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PubMed1993

Optimization of the Hamilton-Thorn computerized sperm motility analysis system for use with rat spermatozoa in toxicological studies.

To optimize the Hamilton-Thorn Motility Analyzer (HTM; Hamilton-Thorn Research, Beverly, MA) for use in reproductive toxicology studies with rat spermatozoa, the accuracy and precision of the instrument were assessed under a variety of instrument settings. Videotapes of both fast- and slow-swimming sperm were analyzed repeatedly to obtain data across a range of sperm velocities as might be encountered as a consequence of exposure to reproductive toxicants. Acquisition rates were varied across the HTM menu choices (30, 19, 10, or 7 frames/sec) as were the number of frames analyzed (5 to 20) at each framing rate. For fast-swimming samples (mean straight-line velocity (VSL) approximately 130 microns/sec) generally good agreement between computer-assisted sperm analysis (CASA) and manually obtained data was found for percentage of motile sperm and straight-line velocity; i.e., CASA values were within 10% of manual values for most frame/rate combinations. The accuracy of these measures held true over a wide range of sperm concentrations and percentage motilities. However, CASA measures were less accurate for sperm samples of lower velocities (mean VSL approximately 50 microns/sec and mean VSL approximately 30 microns/sec) in that the velocity of very slow sperm was overestimated (particularly at 30 frames/sec). A soft-ware change (6.5R) and performing analyses at 19 instead of 30 frames/sec improved straight-line accuracy for the slow sperm and enhanced the discrimination between fast (presumably control) and slow (presumably treated) sperm samples. These data show that this motility analyzer could be successfully configured to evaluate rodent sperm samples. The use of such CASA systems in toxicology studies will provide valuable information that may improve human reproductive risk assessment.

Animals↗
PubMed1981

Inhibition of human sperm motility by inhibitors of choline acetyltransferase.

Human spermatozoa contain choline acetyltransferase (ChA), acetylcholinesterase and acetylcholine (ACh). There is no storage pool for ACh in spermatozoa. Therefore, ChA inhibitors should exhibit dramatic effects in the alteration of levels and turnover of ACh and sperm motility. The effects of two groups of ChA inhibitors, 2-benzoylethyltrimethylammonium (BETA) and related compounds and halogenoacetylcholines (cholinesters of iodo-, bromo- and chloroacetic acids; XACh, where X = l, Br or Cl), on the motility index of human ejaculated sperm were studied. These investigations gave the following results: 1) BETA was a potent inhibitor of ChA from monkey brain (I50, 4.8 x 10(-6) M), homogenates of rat spermatozoa (I50, 6.5 x 10(-5) M) and homogenates of human spermatozoa (I50, 5.6 x 10(-5) M). It decreased the motility index of human spermatozoa (I50, 8.5 x 10(-8) M) at concentrations higher than 10(-8) M after a contact time of 5 to 60 min. It decreased the motility index of human spermatozoa by about 80% after 5 min and by 95% after 1 hr at a concentration of 10(-6) M. 2) There was a positive relationship between the inhibition of ChA and the depression of the motility index of human spermatozoa among these inhibitors. Both the number of motile cells and the graded motility were decreased. 3) All ChA inhibitors studied are quaternary ammonium compounds that do not significantly cross membrane barriers. 4) Both human sperm cells and human sperm cell homogenates had the same ChA activity. 5) Seventy-five percent of ChA activity could be washed away from human spermatozoa. 6) The same amount of ChA inhibition was observed when BETA was added to the homogenate of sperm cells or whole sperm cells. All of these observations indicate that sperm cell ChA is accessible to BETA and related quaternary ammonium compounds. These studies indicate that ACh is possibly synthesized by the tail and is a local hormone in the coordination of contraction and relaxation cycles of spermatozoan flagella.

Acetylcholine↗
PubMed1991

Identification of apolipoprotein A1 and immunoglobulin as components of a serum complex that mediates activation of human sperm motility.

