Testing human sperm chemotaxis: how to detect biased motion in population assays

Biased motion of motile cells in a concentration gradient of a chemoattractant is frequently studied on the population level. This approach has been particularly employed in human sperm chemotactic assays, where the fraction of responsive cells is low and detection of biased motion depends on subtle differences. In these assays, statistical measures such as population odds ratios of swimming directions can be employed to infer chemotactic performance. Here, we report on an improved method to assess statistical significance of experimentally determined odds ratios and discuss the strong impact of data correlations that arise from the directional persistence of sperm swimming.     

Increased velocity and induction of chemotactic response in mouse spermatozoa by follicular and oviductal fluids.

The dynamic parameters of mouse sperm cells exposed to follicular and oviductal fluids were assessed. Spermatozoa were tracked on a chemotactic Zigmond chamber and recorded using a videomicroscopy system. The results were evaluated with computer-supported image analysis. Follicular fluid at a dilution of 10(-4) markedly increased the proportion of spermatozoa with high velocity, and stimulated chemotactic behaviour. The highest velocities were observed in sperm cells exposed to oviductal fluid, and a greater proportion of these cells had high velocity compared with those exposed to follicular fluid. Chemotaxis was induced in spermatozoa exposed to oviductal fluid at dilutions of 10(-3) and 10(-5). These results suggest the presence of temporal subpopulations of responsive spermatozoa, considering the distance travelled towards both follicular and oviductal fluids and the proportion of sperm cells migrating towards the gradient in the highest distance ranges. This is the first report on the effect of isolated follicular and oviductal fluids on dynamic parameters and chemotaxis of mouse spermatozoa. The findings support previous work showing that the motility and directionality of mouse sperm cells is increased by factors in the microenvironment of the egg. Although the significance of these factors in vivo is unknown, it is possible that there is a relay mechanism involving sequential activity of both oviductal and follicular fluids to direct the male gametes towards the egg.     

Chemotaxis of capacitated rabbit spermatozoa to follicular fluid revealed by a novel directionality-based assay

Precontact communication between gametes is established by chemotaxis. Sperm chemotaxis toward factor(s) in follicular fluid (FF) has been demonstrated in humans and mice. In humans, the chemotactic responsiveness is restricted to capacitated spermatozoa. Here, we investigated whether sperm chemotaxis to factor(s) present in FF also occurs in rabbits and, if so, whether only capacitated spermatozoa are chemotactically responsive. Chemotaxis assays were performed by videomicroscopy in a Zigmond chamber. We measured chemotactic responsiveness as a function of FF dilution by means of a novel directionality-based method that considers the ratio between the distances traveled by the spermatozoa both parallel to the chemoattractant gradient and perpendicular to it. A peak of maximal response was observed at 10(-4) dilution of FF, resulting in a typical chemotactic concentration-dependent curve in which 23% of the spermatozoa were chemotactically responsive. In contrast, the percentage of cells exhibiting FF-dependent enhanced speed of swimming increased with the FF concentration, whereas the percentage of cells maintaining linear motility decreased with the FF concentration. The percentages of chemotactically responsive cells were very similar to those of capacitated spermatozoa. Depletion of the latter by stimulation of the acrosome reaction resulted in a total loss of the chemotactic response, whereas the reappearance of capacitated cells resulted in a recovery of chemotactic responsiveness. We conclude that rabbit spermatozoa, like human spermatozoa, are chemotactically responsive to FF factor(s) and acquire this responsiveness as part of the capacitation process.     

