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.     

Detection of immunosuppressive activity in human seminal plasma by an immunobioassay

Recent evidence has shown that under in-vitro conditions human seminal plasma can interfere directly or indirectly with the function of cells of the immune system. It is however, questionable whether the results generated in vitro can be related directly to in-vivo activity. We have therefore standardized an in-vivo immunobioassay to detect the immunosuppressive property of human seminal plasma using adoptive transfer of contact sensitivity to a specific antigen such as dinitrofluorobenzene. Our results indicate that when sensitized lymphoid cells were incubated in vitro with human seminal plasma, their ability to transfer the delayed hypersensitivity in non-sensitized mice was suppressed or inhibited in comparison with the controls. The percentage suppression varied with different samples but the results indicate clearly that the immunosuppressive properties of human seminal plasma can be demonstrated using an in-vivo immunoassay.     

Heme oxygenase enzyme activity in seminal plasma of oligoasthenoteratozoospermic males with varicocele

This work aimed to assess seminal plasma heme oxygenase (HO) enzyme activity in oligoasthenoteratozoospermia (OAT) males with varicocele. Ninety‐three men were divided according to their sperm count and clinical examination into: healthy fertile controls (n = 34), OAT without varicocele (n = 37) and OAT associated with varicocele (n = 22). They were subjected to semen analysis and estimation of seminal plasma HO enzyme activity in the form of bilirubin concentration. Seminal plasma HO enzyme activity decreased significantly in OAT cases compared with controls. Seminal plasma HO in OAT cases associated with varicocele decreased significantly compared with OAT cases without varicocele and healthy controls (mean ± SD; 109.2 ± 29.5, 283.6 ± 88.4, 669.5 ± 236.1 nMol bilirubin/mg ptn/min, P < 0.001). There was positive correlation between seminal plasma HO enzyme activity and sperm concentration, per cent of motile spermatozoa, number of motile spermatozoas ml^?1 and significant negative correlation with sperm abnormal forms per cent. It is concluded that varicocele has a negative impact on seminal HO enzyme activity. Therefore, improved seminal picture after correcting varicocele repair might be related, in part, to improved HO action(s).     

Phospholipase A2 activity in prostasomes from human seminal plasma

Phospholipase A2 (PLA2) activity was measured and partially characterized in prostasomes isolated from human seminal plasma. The results show high PLA2 activity in seminal plasma with a threefold enrichment in the isolated prostasomes. Highest activity was found at pH 8.0 and 10.5, and there was an absolute requirement for calcium with almost total inhibition of PLA2 activity in the presence of 1 mM EDTA. Analysis with two-dimensional gel electrophoresis of prostasome material revealed a complex protein pattern with most of the proteins in the molecular weight interval of 10,000-90,000 daltons. The possible role of PLA2 in prostasomes is discussed.     

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.     

人精浆中端粒酶活性测定及其临床意义

目的 观察精浆端粒酶活性(TA)在不同类型男性不育症患者中的变化,以及与精浆生殖激素浓度、精液中精子数量和质量之间的相互关系.方法 随机选取110例男性不育症患者与30例生育者,应用酶联免疫吸附(ELISA)方法,对其精浆TA进行检测;应用放射免疫分析(RIA)方法对其精浆T、FSH、LH浓度进行检测;按WHO规定的方法,定量分析精液中的精子密度、精子活率、(a+b)级精子活力百分率、精液中白细胞数量.结果 不育症组精浆TA和T浓度均明显低于生育组(P均<0.01);不育症组精浆FSH和LH浓度均明显高于生育组(P均<0.05).在不育症组中,随着精液中精子数量的递减,TA水平亦呈现明显的逐渐下降趋势;精浆中TA与T浓度之间存在明显的相关性(P<0.01),.与FSH和LH浓度之间无相关性(P>0.05);精子活力、活率正常组精浆TA均高于不良组(P<0.01);WBC精液组精浆TA高于非WBC精液组(P<0.05).结论 精浆TA水平与精子数量和质量存在一定的相关性,同时与精子发育密切相关的睾酮浓度有协同促进作用,端粒酶在人类生殖发育中发挥着重要作用.检测精浆TA水平对于进一步了解、预防和治疗某些病理状态下所致的生殖能力低下有重要意义.     

