.

Heme oxygenase enzyme activity in human seminal plasma of fertile and infertile males

This work aimed to assess heme oxygenase (HO) enzyme activity relationship with different human semen parameters. One hundred and twenty men were divided according to their sperm count and clinical examination into: obstructive azoospermia (n = 20), nonobstructive azoospermia (NOA) (n = 25), oligozoospermia (n = 35) and normozoospermia (n = 40). Semen analysis, western blot for HO-1 and HO-2, and estimation of seminal plasma HO enzyme activity chemically in the form of bilirubin concentration were carried out. Seminal plasma HO enzyme activity was very low in OA specimens, low in NOA, moderate in oligozoospermia while higher in normozoospermia (mean +/- SD; 6.26 +/- 2.2, 81.4 +/- 35.5, 283.8 +/- 90.1, 657.4 +/- 227.6 pmol ml(-1) min(-1)) with significant differences. Western blot analysis demonstrated HO-2 expression in all studied groups whereas HO-1 was highly expressed in fertile normozoospermic group compared with other groups. There was positive correlation between seminal plasma HO enzyme activity and sperm concentration, sperm motility percentage, motile spermatozoa ml(-1) and sperm normal morphology per cent. It is concluded that HO enzyme activity in the human seminal plasma is related to spermatogenesis and sperm-motility processes.

The Major Bactericidal Activity of Human Seminal Plasma Is Zinc-Dependent and Derived from Fragmentation of the Semenogelins

One of the major roles of seminal plasma is to provide antimicrobial protection for the spermatozoa in the female reproductive tract. We found that the bactericidal activity of seminal plasma was highest after resolution of the seminal clot and that this antibacterial activity subsequently became greatly diminished. The antibacterial activity was derived from peptides generated by fragmentation of the semenogelins while the semenogelin holoproteins displayed no antibacterial activity. After ejaculation the semenogelin-derived peptides were fragmented to smaller and smaller fragments over time and thereby lost antibacterial activity. This paralleled the loss of antibacterial activity of whole seminal plasma both in vitro and after sexual intercourse. Moreover, the antibacterial activity of the semenogelin-derived peptides generated in seminal plasma was strictly zinc-dependent both at neutral and low pH. These data provide novel roles for the resolution of seminal clots and for the high zinc concentration in human seminal plasma.

Interferon-alpha-like biological activity in human seminal plasma, follicular fluid, embryo culture medium, amniotic fluid and fetal blood.

Interferons (IFNs) are a group of cytokines exhibiting antiviral, antiproliferative and immunoregulatory properties. The principal stimulus for the synthesis of IFNs is the presence of viral double-stranded RNA, although rare examples of constitutive synthesis have also been described. The aim of the present study was to determine IFN-alpha-like biological activity in the seminal plasma, follicular and amniotic fluid, embryo culture medium, and fetal blood obtained from patients without apparent viral or bacterial infections. Interferon-alpha-like biological activity was determined by a standard cytopathic effect inhibition bioassay. The study included two groups of patients. The first group consisted of 30 married couples participating in the programme for assisted reproduction and the second group consisted of 23 patients scheduled for prenatal diagnosis (15 for amniocentesis and eight for cordocentesis). The seminal plasma of infertile men (asthenozoospermia, oligoasthenozoospermia) contained a high titre of IFN-alpha-like antiviral activity. Asthenozoospermia was diagnosed in men with a normal sperm concentration but less than 50% progressively motile sperm and oligoasthenozoospermia was diagnosed in men with a sperm count less than 1 x 10(6) mL(-1). Despite slightly higher antiviral titres in the seminal plasma obtained from asthenozoospermic patients, no clear association between IFN-alpha-like biological activity and sperm concentration was found. Interferon-alpha-like biological activity was found in all samples of follicular and amniotic fluid and in fetal blood of patients with intrauterine growth retardation and trisomy 18. Antiviral titres from seminal plasma and follicular fluids were significantly higher compared with amniotic fluids and fetal blood. Embryo culture medium did not contain IFN-alpha-like biological activity. Our results demonstrate that IFN-alpha-like activity in biological fluids is relevant for reproduction, even in the absence of infection.

Glutathione peroxidase (GPX) activity in seminal plasma of healthy and infertile males.

Human spermatozoa are more dependent on glutathione peroxidase/glutathione reductase (GPX/GR) system, via reduced glutathione (GSH), to inactivate reactive oxygen metabolites (ROMs) such as hydrogen peroxide and organic hydroperoxides. To demonstrate whether there is a substantial difference in GPX activity between normal and pathological seminal plasma, we decided to evaluate the activity of this enzyme in healthy subjects and infertile males with normal hormonal patterns. Our results demonstrate that in healthy subjects the seminal plasma contains a GPX activity that is about 10 times greater than the GPX activity detected in the seminal plasma of infertile males. By using specific antibodies against plasmatic GPX (GPX3), we also demonstrate that enzymatic activity, detected in the seminal plasma of both healthy and infertile males is GPX3. Therefore, the evaluation of GPX activity in human seminal plasma could be a new useful marker of gonadal function in men.