Behavioral mechanism during human sperm chemotaxis: involvement of hyperactivation

When mammalian spermatozoa become capacitated they acquire, among other activities, chemotactic responsiveness and the ability to exhibit occasional events of hyperactivated motility--a vigorous motility type with large amplitudes of head displacement. Although a number of roles have been proposed for this type of motility, its function is still obscure. Here we provide evidence suggesting that hyperactivation is part of the chemotactic response. By analyzing tracks of spermatozoa swimming in a spatial chemoattractant gradient we demonstrate that, in such a gradient, the level of hyperactivation events is significantly lower than in proper controls. This suggests that upon sensing an increase in the chemoattractant concentration capacitated cells repress their hyperactivation events and thus maintain their course of swimming toward the chemoattractant. Furthermore, in response to a temporal concentration jump achieved by photorelease of the chemoattractant progesterone from its caged form, the responsive cells exhibited a delayed turn, often accompanied by hyperactivation events or an even more intense response in the form of flagellar arrest. This study suggests that the function of hyperactivation is to cause a rather sharp turn during the chemotactic response of capacitated cells so as to assist them to reorient according to the chemoattractant gradient. On the basis of these results a model for the behavior of spermatozoa responding to a spatial chemoattractant gradient is proposed.     

Quantification and identification of sperm subpopulations using computer-aided sperm analysis and species-specific cut-off values for swimming speed

Abstract

Motility is an essential characteristic of all flagellated spermatozoa and assessment of this parameter is one criterion for most semen or sperm evaluations. Computer-aided sperm analysis (CASA) can be used to measure sperm motility more objectively and accurately than manual methods, provided that analysis techniques are standardized. Previous studies have shown that evaluation of sperm subpopulations is more important than analyzing the total motile sperm population alone. We developed a quantitative method to determine cut-off values for swimming speed to identify three sperm subpopulations. We used the Sperm Class Analyzer^® (SCA) CASA system to assess the total percentage of motile spermatozoa in a sperm preparation as well as the percentages of rapid, medium and slow swimming spermatozoa for six mammalian species. Curvilinear velocity (VCL) cut-off values were adjusted manually for each species to include 80% rapid, 15% medium and 5% slow swimming spermatozoa. Our results indicate that the same VCL intervals cannot be used for all species to classify spermatozoa according to swimming speed. After VCL intervals were adjusted for each species, three unique sperm subpopulations could be identified. The effects of medical treatments on sperm motility become apparent in changes in the distribution of spermatozoa among the three swimming speed classes.     

A simple expression for quantifying bacterial chemotaxis using capillary assay data: application to the analysis of enhanced chemotactic responses from growth-limited cultures.

An individual cell-based mathematical model of Rivero et al. provides a framework for determining values of the chemotactic sensitivity coefficient chi 0, an intrinsic cell population parameter that characterizes the chemotactic response of bacterial populations. This coefficient can theoretically relate the swimming behavior of individual cells to the resulting migration of a bacterial population. When this model is applied to the commonly used capillary assay, an approximate solution can be obtained for a particular range of chemotactic strengths yielding a very simple analytical expression for estimating the value of chi 0, [formula: see text] from measurements of cell accumulation in the capillary, N, when attractant uptake is negligible. A0 and A infinity are the dimensionless attractant concentrations initially present at the mouth of the capillary and far into the capillary, respectively, which are scaled by Kd, the effective dissociation constant for receptor-attractant binding. D is the attractant diffusivity, and mu is the cell random motility coefficient. NRM is the cell accumulation in the capillary in the absence of an attractant gradient, from which mu can be determined independently as mu = (pi/4t)(NRM/pi r2bc)2, with r the capillary tube radius and bc the bacterial density initially in the chamber. When attractant uptake is significant, a slightly more involved procedure requiring a simple numerical integration becomes necessary. As an example, we apply this approach to quantitatively characterize, in terms of the chemotactic sensitivity coefficient chi 0, data from Terracciano indicating enhanced chemotactic responses of Escherichia coli to galactose when cultured under growth-limiting galactose levels in a chemostat.     

Tracking sperm in three-dimensions

Sperm motility, crucial for fertilization, has been mostly studied in two dimensions (2D) by recording their swimming trajectories near a flat surface. However, spermatozoa swim in three-dimensions (3D) to find eggs, with their speed being the main impediment to track them under realistic conditions. Here, we describe a novel method allowing 3D tracking and analysis of the trajectories of multiple free-swimming sperm. The system uses a piezo-electric device displacing a large focal distance objective mounted on a microscope to acquire 70 image stacks per second, each stack composed of 60 images that span a depth of 100 μm. With this method, 3D paths of multiple sperm in the same field could be visualized simultaneously during 1 s . Within the same sample we found that surface-confined sperm swam 25% slower, produced 3-fold fewer circular revolutions per second, and had trajectories of 134% greater radius of curvature than those sperm swimming freely in 3D.     