Quantification of seminal plasma motility inhibitor/semenogelin in human seminal plasma

Semenogelin I and II (Sg I and II) are the major components of human semen coagulum. The protein is rapidly cleaved after ejaculation by the chymotrypsin-like protease prostate-specific antigen (PSA), which results in the liquefaction of the semen coagulum and the progressive release of motile spermatozoa. One of the cleavage products of the protein, a 14-kDa protein, is a sperm motility inhibitor (seminal plasma motility inhibitor [SPMI]). We developed a monoclonal antibody (mAb) that is specific to the fragment of Sgs, SPMI, and a sandwich enzyme-linked immunosorbent assay (ELISA) system for the quantification of Sgs using this mAb. Then, we measured SPMI/Sg levels in human seminal plasma from healthy male volunteers (n = 100, aged 18-24 years). The mean level of SPMI/Sg in seminal plasma was 19 +/- 13 mg/mL (range, 4-68 mg/mL). Log-transformed SPMI/Sg levels were negatively correlated with the sperm motility (r = -0.229, P =.0220) and positively correlated with the total protein concentration (r = 0.793, P <.0001). This result supports that SPMI, one of the fragments of Sg, has its inhibitory effect on ejaculated spermatozoa in liquefied semen under physiological conditions.     

Origin of an 84-kDa protein with ABH blood-typing antigen activity in human seminal plasma.

In order to investigate the origin of an 84-kDa protein with ABH blood-typing antigen activity (p 84) and its concentration in human seminal plasma, a monoclonal antibody (mAb p 84) was produced. The protein was recognized in breast milk as well as in seminal plasma by an indirect, enzyme-linked immunosorbent assay (ELISA) using this mAb. mAb p 84 identified 84-kDa and 83-kDa forms of the protein in seminal plasma and breast milk, respectively, on immunoblotting. The mean concentration of p 84 in seminal plasma was 949 microg/ml (n = 54 subjects). There was no significant difference in the concentration of p 84 between individuals who secreted (Se) or did not secrete (non-se) the ABH antigen into their seminal plasma, nor were there any significant correlations between the concentration of p 84 and the total seminal protein concentration. An immunohistochemical study using mAb p 84 with light microscopic detection showed that p 84 was located in the cytoplasm of the inner layer of pseudostratified cuboidal epithelial cells of the seminal vesicles, but no immunoreactivity was found in the prostate. These data indicate that p 84 originates from a single tissue, the seminal vesicles, and suggest that p 84 is an ABH epitope-bearing protein that has not previously been identified but possesses some immunological properties similar to those of lactotransferrin.     

Free L-carnitine in human seminal plasma

It has often been suggested that determination of free L(—)-carnitine in seminal plasma may provide a good indication of epididymal function. However, there has been disagreement regarding the origin of L(—)-carnitine (epididymis and seminal vesicles) and its concentration in human seminal plasma. In this study, free L(—)-carnitine was determined after deproteinization with an enzymatic spectrophotometric method. In 29 semen samples from fathers and with normal spermiograms (semen volume between 2 and 6 ml, sperm count over 20.10⁶/ml, more than 50% motile spermatozoa), the total free L(—)-carnitine in the seminal plasma was 1010 nmoles (SD:±480), in 16 samples from vasectomized men it was 131 nmoles (SD:±77), and in 5 from men with agenesis of the vas deferens and seminal vesicles it was 21 nmoles (SD:±25). These results suggest that free L(—)-carnitine in the seminal fluid is predominantly of epididymal origin. The results of free L(—)-carnitine determinations in split ejaculates and the absence of a correlation between L(—)-carnitine and fructose concentrations in semen from normal subjects indicate that the seminal vesicles make only on minor contribution to L(—)-carnitine in the seminal plasma.     

