Superoxide dismutase isoenzymes in human seminal plasma and spermatozoa

Superoxide radicals may exert both toxic and physiological regulating actions on spermatozoa. The objective of the present study was to examine the occurrence and distribution of the three superoxide dismutase (SOD) isoenzymes in human seminal plasma and spermatozoa. Human seminal plasma has previously been reported to possess high SOD activity. Here we show that the normally cytosolic CuZn-SOD remarkably accounts for 75% of the activity while the secretory extracellular SOD (EC-SOD) accounts for 25%. Studies of split ejaculates suggest that both these SOD isoenzymes are of primarily prostatic origin. The Mn-SOD activity was negligible. The total SOD activity of seminal plasma was 20 times higher than that of human blood plasma. While native EC-SOD shows high affinity for heparin and heparan sulphate, 90% of the EC-SOD in seminal plasma lacks the high affinity at ejaculation. Thus only a minor part of the seminal plasma EC-SOD has the potential to bind to cell surfaces. Human spermatozoa were found to contain exceptionally large amounts of CuZn-SOD. There was little Mn-SOD activity and the amount of EC-SOD was negligible. We conclude that spermatozoa in semen are exceptionally well protected against superoxide radicals both internally and externally. This should be of importance for both their survival and the integrity of DNA, and may also have physiological effects such as influencing capacitation.     

The lipids of buffalo spermatozoa and seminal plasma

An allometric method for estimating the volume change in inflamed paw of rats is described. The technique is effective and advantageous for long term observation of adjuvant arthritis which lacks a suitable reference criterion due to the simultaneous swelling of the control paw. In the present method, the inflammatory intensity (IF) of paw edema is estimated by means of the formula; IF(%)=(2Vr/cXd(I+Wt/aXb)-I) X 100 where Vt is the paw volume, Wt is the body weight weight and X is the tail length of the inflamed rat. The constants a, b, c, and d are obtained from tthe normal rats using the relative growth law, W=aXb and V=cXd (where W is the body weight, V is the paw volume and X is the tail length).     

Extracellular signal-regulated kinases modulate capacitation of human spermatozoa

Recent evidence indicates the presence of p21 Ras and of a protein with characteristics similar to mitogen-activated protein kinases (MAPKs), also known as extracellular signal-regulated kinases (ERKs), in mammalian spermatozoa, suggesting the occurrence of the Ras/ERK cascade in these cells. In the present study we investigated the subcellular localization of ERKs and their biological functions in human spermatozoa. Immunohistochemistry, immunofluorescence, confocal microscopy, and immunoelectron microscopy demonstrated localization of ERKs in the postacrosomal region of spermatozoa. After stimulation of acrosome reaction with the calcium ionophore A23187 and progesterone, ERKs were mostly localized at the level of the equatorial region, indicating redistribution of these proteins in acrosome-reacted spermatozoa. Two proteins of 42 and 44 kDa that are tyrosine phosphorylated in a time-dependent manner during in vitro capacitation were identified as p42 (ERK-2) and p44 (ERK-1) by means of specific antibodies. The increase in tyrosine phosphorylation of these proteins during capacitation was accompanied by increased kinase activity, as determined by the ability of ERK-1 and ERK-2 to phosphorylate the substrate myelin basic protein. The role of this activity in the occurrence of sperm capacitation was also investigated by using PD098059, an inhibitor of the MAPK cascade. The presence of this compound during in vitro capacitation inhibits ERK activation and significantly reduces the ability of spermatozoa to undergo the acrosome reaction in response to progesterone. Since only capacitated spermatozoa are able to respond to progesterone, these data strongly indicate that ERKs are involved in the regulation of capacitation. In summary, our data demonstrate the presence of functional ERKs in human spermatozoa and indicate that these enzymes are involved in activation of these cells during capacitation, providing new insight in clarifying the molecular mechanisms and the signal transduction pathways of this process.     

Inhibition of the oxidative metabolism of human spermatozoa by a heat-labile factor in seminal plasma

The effect of different media on the oxidative and glycolytic metabolism and on the motility of human spermatozoa has been investigated. Sperm resuspended in Ringer's solution produce more carbon dioxide than sperm in the original seminal plasma. In addition, sperm resuspended in heated seminal plasma have a higher carbon dioxide production and higher oxygen uptake than sperm in untreated seminal plasma. The mean lactic acid production was not significantly affected by different media. The motility of sperm in heated seminal plasma was poorer than that in untreated seminal plasma, although the percentage of cells that stained with eosin did not differ in the two media. After being heated, seminal plasma not only continued to take up oxygen but did so at a higher rate. The results of the present study suggest that seminal plasma contains a heat-labile component that inhibits the oxidative metabolism of human sperm but does not significantly alter sperm glycolysis.     