Serum is superior to other body fluids in activating the progressive motility of human spermatozoa and is used in connection with sperm separation for fertilization in vitro. The major activating capacity is localized to a macromolecular fraction, purified to homogeneity by a four-step protocol with ion-exchange chromatography, chromatofocusing, exclusion FPLC (elution corresponding to a molecular mass of about 250 kDa), and Blue Sepharose chromatography (no binding but elimination of albumin). The pure protein, at a concentration of 20-70 nmol/L, activated the motility to the same extent as serum. SDS-polyacrylamide gel electrophoresis under nonreducing conditions showed one band corresponding to a molecular mass of about 180 kDa. In the presence of mercaptoethanol, two bands are obtained corresponding to 50 kDa and about 25 kDa, respectively. Without the Blue Sepharose step, the sample after reduction revealed an additional band at about 67 kDa, suggesting that the fraction is then in complex also with albumin. Amino acid sequence analysis of the Blue Sepharose eluate identified three protein chains--those of apolipoprotein A1 and immunoglobulin heavy and light chains--suggesting that the preparation is an apolipoprotein A1-immunoglobulin complex. Antiserum raised toward the pure preparation in a rabbit inhibited human sperm motility, when added directly to spermatozoa. Pretreatment of human serum with rabbit antiserum significantly reduced its ability to activate sperm motility. The sperm activating capacity of the protein complex was destroyed by heating at 100 degrees C for 5 min, suggesting that the activity was dependent on intact protein conformations. Albumin, apolipoprotein A1, and immunoglobulins by themselves had only minor effects on sperm motility.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2006

The production of peroxynitrite by human spermatozoa may affect sperm motility through the formation of protein nitrotyrosine.

OBJECTIVE: To detect peroxynitrite and 3-nitrotyrosine production in human spermatozoa of asthenozoospermic infertile patients and normospermic donors, and evaluate any influence on kinetic sperm features. DESIGN: Basic study. SETTING: Andrology Unit, Dept of Internal Medicine and Biochemistry Institute, Polytechnic University of Marche, Italy. PATIENT(S): Sixty-nine infertile patients affected by idiopathic asthenozoospermia and 29 normal fertile donors. INTERVENTION(S): No therapeutic intervention was performed on patients. MAIN OUTCOME MEASURE(S): Production of peroxynitrite (ONOO-) and 3-nitrotyrosine by human spermatozoa; kinetic sperm cells parameters. RESULT(S): Normospermic fertile donors exhibited ONOO- concentrations significantly lower than those of asthenozoospermic infertile men (9.11 +/- 3.37 vs. 27.46 +/- 5.77 nmol/10(6) cells); confocal microscopy showed that ONOO- was more evident in spermatozoa of patients than in healthy donors. Moreover, a significant negative correlation was evident between ONOO- concentration and total sperm motility, curvilinear velocity (VCL), straight progressive velocity (VSL), and linearity coefficient. Finally, an increase was found in the nitration of the tyrosine residues in asthenozoospermic samples compared to controls. CONCLUSION(S): Spontaneous tyrosine nitration occurs in human spermatozoa. This post-translational protein modification is enhanced by an overproduction of peroxynitrite, which is more evident in asthenozoospermic infertile patients when compared with normospermic fertile donors. Motility parameters are negatively affected, suggesting that ONOO- may be involved in defective sperm function.

Adult↗
PubMed2003

Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate.

Antioxidant supplementation during cooling was assayed to improve the motility of frozen-thawed (FT) boar spermatozoa from two different fractions of the ejaculate, the first component of the sperm-rich fraction (Fraction I) and the rest of the bulk ejaculate (Fraction II). Using a split-sample design, addition of two different concentrations (100 and 200 microMl(-1)) of the water-soluble Vitamin E analogue Trolox (6-hydroxy -2,5,7,8-tetramethylchroman -2-carboxylic acid) was evaluated for an effect on sperm motility (measured both subjectively and by means of a computer assisted motility assessment (CASA)), and on mitochondrial membrane potential using flow cytometry after cell-loading with JC-1. The effect of the Vitamin E analogue was clearly dose-dependent and varied with the fraction of the ejaculate considered. Motility was significantly higher in Trolox-treated spermatozoa (200 microm), from either ejaculate fraction, albeit the effect was more evident in spermatozoa from Fraction II (P<0.05) for any Trolox-concentration. Antioxidant supplementation resulted, also dose-dependent, in a higher number of spermatozoa showing high mitochondrial activity as assessed by the JC-1 staining, in both ejaculate fractions. In the present trial, exogenous Trolox positively affected post-thaw sperm viability (as motility and mitochondrial membrane potential) in both fractions of the ejaculate. The magnitude of the effect appeared, however, to be dependent of the fraction of the ejaculate considered.

Animals↗
PubMed2006

Development of a novel CASA system based on open source software for characterization of zebrafish sperm motility parameters.