Phospholipase A activity in seminal plasma of men attending an infertility clinic

Seminal plasma from 40 men attending an infertility clinic was analyzed for phospholipase A activity and compared with serum from 20 healthy blood donors. The imprecision of the method was acceptable and the overall coefficient of variation was 8.1%. A huge discrepancy existed between seminal plasma and blood serum with regard to phospholipase A activity, the former fluid displaying on average a 180-fold higher activity than the latter. Preincubation of seminal plasma for 1 h at 37 degrees C resulted in an even higher phospholipase A activity while the corresponding activity in serum remained unchanged by such a treatment. This suggested the existence of a zymogen form of phospholipase A in seminal plasma. A significant correlation existed between sperm concentration and phospholipase A activity in seminal plasma (r = 0.46; p less than 0.01) and preincubated seminal plasma samples from normozoospermic men displayed a significantly higher mean value than those of azoospermic men. Phospholipase A activity also correlated significantly to zinc concentration in seminal plasma (r = 0.67; p less than 0.001). 4-Bromophenacyl bromide was inhibitory to a certain extent, the inhibition being most evident in the samples with high phospholipase A activity. Dibucaine and quinacrine, known phospholipase inhibitors in other systems, had no inhibitory effects. Only 58% of the seminal plasma samples contained measurable amounts of triglycerides. No significant correlation existed between triglyceride concentration and phospholipase A activity, r = 0.11.     

Zinc and magnesium in human seminal plasma

Several hundreds of specimens of human seminal plasma have been analyzed for zinc, magnesium, acid phosphatase activity, cholesterol and fructose. The semen was also analyzed for sperm density, motility, viability and morphology. The mean concentrations of zinc and magnesium were 129 and 106 g per ml, respectively. Zinc and magnesium concentrations were correlated (r=0.84) and both ions appear suitable for evaluation of the secretory activity of the prostate gland. Three out of four semen samples with less than 50 g Zn and/or Mg per ml had pathological spermiograms compared to 40 per cent among those with more than 100 g/ml.Presented at the VIth World Congress on Fertility and Sterility, Tel Aviv, 1968.     

Deoxyribonuclease activity in human seminal fluid

Deoxyribonuclease (DNase) activity was examined in the whole and two split fractions of human seminal fluid from normospermic, oligozoospermic, and azoospermic origins as well as in sonicates of isolated sperm after freezing and thawing of samples and at various pH values of substrates. The method consisted in the measurement of digested areas in plates containing herring sperm deoxyribonucleic acid (DNA). No correlation was found between DNase activity (875 +/- 22 (SE) ng/ml) and seminal fluid quality. The enzyme activity was significantly lower in the second split portion (764 +/- 43 ng/ml) as compared to the first (971 +/- 41 ng/ml). Sonicates of washed sperm were inactive. It is suggested that DNase activity derives from the epididymis and possibly from the vas deferens where it participates in the decomposition of DNA from dead cells.     

Inhibition of sialyl transferase activity by gossypol acetic acid in human seminal plasma

Seventeen aliquots of 15 microliters seminal plasma of human origin were incubated with 25 micrograms, 15 micrograms, 5 micrograms and 2.5 micrograms gossypol acetic acid (GAA), dissolved in BWW containing 0.9% sodium chloride and 0.9% benzyl alcohol, at 37 degrees C for 60 minutes. Following incubation, activity of sialyl transferase (S.T.) was determined by a procedure involving incorporation of radioactive sialic acid into asialofetuin. The activity of S.T., expressed as cpm per hour per 15 microliters seminal plasma was compared to controls consisting in incubation of sperm in BWW containing 0.9% benzyl alcohol and 0.9% sodium chloride. The activity of S.T. in controls represented 61.3 +/- 8.0%-67.7 +/- 8.9% of the activity obtained by incubation with BWW only. GAA was found to exert a dose-dependent inhibition of S.T. activity, ranging from 38.3 +/- 20.6% to 53.4 +/- 19.4% (with 25 micrograms) and from 11.3 +/- 14.8% to 21.9 +/- 14.8% (with 2.5 micrograms).     