Major proteins of bovine seminal plasma modulate sperm capacitation by high-density lipoprotein

Bovine seminal plasma (BSP) contains four similar proteins secreted by the seminal vesicles, designated BSP-A1, -A2, -A3, and -30 kDa. These proteins bind to choline phospholipids on the surface of the sperm after ejaculation. These BSP proteins also interact with heparin, apolipoprotein A-I (apoA-I) and apoA-I associated with high-density lipoprotein (HDL). The HDL and heparin present in the female reproductive tract have been implicated in sperm capacitation and the acrosome reaction (AR). This study was undertaken to determine whether or not these BSP proteins and HDL could modulate the capacitation of sperm, and to determine the combined effect of HDL and heparin on capacitation. Washed bovine epididymal sperm were preincubated in buffer containing BSP proteins, washed, and incubated with lipoproteins (HDL, and low- and very low-density lipoproteins) or liposomes with or without apoA-I in the presence or absence of heparin. The percentage of capacitated sperm was evaluated after the AR was induced with lysophosphatidylcholine. HDL alone (160 microg/ml) after an 8-h incubation stimulated the AR of epididymal sperm. The percentage of HDL-enhanced AR further increased when sperm were preincubated with BSP proteins. ApoA-I-liposomes stimulated the AR more rapidly (5 h, 160 microg/ml) than HDL. When sperm were preincubated with BSP proteins, the percentage of apoA-I-enhanced AR further increased. In contrast, when liposomes without apoA-I or when low- or very low-density lipoproteins or lipoprotein-depleted serum was used, no significant increase in the AR was detected with or without BSP proteins. When heparin and HDL or apoA-I-liposomes were used together, their combined effects on the AR were not additive. These results indicate that BSP proteins modulate the process of capacitation induced by heparin, HDL, and apoA-I-liposomes.     

Effect of seminal plasma on implantation rates

Of 2774 consecutive, prospectively documented fractures of the distal radius, 225 (8 per cent) occurred as sports injuries, mainly in young men. Soccer produced the greatest number of wrist fractures with 112 cases (50 per cent). Skiing, dancing and rugby caused 12 per cent, 9 per cent and 7 per cent of all sporting wrist fractures respectively. Skiing, horseriding and dancing consistently resulted in more complex fractures. In soccer, synthetic pitches increased the likelihood of a fracture following a fall by a factor of five. Twelve per cent of fractures required further treatment because of instability leading to redisplacement. The complication rate was 14 per cent with the majority being cases of malunion (12 per cent). Of the 131 patients who returned a questionnaire, 72.5 per cent had returned to their original sport. This was influenced mainly by the patient's age and pre-injury standard of competition.     

Induction of capacitation in human spermatozoa in vitro by an endometrial sialic acid-binding protein

A Ca(2+)-dependent sialic acid-binding protein (SABP) of human endometrium, which specifically bound to human sperm head plasma membrane in vitro, was found to increase the percentage motility and acrosome-reacted pattern of uncapacitated spermatozoa. The protein was synthesized in the endometrium and secreted into the uterine fluid. This intra-uterine factor, which is apparently advantageous in vitro in inducing human sperm capacitation, may play a significant role in promoting the post-release maturation of ejaculated spermatozoa by enhancing 45Ca uptake into spermatozoa by a pathway which is insensitive to calcium-channel blockers. However, the 45Ca uptake could be enhanced on exposure to the divalent cation ionophore A23187 and inhibited in the presence of the calmodulin inhibitor trifluoperazine. The SABP also induces an increase in intracellular Ca2+ in spermatozoa, as seen by FURA-2 AM studies. Furthermore, overlay studies show human SABP to be a Ca(2+)-binding protein. The data presented here suggest that SABP induces in-vitro sperm capacitation and the subsequent acrosome reaction by increasing intracellular Ca2+ concentration.     