Chemotactic response of frozen-thawed bovine spermatozoa towards follicular fluid

The aim of this study was to verify whether cattle spermatozoa respond by chemotaxis to follicular fluid (FF). The experimental conditions were defined to maintain a frozen-thawed sperm population with great motility and capacitation, and lesser sperm agglutination. Several sperm preparation conditions were studied: sperm separation from the seminal plasma by Sephadex column or migration-sedimentation, incubation under capacitating conditions in the presence or absence of a superficial layer of mineral oil, and different pH of the culture medium. The percentage of motile and agglutinated spermatozoa was determined in plate dishes under inverted phase contrast microscope. The percentage of capacitated spermatozoa was calculated as the difference between the percentages of acrosome reacted spermatozoa with and without lysophosphatidylcholine stimulation. The most ideal experimental conditions to evaluate chemotaxis in frozen-thawed cattle spermatozoa were: to separate the cells from the seminal plasma by migration-sedimentation and to incubate them under oil, in culture medium at pH 7.2, for less than 2h. The chemotaxis assays were conducted with spermatozoa treated as mentioned above which were confronted to several dilutions of FF (1:10(3), 1:10(4), 1:10(5), 1:10(6)) in a chemotaxis chamber by videomicroscopy and computer image analysis. A subpopulation of capacitated spermatozoa ( approximately 10%) that responded chemotactically to a concentration gradient generated by FF (1:10(4) to 1:10(5)) was observed. Since cryopreserved spermatozoa are regularly used to artificially inseminate the cows, the sperm chemotactic response towards FF would be potentially used to diagnose the bull sperm sample or to select the spermatozoa in the most functional state.     

Tuning sperm chemotaxis by calcium burst timing

Marine invertebrate oocytes establish chemoattractant gradients that guide spermatozoa towards their source. In sea urchin spermatozoa, this relocation requires coordinated motility changes initiated by Ca²⁺-driven alterations in sperm flagellar curvature. We discovered that Lytechinus pictus spermatozoa undergo chemotaxis in response to speract, an egg-derived decapeptide previously noted to stimulate non-chemotactic motility alterations in Strongylocentrotus purpuratus spermatozoa. Sperm of both species responded to speract gradients with a sequence of turning episodes that correlate with transient flagellar Ca²⁺ increases, yet only L. pictus spermatozoa accumulated at the gradient source. Detailed analysis of sperm behavior revealed that L. pictus spermatozoa selectively undergo Ca²⁺ fluctuations while swimming along negative speract gradients while S. purpuratus sperm generate Ca²⁺ fluctuations in a spatially non-selective manner. This difference is attributed to the selective suppression of Ca²⁺ fluctuations of L. pictus spermatozoa as they swim towards the source of the chemoattractant gradient. This is the first study to compare and characterize the motility components that differ in chemotactic and non-chemotactic spermatozoa. Tuning of Ca²⁺ fluctuations and associated turning episodes to the chemoattractant gradient polarity is a central feature of sea urchin sperm chemotaxis and may be a feature of sperm chemotaxis in general.     

A stochastic description of Dictyostelium chemotaxis

Chemotaxis, the directed motion of a cell toward a chemical source, plays a key role in many essential biological processes. Here, we derive a statistical model that quantitatively describes the chemotactic motion of eukaryotic cells in a chemical gradient. Our model is based on observations of the chemotactic motion of the social ameba Dictyostelium discoideum, a model organism for eukaryotic chemotaxis. A large number of cell trajectories in stationary, linear chemoattractant gradients is measured, using microfluidic tools in combination with automated cell tracking. We describe the directional motion as the interplay between deterministic and stochastic contributions based on a Langevin equation. The functional form of this equation is directly extracted from experimental data by angle-resolved conditional averages. It contains quadratic deterministic damping and multiplicative noise. In the presence of an external gradient, the deterministic part shows a clear angular dependence that takes the form of a force pointing in gradient direction. With increasing gradient steepness, this force passes through a maximum that coincides with maxima in both speed and directionality of the cells. The stochastic part, on the other hand, does not depend on the orientation of the directional cue and remains independent of the gradient magnitude. Numerical simulations of our probabilistic model yield quantitative agreement with the experimental distribution functions. Thus our model captures well the dynamics of chemotactic cells and can serve to quantify differences and similarities of different chemotactic eukaryotes. Finally, on the basis of our model, we can characterize the heterogeneity within a population of chemotactic cells.     