Lipid composition of human seminal plasma

This study aims at determining the amounts of cholesterol, triglycerides, phospholipids, and nonesterified fatty acids in man's seminal liquid and determining their possible variations linked with the ways of taking and congealing samples. It concludes the determinations of lipids in human seminal liquid are reproducible; the way of taking samples has no real influence; however, it seems best to centrifuge sperm immediately after liquefaction to avoid use of triglycerides and NEFA by the spermatozoa.     

Lack of HLA-molecules on human spermatozoa and in seminal plasma

Summary. The expression of human leukocyte antigens (HLA) on ejaculated spermatozoa and on lymphocytes was compared by flow cytometry using monoclonal antibodies towards HLA class I (pan-HLA-A, -B, -C) and class II (DR) antigens. Soluble antigens of HLA class I (s HLA-A, -B, -C) in seminal plasma and in blood plasma were monitored with an elisa technique. Lymphocytes showed specific fluorescence after incubation with the antibodies against HLA class I and class II (DR), whereas, on spermatozoa no positive immunofluorescence could be detected. No antibodies were bound to any significant extent either after modifications of sperm preparation (density gradient centrifugation, swim up-technique, addition of azide, foetal calf serum or benzamidine chloride) or after treatment of spermatozoa with detergens. Furthermore, different concentrations of soluble HLA-A, -B, -C in seminal plasma and in blood plasma were detected. The latter one showed soluble HLA about four-fold more concentrated than the seminal plasma (x ± SD: 262.5 ± 144.4 nmol 1-¹vs. 62.5 ± 27.1 nmol 1-¹). These results suggest, that the HLA-expression differs between human spermatozoa and somatic cells.     

Angiotensin converting enzyme in human seminal plasma is synthesized by the testis, epididymis and prostate

The activity of angiotensin converting enzyme (ACE) was assessed in human body fluids (serum, seminal plasma, prostatic secretions), in tissue extracts of the testis, epididymis, prostate and skeletal muscle, in split ejaculates and in seminal plasma obtained from patients before and after vasectomy. To ensure the specificity of the results the dependence of ACE activity on specific inhibitors was evaluated. Enzyme activity found in tissues of the male genital tract was considerably higher than that in serum and other tissues. ACE in human seminal plasma is synthesized by the testis, epididymis and prostate in different amounts.     

Studies of immunosuppressive substances in human seminal plasma

Endogenous opioids, which have been shown to exhibit pharmacological and physiological activity on the immune system, have recently been reported to inhibit rosette formation between human T lymphocytes and sheep red blood cells. Because those opioids were found at a higher concentration in seminal plasma than in plasma, we studied whether seminal plasma could influence E rosette formation in 23 patients who complained of infertility. Seminal plasma significantly inhibited E rosette formation. With the dilution of seminal plasma, the percentage of E rosette formation increased. The incubation mixture of naloxone prevented the inhibition of E rosette formation produced by seminal plasma. The percentage of E rosette formation was significantly inhibited in azoospermia and oligozoospermia than in normozoospermia. In conclusion, these results suggest that opioids exert a suppressive effect on E rosette formation and may play a role in human spermatogenesis.     

Prostasomes as zinc ligands in human seminal plasma

Prostasomes are small vesicles, containing zinc, secreted by prostate in human seminal plasma and showing a physiological role on sperm properties. In this study, the possible correspondence between prostasomes and a prostatic high molecular weight protein complex, recently indicated as zinc ligand, has been investigated. Isolated prostasomes, examined by scanning electron microscopy, were dialysed to evaluate their zinc binding capacity. Furthermore, seminal plasma Sephadex G-75 elution was carried out before and after prostasome removal. Prostasome preparations, containing typical vesicles of 50-500 nm, showed a positive correlation between their zinc and protein levels. They were able to take up zinc against gradient. Furthermore, the seminal zinc amount, bound to the high molecular weight proteins, was strongly reduced in the free-prostasome sample with respect to the total seminal plasma. This study suggested the correspondence between the prostasomes and a high-sized zinc ligand complex of prostatic origin. Therefore, it demonstrated, for the first time, the zinc binding capacity of prostasomes, a new property which could be related to their biological functions.