Effect of natural antioxidants, superoxide dismutase and hydrogen peroxide on capacitation of frozen-thawed bull spermatozoa

This study comprised 100 persons with antibodies to hepatitis C virus (HCV), including 77 intravenous drug users (IVDUs). They were tested with serological HCV typing assays (Murex HCV serotyping 1-6 assay; Chiron RIBA HCV Serotyping SIA). Patients with a positive polymerase chain reaction (PCR) for HCV (n = 66) were tested with genotyping molecular assays (Inno-Lipa HCV II test; Sorin GEN-ETI-K HCV typing assay). Comparison of the results of these tests showed that (a) 92% of samples could be typed by one test at least; 44% could be typed by all four tests; 88% could be typed by one serological test at least and 66% by one molecular test at least; (b) 81% of the samples successfully tested with both serological tests gave comparable results; 95% of the samples successfully tested with both molecular tests gave comparable results; (c) when serological and molecular tests yielded different results, sequences in the 5'-non-coding (5' NC) or E1 regions always confirmed the results of the molecular tests; (d) in case of discrepancy between the results of the molecular tests the E1 region sequences confirmed the Sorin test results. It is concluded that the molecular tests compared gave similar results. The fact that the Murex serological test gave comparable results in more than 80% of cases indicates that it is an alternative to the molecular tests for routine diagnosis. However, comparison of the results of this test with those obtained in patients consulting a hepatology department showed that it gave the best results in a population of patients not exposed repeatedly to HCV.     

Treatment of human spermatozoa with follicular fluid can influence lipid content and motility during in vitro capacitation

In order to evaluate the role of human follicular fluid (HFF) on the fertilizing capacity of spermatozoa, we studied the effect of HFF on the lipid composition and on the movement characteristics of human spermatozoa. Spermatozoa (spz) from normospermic patients were prepared with a discontinuous Percoll gradient and incubated in Ménézo B2 medium with or without a supplement of 20% HFF (HFF-Percoll spz and B2-Percoll spz respectively) for 2 and 24 h. After 2 h HFF incubation, percentage progressive motility, straight line velocity (VSL), and amplitude of lateral head displacement (ALH) were improved in HFF-Percoll spz as compared to B2-Percoll spz (P < or = 0.05). After a longer incubation period (24 h), lipid changes appeared in HFF-Percoll spz with lower levels of cholesterol (P = 0.02) and phospholipids (P = 0.05). No modification of the cholesterol/phospholipid ratio after 2 and 24 h of incubation in either B2-Percoll spz or HFF-Percoll spz was observed. Such decreases in lipid content of HFF-Percoll spz may be factors which could be taken into account as constituting part of membrane modifications during the capacitation process.     

Identification of insulin-like growth factor I in bovine seminal plasma and its receptor on spermatozoa: influence on sperm motility

UDP-N-acetylglucosamine-3-O-acyltransferase (UDP-GlcNAc acyltransferase) catalyzes the first step of lipid A biosynthesis (M. S. Anderson and C. R. H. Raetz, J. Biol. Chem. 262:5159-5169, 1987). We here report the isolation of the lpxA gene of Pseudomonas aeruginosa from a library of Pseudomonas strain PAO1 expressed in Escherichia coli LE392 (J. Lightfoot and J. S. Lam, J. Bacteriol. 173:5624-5630, 1991). Pseudomonas lpxA encodes a 10-carbon-specific UDP-GlcNAc acyltransferase, whereas the E. coli transferase is selective for a 14-carbon acyl chain. Recombinant cosmid 1137 enabled production of a 3-hydroxydecanoyl-specific UDP-GlcNAc acyltransferase in E. coli. It was identified by assaying lysozyme-EDTA lysates of individual members of the library with 3-hydroxydecanoyl-acyl carrier protein (ACP) as the substrate. Cosmid 1137 contained a 20-kb insert of P. aeruginosa DNA. The lpxA gene region was localized to a 1.3-kb SalI-PstI fragment. Sequencing revealed that it contains one complete open reading frame (777 bp) encoding a new lpxA homolog. The predicted Pseudomonas LpxA is 258 amino acids long and contains 21 complete hexapeptide repeating units, spaced in approximately the same manner as the 24 repeats of E. coli LpxA. The P. aeruginosa UDP-GlcNAc acyltransferase is 54% identical and 67% similar to the E. coli enzyme. A plasmid (pGD3) containing the 1.3-kb SalI-PstI fragment complemented E. coli RO138, a temperature-sensitive mutant harboring lpxA2. LpxA assays of extracts of this construct indicated that it is > 1,000-fold more selective for 3-hydroxydecanoyl-ACP than for 3-hydroxymyristoyl-ACP. Mass spectrometry of lipid A isolated from this strain by hydrolysis at pH 4.5 revealed [M-H]- 1,684.5 (versus 1,796.5 for wild-type lipid A), consistent with 3-hydroxydecanoate rather than 3-hydroxymyristate at positions 3 and 3'.     