Although computer-assisted sperm analysis (CASA) outperforms manual techniques, many investigators rely on non-automated analysis due to the high cost of commercial options. In this study, we have written and validated a free CASA software primarily for analysis of fish sperm. This software is a plugin for the free National Institutes of Health software ImageJ and is available with documentation at . That it is open source makes possible external validation, should improve quality control and enhance the comparative value of data obtained among laboratories. In addition, we have improved upon the traditional velocity straight line (VSL) algorithm, eliminating inaccurate characterization of highly curved fish sperm paths. Using this system, the motion of zebrafish (Danio rerio) sperm was characterized relative to time post-activation and the impact of acquisition conditions upon data analysis determined. There were decreases in velocity and path straightness (STR), but not linearity (LIN), relative to time. From 30 to 300 frames/s, frame rate significantly affected curvilinear velocity (VCL) and STR measurements. Sperm density in the field of view did not affect any measured parameter. There was significant inter-male variation for VCL, VSL, velocity average path (VAP), percent motility, path character (STR, LIN), and duration of motility. Furthermore, relative sperm output (a measure reflecting both semen volume and concentration) was positively correlated to percent motility. For all motion parameters measured (except duration), the average CV was < or =10%, comparable to values obtained using commercial systems.

Algorithms↗
PubMedDate not supplied

Laser light-scattering study of the toxic effects of methylmercury on sperm motility.

An in vitro study was designed using the laser light-scattering technique to obtain further information on the dose-effect relationship of methylmercury on sperm motility. The technique provided a quantitative evaluation of sperm swimming speed. Semen samples were collected from normal male Macaca fascicularis monkeys by anal electroejaculation. Methylmercury was added to aliquots of sperm suspensions in BWW medium in doses of 10, 5, 2, and 1 ppm. After 3 hours, the relative speed was 35%, 59%, 69%, and 92% of the corresponding controls at doses of 10, 5, 2, and 1 ppm, respectively. The percentage of motile spermatozoa decreased significantly at 10 ppm. By microscopic observation abnormal motility was detected at 5 and 10 ppm, especially after 20 to 40 minutes. Head movement increased from side to side, and many spermatozoa developed coiled tails. The technique proved useful for defining the dose-effect relationship of methylmercury and sperm swimming speed.

Animals↗
PubMed1988

Capacity of objectively assessed sperm motility characteristics in differentiating between semen of fertile and subfertile men.

A simple and inexpensive computer-assisted method for the objective assessment of sperm motility characteristics was employed to evaluate semen samples of 42 fertile men and 70 subfertile patients. The capacity of each motility parameter to discriminate between semen of the two groups was evaluated by receiver operating characteristic curve analysis. Velocity, linear velocity, and "angular" velocity are reasonably accurate, whereas linearity and angularity index had a very poor discriminating power. The best discrimination between the two groups was the proportion and concentration of sperm with rapid linear progressive motility, based on the cut-off value of linear velocity greater than or equal to 22 microns/sec. This parameter was 90% accurate in discriminating semen of infertile men from that of subfertile patients.

Diagnosis, Computer-Assisted↗
PubMedDate not supplied

Sperm motile efficiency (SME) of fresh spermatozoa in a hypotonic medium correlates with their cryosurvival in fertile men and infertility patients (predicting test).

The sperm motile efficiency (SME) of fresh spermatozoa in a hypotonic medium was investigated concerning the prediction of post thaw motility and the differentiation between fertile men and infertility patients. A total of 95 semen samples were collected for this study. Twenty of these samples were obtained from semen donors of proven fertility and 75 samples were collected from male partners of infertile couples. There were no significant differences between the SME after mixture of the fresh semen with a 140 mOsm saline in equivalent volumes and after cryopreservation (Wilcoxon-matched pairs signed ranks-test) but a significant positive correlation between the SME after both treatments of semen samples was notable. Significant differences between the fertile control group and group of infertility patients could be determined concerning the SME after mixing of fresh semen with 140 mOsm saline as well as after cryopreservation (Mann-Whitney-U-test). The discriminant analysis revealed a most discriminating effectiveness for SME immediately after the addition of hypoosmotic saline.

Freezing↗
PubMed1994

[Tumor necrosis factor in peritoneal fluid of infertile women with endometriosis and its relation to sperm motility].