Automated analysis of rabbit sperm motility and the effect of chemicals on sperm motion parameters.

Appropriate software settings and optimum procedures were determined for the measurement of the motion parameters of rabbit spermatozoa by the CellSoft (Cryo Resources Ltd., Montgomery, NY) computer-assisted digital image analysis system. The system was used to follow motion parameter changes occurring in spermatozoa incubated for 6 hr with or without exposure to chemicals. Mean amplitude of lateral head displacement (AALH) increased over the 6 hr period, while curvilinear velocity (Vc) first increased and then decreased. Values for linearity (Lin), or beat cross frequency (BCF), were unchanged. The majority of spermatozoa progressed linearly, with rapid rotation of the sperm head, but subpopulations of spermatozoa with different swimming patterns appeared after 1-3 hr of incubation. Percentage motile sperm and Vc were most sensitive to the action of the compounds (pyrogallol, hydroquinone, ammonium oxalate, triethyl phosphite, and pinocolyl alcohol), while BCF was least affected. The decline in percentage of motile sperm was dependent on duration of exposure and chemical concentration. Mean Vc of the sperm population decreased rapidly upon chemical exposure and remained at a low value until motility ceased. The initial decrease in Vc was dependent on the concentration of the added compound. Motion-based indices--motility concentration (MCI50), motility time (MTI50), and velocity (VI)--were defined and used as toxicological endpoints. The rank order of these indices, the end point of the neutral red in vitro assay for cytotoxicity, and LD50 values for the five compounds were the same, suggesting that chemical inhibition of sperm motility may be useful as a method for the in vitro assessment of chemical cytotoxicity.     

Rotational and translational swimming of human spermatozoa: a dynamic laser light scattering study

The rotational swimming motion of human spermatozoa is evaluated from measurements of depolarized dynamic laser light scattering at zero angle. The analysis is based on a Maxwellian angular velocity distribution and yields a rotational frequency of about 4 Hz that is ascribed to the rotation of the sperm head. From comparison with the translational swimming motion, a propelling efficiency of about 10 micron per turn is deduced. This parameter describes the linkage between the rotational and translational swimming motion and is likely to be discriminatory in the analysis of physiological and pathological sperm motions.     

INHIBITION OF RESPIRATION IN SEA URCHIN SPERMATOZOA FOLLOWING INTERACTION WITH FIXED UNFERTILIZED EGGS

The respiration of spermatozoa of the sea urchin, Hemicentrotus pulcherrimus , was found to be sensitive to rotenone, antimycin A, and cyanide. This suggests that sperm respiration results from electron transport which spans the whole mitochondrial respiratory chain. The sperm respiration was inhibited by oligomycin and this inhibition was released by 2, 4-dinitrophenol (DNP). DNP did not stimulate the respiration of spermatozoa in a diluted suspension (2 × 10⁸/ml), where they were swimming vigorously. The ADP level of spermatozoa in the diluted suspension was markedly higher than that in dry sperm. The spermatozoa, which had reacted with unfertilized eggs fixed with glutaraldehyde, were immotile with a quite low respiratory rate. The respiratory rate of the immotile spermatozoa was enhanced by DNP. In the immotile spermatozoa, ADP level was markedly low and the ATP level was as high as that in dry sperm. From these findings, it is concluded that in the swimming spermatozoa respiration coupled with oxidative phosphorylation occurs at the maximum rate. State 3 respiration probably occurs in the swimming spermatozoa. The low respiratory rate of the immotile spermatozoa is assumed to be due to a shortage of ADP and is practically regarded as state 4 respiration.     