Sperm-immobilizing monoclonal antibody to human seminal plasma antigens

Rat spleen cells immunized to human azoospermic semen (a mixture of seminal plasma components) and mouse myeloma cells (P3/X63 Ag8U1; P3U1) (Marguilies et al., 1976) were successfully fused with polyethylene glycol (PEG 1500) and 19 of 89 fused cell cultures were found to produce sperm-immobilizing antibody. The cells that produced antibody indicating the highest sperm-immobilizing activity were distributed into wells for further recloning and 10 clones producing sperm-immobilizing antibody were established. The clone (1C4) producing the highest antibody titre was found to produce a large amount of IgG in culture supernatants and to contain a mixture of rat and mouse chromosomes. It was proved by immunodiffusion test that the monoclonal antibody was produced to the human seminal plasma antigen No. 7 which is common to human milk protein. Using this hybridoma which produced a large amount of monoclonal sperm-immobilizing antibody, a new method could be developed for purifying human seminal plasma antigen by immunoaffinity chromatography with bound antibody from the hybridoma.     

Movements of sodium and potassium into ejaculated boar spermatozoa suspended in seminal plasma and a biological salt solution

Uptake of 22Na and 42K into ejaculated boar spermatozoa was measured in vitro. Cells were suspended either in seminal plasma or in a biological salt solution of essentially the same composition as boar seminal plasma. Samples were incubated at 30 degrees C. Correction was made for extracellular space in the centrifuged sperm pellet. This was determined as 22Na-space, which was less (P less than 0.001) than [14C] carboxyinulin space. Large differences were observed among individual ejaculates. The half-time for potassium uptake into the spermatozoa averaged 11.5 min, which is much faster than that for leukocytes or erythrocytes. When the spermatozoa were suspended in the biological salt solution, the initial rate of 42K uptake was significantly decreased. This may be due to disturbances of the protein components of the sperm membrane. The uptake of 22Na into the spermatozoa was slow. Sodium and potassium transport appeared not to be coupled in the 3/2 ratio which has been reported for erythrocyte membranes. The average concentration of sodium was 108 mM in seminal plasma and 26 mM in the spermatozoa (112 mmol/kg water and 38 mmol/kg water, respectively). The corresponding figures for potassium were 26 mM and 51 mM (27 mmol/kg water and 74 mmol/kg water). The random error for a single determination for the various methods used varied between 2.4 and 13.3% of the mean.     

Effect of prolactin on metabolism of human spermatozoa

The effect of prolactin on adenyl cyclase, rate of fructose utilization, and glucose oxidation by human spermatozoa was studied. Prolactin stimulated all of these processes at a concentration generally available in seminal plasma. These results suggest that prolactin plays an important role in the energy metabolism of human spermatozoa.     

Methods for induction of capacitation and the acrosome reaction of stallion spermatozoa

Taking into consideration the traditional official health assistance forms, the present paper discusses questions related to dependence and independence of nurse and patient in the health/illness process. Under that perspective it proposes a relationship that preserves the autonomy of one and the other, so that both can participate in the process of nursing and self-care.     

Regionalization and redistribution of membrane phospholipids and cholesterol in mouse spermatozoa during in vitro capacitation

Fracture-label, surface-replica, and routine freeze-fracture techniques were used in combination with phospholipase A2-colloidal gold (PLA2-CG) and filipin as probes to study changes in the distribution of phospholipids and cholesterol, respectively, in morphologically defined plasma membrane domains of mouse spermatozoa during in vitro capacitation. In noncapacitated spermatozoa, quantitative analysis revealed that the fractured plasma membrane overlying the equatorial segment carried the highest PLA2-CG labeling density. The next highest labeling densities were found in the anterior acrosome region and the post-acrosomal region. On the external surface of the plasma membrane revealed by surface replicas, a uniform distribution of PLA2-CG was confined mainly to the acrosomal region of the head. The plasma membrane of the sperm tail had a relatively low labeling density for PLA2-CG. In freeze-fracture replicas of filipin-treated spermatozoa, the labeling density of filipin/sterol complexes (FSCs) was high in the plasma membrane over the acrosomal region where the FSCs were uniformly distributed. The postacrosomal region was weakly labeled. After in vitro capacitation, the densities of PLA2-CG and FSCs were significantly reduced in the fractured plasma membrane of the sperm head and the middle piece of the tail. However, surface replicas revealed an increased PLA2-CG labeling on the external surface of the plasma membrane covering the postacrosomal region, the middle piece, and the principal piece. Another major change detected in capacitated spermatozoa was the presence of small aggregates and patches of elevated, membrane-associated particles on the surface-replicated plasma membrane in the upper portion of the postacrosomal domain. Here the PLA2-CG labeling density was found to be higher than in noncapacitated spermatozoa. These results provide new information with respect to the reorganization and redistribution of phospholipids in specific regions of the plasma membrane during capacitation and provide further support for the concept that removal or loss of antifusigenic sterol from the sperm plasma membrane constitutes an important step of the capacitation process.     