Tumor necrosis factor (TNF) of 16 infertile women with endometriosis was measured in their peritoneal fluid (PF) with double monoclonal antibody ELISA method, and its effect on sperm motility and membrane integrity was evaluated by semen analysis and hypoosmotic swelling test (HOS). Comparing with that of infertile women without endometriosis (n = 11) and normal fertile women (n = 7), the level of PF-TNF was significantly higher in women with endometriosis (P < 0.01), in accord with the stage of the disease. In the PF of these patients after incubation with the donor sperms, the percentages of forward movement, total motility and hypoosmotic swelling of the donor sperms were significantly decreased (P < 0.01), showing a negative correlation to TNF levels. These results indicate that the elevation of PF-TNF in endometriosis has detrimental effect on sperm function.

Adult↗
PubMed1989

Transmembrane migration technique: reexamination of its usefulness in sperm motility assessments.

A study was carried out using time-exposure photomicrography to investigate the potential usefulness of the transmembrane migration technique in the assessment of drug effects on human sperm motility. Significant but weak correlations were evident between the transmembrane migration ratio (TMMR%) and both % motility (r = 0.43; p less than 0.05) and the amplitude of lateral sperm head displacement (r = 0.40; p less than 0.05). However, no significant correlations were evident between TMMR% and the concentration of motile spermatozoa, mean path velocity, or frequency of sperm head rotation. Exposure to 2.5 mM 2-deoxyadenosine caused significant increases in % motility, mean path velocity, and frequency of sperm head rotation, with no concomitant rise in TMMR%, while 5.0 mM caffeine caused significant elevations in both TMMR% and in % motile cells. It is concluded that the transmembrane migration technique is a relatively insensitive method for detecting subtle but important changes in the movement characteristics of human spermatozoa.

Caffeine↗
PubMed1995

Hyaluronan mediates sperm motility by enhancing phosphorylation of proteins including hyaluronan binding protein.

The present study was carried out to explore the mechanisms underlying hyaluronan-induced sperm motility. We demonstrate for the first time the enhanced cellular protein phosphorylation in hyaluronic acid stimulated cauda spermatozoa. Labeling of spermatozoa with [Pi] orthophosphoric acid yielded a wide range of labeled phosphoproteins in presence of hyaluronan. Under these experimental conditions, we further show tyrosine specific phosphorylation of proteins. In addition, this is also the first report showing enhanced phosphorylation of 34 kDa hyaluronan binding protein in response to hyaluronan. The role of 34 kDa hyaluronan binding protein in hyaluronan-induced spermatozoa is supported by elevated production of inositol triphosphate accompanied by increased phosphorylation in Triton X-100 insoluble cytoskeletal proteins. The evidence strongly suggests the biological importance of hyaluronan binding protein phosphorylation in the transduction of signals resulting from the interaction of hyaluronate with the sperm surface.

Animals↗
PubMed1998

Xenopus laevis fertilisation: analysis of sperm motility in egg jelly using video light microscopy.

Xenopus laevis eggs are surrounded by an extracellular matrix consisting of a vitelline envelope, and three jelly layers, J1, J2, and J3 (from egg surface outward). The jelly layers vary in thickness (about 150, 15 and 200 microns for J1, J2 and J3 respectively) but all are translucent allowing observation of sperm penetration. Video microscopy demonstrated that sperm are able to penetrate and traverse J3 at velocities approaching 30 microns/s. Sperm swim through jelly in a corkscrew-like manner with their rotational and forward velocities being tightly coupled at about 30 degrees/micron forward travel. They are propelled by whip-like power strokes involving hairpin bends in the flagellum that are generated every 180 degrees of rotation and which are propagated from base to tip. The overall trajectories of individual sperm are quite variable. Many sperm head directly for J2 but some do not, these swimming circumferentially, or even away from the egg surface. Most sperm (over 97%) that enter the jelly do not get to the egg surface but are stopped at a variety of positions within J3 or at the outer surface of J2. Efficient sperm penetration and passage through the jelly layers requires a low electrolyte concentration in the surrounding medium, and is inhibited by the lectin wheat germ agglutin (WGA) in a dose-dependent manner. WGA does not block sperm penetration of J3 but does block further progression towards the egg surface. This observation suggests that sperm motility within the jelly is dependent on the carbohydrate moieties of the large glycoconjugates present, and that their alteration by WGA binding accounts for the inability of sperm to reach the egg surface and fertilise the egg.