Progesterone from the cumulus cells is the sperm chemoattractant secreted by the rabbit oocyte cumulus complex

Sperm chemotaxis in mammals have been identified towards several female sources as follicular fluid (FF), oviduct fluid, and conditioned medium from the cumulus oophorus (CU) and the oocyte (O). Though several substances were confirmed as sperm chemoattractant, Progesterone (P) seems to be the best chemoattractant candidate, because: 1) spermatozoa express a cell surface P receptor, 2) capacitated spermatozoa are chemotactically attracted in vitro by gradients of low quantities of P; 3) the CU cells produce and secrete P after ovulation; 4) a gradient of P may be kept stable along the CU; and 5) the most probable site for sperm chemotaxis in vivo could be near and/or inside the CU. The aim of this study was to verify whether P is the sperm chemoattractant secreted by the rabbit oocyte-cumulus complex (OCC) in the rabbit, as a mammalian animal model. By means of videomicroscopy and computer image analysis we observed that only the CU are a stable source of sperm attractants. The CU produce and secrete P since the hormone was localized inside these cells by immunocytochemistry and in the conditioned medium by enzyme immunoassay. In addition, rabbit spermatozoa express a cell surface P receptor detected by western blot and localized over the acrosomal region by immunocytochemistry. To confirm that P is the sperm chemoattractant secreted by the CU, the sperm chemotactic response towards the OCC conditioned medium was inhibited by three different approaches: P from the OCC conditioned medium was removed with an anti-P antibody, the attractant gradient of the OCC conditioned medium was disrupted by a P counter gradient, and the sperm P receptor was blocked with a specific antibody. We concluded that only the CU but not the oocyte secretes P, and the latter chemoattract spermatozoa by means of a cell surface receptor. Our findings may be of interest in assisted reproduction procedures in humans, animals of economic importance and endangered species.     

Le chimiotactisme des spermatozoïdes a-t-il un rôle dans la fécondation?

Sperm chemotaxis to follicular fluid has been established by a variety of means in human and mouse spermatozoa. It was found that only a small fraction of a given sperm population (averaging around 10%) is chemotactically responsive and that this fraction constitutes capacitated (ripe) spermatozoa. Both the chemotactic responsiveness and the capacitated state are transient (with a lifetime between 50 min and 4 h) and they occur only once in the sperm’s lifetime. It has been proposed that the role of sperm chemotaxis in mammals (at least in man) is selective recruitment of capacitated spermatozoa for fertilizing the egg, and that the role of the continuous replacement of chemotactic/capacitated spermatozoa is to prolong the duration of time over which capacitated spermatozoa would be available in the female reproductive tract. The sperm chemoattractants have not been identified but they appear to be heat-stable peptides. Thein vivo location of sperm chemotaxis is not known; a number of possible locations are discussed.     

DNA profiling of spermatozoa by laser capture microdissection and low volume-PCR

Genetic profiling of sperm from complex biological mixtures such as sexual assault casework samples requires isolation of a pure sperm population and the ability to analyze low abundant samples. Current standard procedure for sperm isolation includes preferential lysis of epithelial contaminants followed by collection of intact sperm by centrifugation. While effective for samples where sperm are abundant, this method is less effective when samples contain few spermatozoa. Laser capture microdissection (LCM) is a proven method for the isolation of cells biological mixtures, even when found in low abundance. Here, we demonstrate the efficacy of LCM coupled with on-chip low volume PCR (LV-PCR) for the isolation and genotyping of low abundance sperm samples. Our results indicate that this method can obtain complete profiles (13-16 loci) from as few as 15 sperm cells with 80% reproducibility, whereas at least 40 sperm cells are required to profile 13-16 loci by standard 'in-tube' PCR. Further, LCM and LV-PCR of a sexual assault casework sample generated a DNA genotype that was consistent with that of the suspect. This method was unable, however, to analyze a casework sample from a gang rape case in which two or more sperm contributors were in a mixed population. The results indicate that LCM and LV-PCR is sensitive and effective for genotyping sperm from sperm/epithelial cell mixtures when epithelial lysis may be insufficient due to low abundance of sperm; LCM and LV-PCR, however, failed in a casework sample when spermatozoa from multiple donors was present, indicating that further study is necessitated.     