Generation of a neutrophil chemotactic agent by spermatozoa: role of complement and regulation by seminal plasma factors

The interactions of polymorphonuclear leukocytes, spermatozoa, seminal plasma, and serum have been examined in an in vitro leukocyte chemotaxis system. Bovine or human spermatozoa have no neutrophil chemotactic activity alone, but generate potent activity when incubated with serum. Generation of this activity requires magnesium, but not calcium ions and is blocked by pre-heating the serum. Chemotactic activity is generated normally in serum deficient in immunoglobulins or C4, but is not produced in serum deficient in C3. Thus, spermatozoa activate complement via the alternative pathway. The chemotactically active agent has been identified as the low m.w. C5 cleavage product (C5a) on the basis of heat stability, gel filtration characteristics, and specific inhibition by anti-serum to C5. The spermatozoal constituents responsible for complement activation are largely heat stable, resistant to diisopropyfluorophosphate and on gel filtration compise a heterogeneous group of large m.w. materials. Bovine seminal plasma has no neutrophil chemotactic activity alone; considerable activity, however, is generated by incubating low, but not high concentrations of seminal plasma with serum. The chemotactic agent produced in serum is C5a. Bovine seminal plasma also contains potent heat labile inhibitors of chemotaxis. Two distinct inhibitors differing in m.w. (50 to 100,000 and 15,000) and heat stability have been isolated. The inhibitors appear to act directly on the neutrophil rather than on the chemotactic factor. Human seminal plasma exhibits slight chemotactic activity alone but does not generate significant additional activity on incubation with serum. In contrast to the inhibitory activity of bovine seminal plasma, the human material contains heat-stable, nondialyzable factors which enhance the neutrophil chemotactic response. These studies document a specific mechanism for the directed migration of polymorphonuclear leukocytes toward spermatozoa. The findings may provide an explanation for the observed in vivo leukocyte accumulation in the female genital tract in association with spermatozoa and seminal plasma. The biologic significance of this phenomenon relates to potential leukocyte-spermatozoa interaction which may influence the likelihood of fertilization.     

Identification of a transforming growth factor alpha-like molecule in human seminal plasma

In a double-blind controlled trial, 91 middle-aged and elderly women with mild to moderate hypertension who were not on antihypertensive medication were randomly assigned to treatment with magnesium aspartate-HCl (20 mmol Mg/d) or placebo for 6 mo. Magnesium aspartate-HCl in the given dose was well-tolerated and was not associated with an increased frequency of diarrhea compared with placebo. At the end of the study, systolic blood pressure had fallen by 2.7 mm Hg (95% CI -1.2, 6.7; P = 0.18) and diastolic blood pressure by 3.4 mm Hg (1.3, 5.6; P = 0.003) more in the magnesium group than in the placebo group. Blood pressure response was not associated with baseline magnesium status, as measured by dietary magnesium intake and urinary magnesium excretion. Urinary magnesium excretion in the magnesium group increased by 50% during the intervention period. No changes were seen in other biochemical indexes, including serum concentrations of total and high-density-lipoprotein cholesterol. The findings suggest that oral supplementation with magnesium aspartate-HCl may lower blood pressure in subjects with mild to moderate hypertension.     

Oxygen consumption of human seminal plasma: relationships with semen characteristics

It is possible to determine oxygen consumption rate in human seminal plasma through voltammetric measurements. The seminal fluid of the sterile patients in this investigation was characterized by an anomalous sperm motility. Also studied was a control group of proven fertile subjects. The mean value of oxygen consumption resulted greater in the reduced sperm motility group than in the control group. The polyaminoxidase system is the most important factor in oxygen consumption in seminal plasma; however, it is likely that the high toxicity of polyamine degradation products is the cause of reduced and/or anomalous sperm motility in some seminal fluids.