Nitric oxide synthase activity in human seminal plasma

Objectives. To determine whether human seminal plasma possesses nitric oxide synthase (NOS) activity and whether, if present, this activity correlates with standard semen analysis parameters. Recent studies have demonstrated the presence of NOS in the epithelium of the male reproductive tract and have shown that nitric oxide can influence human sperm function in vitro. Methods. Semen samples were collected from 30 men presenting for an infertility evaluation at our institution. After a standard semen analysis, the semen samples were centrifuged at 10,000 g, and the seminal plasma was collected and stored at −20°C for later evaluation of the arginine concentration and calcium-dependent and calcium-independent NOS activity. NOS activity was measured by the conversion of [ 3H] l-arginine to [ 3H] l-citrulline. Results. NOS activity was detected in 28 of 30 seminal plasma samples tested. The mean ± SD calcium-dependent NOS activity was 1.6 ± 0.8 nmol/min/mL and the mean calcium-independent NOS activity was 1.0 ± 0.7 nmol/min/mL of the seminal plasma. The mean ± SD arginine concentration in the seminal plasma was 7.0 ± 1.9 mM. No significant correlations were found between the NOS activity in the seminal plasma and sperm concentration, sperm motility, or leukocyte concentration. The 2 men without detectable NOS activity in their semen had documented congenital bilateral absence of the vas deferens. Conclusions. The results of this study demonstrate the presence of NOS activity in human semen and support a role of nitric oxide in sperm function in vivo. Our results also suggest that the seminal vesicles may be an important source of NOS activity in semen.

Inhibitors on an elastase-like enzyme activity catalyzing Suc-Ala-Ala-Pro-Leu-pNA amidolysis in human seminal plasma.

The behavior of some proteinase inhibitors toward the Suc-Ala-Ala-Pro-Leu-pNA amidolytic enzyme activity in human seminal plasma (HSP) was tested. [(2S, 3R)-3-Amino-2-hydroxy-5-methyl-hexanoyl]L-valyl-L-valyl-L-aspartic acid (Amastatin) and 3-[1-[(2-(hydroxymethyl)- -pyrolidinyl)-2-methylpropyl]-carbamoyl] octanohydroxamic acid (Actinonin) showed strong inhibitory effects. No inhibition of this present enzyme activity was seen with anti-human serum (whole), anti-human leukocyte elastase, phenyl-methyl sulfonyl fluoride, Elastatinal, ethyeneglycol bis(beta-aminoethyl ethyl)N,N,N:N'-tetra acetic acid, and [L-3-trans-ethoxycarbonyl-oxirane-2-carbonyl]1-L-leucine(3-methylbutyl)a mido (E-64). No relation was observed between human pancreatic elastase antigen and the Suc-Ala-Ala-Pro-Leu-pNA amidolytic enzyme enzyme activity in HSP. Two peaks of Suc-Ala-Ala-Leu-Pro-pNA amidolytic enzyme activity were separated by Cellulofine GCL-2000 gel filtration and these activities were completely abolished by addition of Amastatin. Suc-Ala-Ala-Pro-Leu-pNA amidolytic enzyme activity in HSP is not an elastase-like metalloproteinase but is rather an acyl amidase-like leucine aminopeptidase.

Nonenzymatic antioxidant activity of human seminal plasma

To examine the nonenzymatic antioxidant properties of human seminal plasma. Determination of total (peroxyl) radical-trapping antioxidant parameter and malondialdehyde concentration in seminal plasma of 112 men attending IVF-ET or intracytoplasmic sperm injection (ICSI) programs. University-based center for reproductive medicine. Normozoospermic men, ages 25 to 50 years, of fertility proved by a pregnancy resulting from IVF-ET, and men ages 25 to 50 years with negative IVF-ET or ICSI outcome, distinguished by their smoking status. Seminal plasma was obtained from ejaculates collected for IVF-ET or ICSI procedures. Total (peroxyl) radical-trapping antioxidant parameter and malondialdehyde concentrations. No significant differences for total (peroxyl)-radical-trapping antioxidant parameter were observed between normozoospermic IVF-ET or asthenozoospermic ICSI patients; neither was a difference revealed for pregnancy-positive or -negative couples within those groups. Age, smoking status, or increased leukocyte count had no effect on total (peroxyl) radical-trapping antioxidant parameter or malondialdehyde concentrations in seminal plasma. Nonenzymatic antioxidant activity in seminal plasma does not reflect sperm fecundity ability.

Gender-specific Glycosylation of Human Glycodelin Affects Its Contraceptive Activity

We have recently demonstrated that a human amniotic fluid-derived glycoprotein, glycodelin-A (GdA; previously known as PP14 or PAEP), potently inhibits gamete binding in an established sperm-egg binding system and expresses immunosuppressive activities directed against a variety of different immune cell types. GdA has high mannose-, hybrid-, and complex-type biantennary oligosaccharides including structures with fucosylated or sialylated N, N'-diacetyllactosediamine (GalNAcbeta1-4GlcNAc) sequences, which are rare in other human glycoproteins. We now report the characterization of glycodelin-S (GdS). This is a human seminal plasma glycoprotein that is immunologically indistinguishable from GdA, but unlike the latter, does not inhibit human sperm-zona pellucida binding under hemizona assay conditions. Analysis of the N-glycans of GdS by mass spectrometry revealed that all glycoforms of GdS are different from those of GdA. GdS glycans are unusually fucose-rich, and the major complex-type structures are biantennary glycans with Lewisx (Galbeta1-4(Fucalpha1-3)GlcNAc) and Lewisy (Fucalpha1-2Galbeta1-4(Fucalpha1-3)GlcNAc) antennae. It is probable that these highly fucosylated epitopes contribute to the immunosuppressive activity of human seminal plasma and to the low immunogenicity of sperm. This study provides the first evidence for gender-specific glycosylation that may serve to regulate key processes involved in human reproduction.

Human leucocyte antigens and nasopharyngeal carcinoma.

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