Characterization of sperm motility in sea bass: the effect of heavy metals and physicochemical variables on sperm motility

Computer assisted sperm analysis (CASA) was used to characterize the motility of sea bass Dicentrarchus labrax spermatozoa and to study the effect of several physicochemical variables and heavy metals on sperm swimming performance. Duration of sperm motility in sea bass was very short (<50 s). During the first 20 s all the motility variables measured remained approximately constant, the velocity and linearity of the movement being maximum during this period, while both variables decreased sharply later. While slight variations in pH did not significantly modify sperm swimming performance, changes in osmolality affected all the measured motility variables. Two of the heavy metals tested, Cu²⁺ and Pb²⁺, did not affect sperm motility when the activating media contained up to 100 ppm of the metal salts. In contrast, Hg²⁺ modified the morphology of post-swimming spermatozoa at 0·4–1 ppm (sperm dilution rate 1:39) and completely arrested sperm motility at concentrations as low as 0·1 ppm (sperm dilution rate 1:2500). Assuming a covalent binding to sperm cells, this revealed a finite number of c. 10 million Hg²⁺ binding sites per spermatozoon. Complementary results using demembranated spermatozoa suggested that the main target of HgCl₂ would be located in the plasma membrane and that HgCl₂ would inhibit water channels, hence preventing sperm motility.     

Bias in the gradient-sensing response of chemotactic cells

We apply linear stability theory and perform perturbation studies to better characterize, and to generate new experimental predictions from, a model of chemotactic gradient sensing in eukaryotic cells. The model uses reaction-diffusion equations to describe 3(') phosphoinositide signaling and its regulation at the plasma membrane. It demonstrates a range of possible gradient-sensing mechanisms and captures such characteristic behaviors as strong polarization in response to static gradients, adaptation to differing mean levels of stimulus, and plasticity in response to changing gradients. An analysis of the stability of polarized steady-state solutions indicates that the model is most sensitive to off-axis perturbations. This biased sensitivity is also reflected in responses to localized external stimuli, and leads to a clear experimental prediction, namely, that a cell which is polarized in a background gradient will be most sensitive to transient point-source stimuli lying within a range of angles that are oblique with respect to the polarization axis. Stimuli at angles below this range will elicit responses whose directions overshoot the stimulus angle, while responses to stimuli applied at larger angles will undershoot the stimulus angle. We argue that such a bias is likely to be a general feature of gradient sensing in highly motile cells, particularly if they are optimized to respond to small gradients. Finally, an angular bias in gradient sensing might lead to preferred turn angles and zigzag movements of cells moving up chemotactic gradients, as has been noted under certain experimental conditions.     

Mammalian sperm chemotaxis and its association with capacitation

Much progress has been made in recent years in establishing mammalian sperm chemotaxis and understanding sperm capacitation. Thus far, chemotaxis to follicular fluid has been established by a variety of means in human and mouse spermatozoa. It was found that only a small fraction of a given sperm population (averaging around 10%) is chemotactically responsive and that this fraction constitutes capacitated (ripe) spermatozoa. Both the chemotactic responsiveness and the capacitated state are transient (with a lifetime of 50 min to 4 h) and they occur only once in the sperm's lifetime. It has been proposed that the role of sperm chemotaxis in mammals (at least in humans) is selective recruitment of capacitated spermatozoa for fertilizing the egg, and that the role of the continuous replacement of chemotactic/capacitated spermatozoa is to prolong the time during which capacitated spermatozoa are available in the female reproductive tract. The sperm chemoattractants have not been identified, but they appear to be heat‐stable peptides. Although the molecular mechanism and the in vivo location of sperm chemotaxis are not known, a number of possible mechanisms and locations are discussed. Dev. Genet. 25:87–94, 1999. © 1999 Wiley‐Liss, Inc.