Animals↗
PubMedDate not supplied

Effect of sperm motility on separation of bovine X- and Y-bearing spermatozoa by means of free-flow electrophoresis.

Free-flow electrophoresis was used on bovine spermatozoa to test the hypothesis that there are surface charge differences between X and Y chromosome-bearing spermatozoa. Spermatozoa were deflected towards the anode by means of tromethamine (THAM) buffers of varying concentrations, and were separated into two populations under specific conditions. Experimental temperature and initial sperm motility had a significant effect on sperm distribution in the electric field. The results of DNA hybridization assays indicated an enrichment for Y chromosome-bearing spermatozoa.

Animals↗
PubMed1981

Mouse sperm motility affected by factors in the T/t complex.

Motility characteristics of epididymal sperm from tw32/+ and T/+ males of several strains were compared to those of sperm from the congenic, wild, type mice. Epididymal sperm from C57BL/6-tw32/+ (B6-tw32/+) males showed a significant decline in vigorous motility and a significant increase in adhesivity to glass during 7.5 hours in vitro, while sperm from B6-+/+ males showed no changes in these characteristics. The apparent mean velocities of epididymal sperm were: B6tw32/+, 32 micro/sec; C3H-tw32/+, 60; B6XC3H hybrids (F2*-tw32/+), 44; and C3HXB6 hybrids (F2-tw32/+), 58. Each of these values was significantly lower than that of sperm from their congenic wild-type strains: B6- +/+, 115; C3H- +/+, 177; F1*-+/+, 168 and F1-+/+, 195 micro/sec. B6-T2J/+ sperm had velocities intermediate to the congenic +/+ and tw32/+ genotypes, whereas C3HT/+ sperm velocity was not significantly different from C3H- +/+. These differences in measured sperm velocity may not reflect real differences in swimming speed, but rather may indicate a difference in the shape of swimming trajectories. Erratic and convoluted swimming trajectories were observed for the majority of sperm from tw32/+ males of all genotypes. Differences in motility patterns of sperm from tw32/+ males could play a role in the abnormally high transmission of tw32 through the male.

Animals↗
PubMed1990

Treatment of spermagglutination with proteolytic enzymes. I. Sperm motility, vitality, longevity and successful disagglutination.

The treatment of antibody-mediated spermagglutination by corticosteroid therapy has a high incidence of side-effects and sperm washing is often followed by re-agglutination. The possibility of enzymatic disagglutination was therefore investigated. In the first part of the study the effects of four proteases on sperm motility, vitality and longevity were evaluated. Subtilisin had prohibitively detrimental effects even at 10 U/ml. However, chymotrypsin (less than or equal to 500 U/ml), trypsin (less than or equal to 500 U/ml) and papain (less than or equal to 50 U/ml) had no adverse effects. In the second series of experiments one or more of these latter three enzymes was found to disagglutinate spermatozoa which had previously been incubated with sperm-agglutinating antibody-positive sera in 87% of cases. Although further investigation is required, enzymatic disagglutination may be beneficial for the treatment of immunologically mediated spermagglutination.

Cell Membrane↗
PubMedDate not supplied

The effects of laser light on sperm motility and velocity in vitro.

The effect of Laser light on the motility and the velocity of human spermatozoa were measured by means of multiple exposure photography. Total sperm motility increased after Laser irradiation at 4 J/cm2, 8 J/cm2 and 32 J/cm2 respectively with respect to control. However, no influence on sperm velocity was demonstrated after Laser irradiation. This observation suggests that Laser light stimulates non-motile live spermatozoa.

Dose-Response Relationship, Radiation↗
PubMed1999

Effect of vitamin E on human sperm motility and lipid peroxidation in vitro.

AIM: To assess the protective efficacy of vitamin E to counteract the reactive oxygen species (ROS) mediated damage on sperm motility, viability and lipid peroxidation. METHODS: Human semen samples were obtained from the local hospital. The split seminal fractions freed of seminal plasma were reconstituted in Ringer-Tyrode and subjected to varied vitamin E concentrations (0.1-2 mmol/L). RESULTS: Dose-dependent improvement in both motility and viability accompanied by concomitant decrease in malondialdehyde (MDA--an end product of lipid peroxidation) following vitamin E supplementation was noticed. CONCLUSION: Vitamin E protects against the ROS mediated damage on spermatozoa. Vitamin E supplementation could be of clinical importance for prolonged spermatozoal storage whenever needed.

Humans↗