Analysis of chemotaxis when the fraction of responsive cells is small--application to mammalian sperm guidance

The detection of chemotaxis-related changes in the swimming behavior of mammalian spermatozoa in a spatial chemoattractant gradient has hitherto been an intractable problem. The difficulty is that the fraction of responsive cells in the sperm population is very small and that the large majority of the cells, though non-responsive, are motile too. Assessment of the chemotactic effects in a spatial gradient is also very sensitive to the quality of sperm tracking. To overcome these difficulties we propose a new approach, based on the analysis of the distribution of instantaneous directionality angles made by spermatozoa in a spatial gradient versus a no-gradient control. Although the use of this parameter does not allow identification of individual responding cells, it is a reliable measure of directionality, independent of errors in cell tracking caused by cell collisions, track crossings, and track splitting. The analysis identifies bias in the swimming direction of a population relative to the gradient direction. It involves statistical chi(2) tests of the very large sample of measured angles, where the critical chi(2) values are adjusted to the sample size by the bootstrapping procedure. The combination of the newly measured parameter and the special analysis provides a highly sensitive method for the detection of a chemotactic response, even a very small one.     

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.     

Fine-Tuning of Chemotactic Response in E. coli Determined by High-Throughput Capillary Assay

In E. coli, chemotactic behavior exhibits perfect adaptation that is robust to changes in the intracellular concentration of the chemotactic proteins, such as CheR and CheB. However, the robustness of the perfect adaptation does not explicitly imply a robust chemotactic response. Previous studies on the robustness of the chemotactic response relied on swarming assays, which can be confounded by processes besides chemotaxis, such as cellular growth and depletion of nutrients. Here, using a high-throughput capillary assay that eliminates the effects of growth, we experimentally studied how the chemotactic response depends on the relative concentration of the chemotactic proteins. We simultaneously measured both the chemotactic response of E. coli cells to L: -aspartate and the concentrations of YFP-CheR and CheB-CFP fusion proteins. We found that the chemotactic response is fine-tuned to a specific ratio of [CheR]/[CheB] with a maximum response comparable to the chemotactic response of wild-type behavior. In contrast to adaptation in chemotaxis, that is robust and exact, capillary assays revealed that the chemotactic response in swimming bacteria is fined-tuned to wild-type level of the [CheR]/[CheB] ratio.     

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.     

Implications of diversity in sperm size and function for sperm competition and fertility

Sperm competition is now recognised as a potent selective force shaping many male reproductive traits. While the influence of sperm competition on sperm number is widely accepted, its effects upon sperm size remain controversial. It had been traditionally assumed that there is a trade-off between sperm number and sperm size, so that an increase in sperm number would result in a decrease in sperm size, under conditions of sperm competition. Contrary to this prediction, we proposed some time ago that sperm competition favours an increase in sperm size, because longer sperm swim faster and are more likely to win the race to fertilize ova. Comparative studies between species show that in many taxa such a relationship exists, but the consequences of an increase in sperm size may vary between taxa depending on the environment in which spermatozoa have to compete. We present new evidence showing that in mammals longer sperm swim at higher speeds. We also show that mean swimming speed is highly correlated with maximum swimming speed, so even if the fastest swimming sperm are more likely to fertilize, both measures are informative. When individuals of the same species are compared, ratios between the dimensions of different sperm components, as well as the shape of the head, seem better at explaining sperm swimming velocity. Finally, we show that mean and maximum sperm swimming speed determine male fertility. Other studies have shown that in competitive contexts, males with faster swimming sperm have higher fertilization success. We conclude that the available evidence supports our original hypothesis.     

Ca2+ spikes in the flagellum control chemotactic behavior of sperm

The events that occur during chemotaxis of sperm are only partly known. As an essential step toward determining the underlying mechanism, we have recorded Ca2+ dynamics in swimming sperm of marine invertebrates. Stimulation of the sea urchin Arbacia punctulata by the chemoattractant or by intracellular cGMP evokes Ca2+ spikes in the flagellum. A Ca2+ spike elicits a turn in the trajectory followed by a period of straight swimming ('turn-and-run'). The train of Ca2+ spikes gives rise to repetitive loop-like movements. When sperm swim in a concentration gradient of the attractant, the Ca2+ spikes and the stimulus function are synchronized, suggesting that precise timing of Ca2+ spikes controls navigation. We identified the peptide asterosap as a chemotactic factor of the starfish Asterias amurensis. The Ca2+ spikes and swimming behavior of sperm from starfish and sea urchin are similar, implying that the signaling pathway of chemotaxis has been conserved for almost 500 million years.     

Molecular Reproduction & Development Volume 78, Issue 6 cover image

Artist's rendition of Xenopus sperm swimming in the jelly layers as they approach the egg surface. Burnett et al. (this issue) fi nd that water soluble components of this jelly layer directly interact with sperm, thereby initiating chemotactic motility towards the egg. Original art by Douglas Chandler.     

Helical nature of sperm swimming affects the fit of fertilization-kinetics models to empirical data

Models of fertilization kinetics rely upon estimates of the swimming velocity of sperm to predict collision rates between egg and sperm. Most investigators measure sperm swimming velocity without accounting for the helical motion of sperm, thereby obtaining an inflated estimate of the velocity with which sperm approach eggs. In turn, models of fertilization predict inflated rates of sperm/egg collision. I observed sea urchin sperm colliding with eggs, quantified the rate of sperm/egg collision, and measured sperm velocity as a component of the helix through which they swim. I also adjusted the "target size" of eggs to reflect the diameter of the helix. My estimate of sperm swimming velocity is an order of magnitude lower than other estimates for the same species. By using helical parameters in fertilization kinetics models and accounting for dead sperm in laboratory trials, I was able to accurately predict lower rates of sperm/egg collision. Moreover, making these adjustments in the model increased the estimated proportion of sperm that initiate fertilization by 6- to 7-fold, suggesting that a better understanding of sperm swimming might lead to a more complete understanding of fertilization biology and natural selection on gamete traits.     

Two types of assays for detecting frog sperm chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm. Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg. Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 μm diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer. The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.     

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.     

Gametic interactions promote inbreeding avoidance in house mice

Reproduction among related individuals is generally maladaptive. Inbreeding imposes significant costs on individual reproductive success, and can decrease population fitness. Theory predicts that polyandrous females can avoid inbreeding by exploiting paternity‐biasing mechanisms that enable differential sperm ‘use’. Evidence of sperm selection is difficult to demonstrate because patterns of non‐random paternity can be generated by a variety of different mechanisms. Here, using in vitro fertilisation in mice, we provide evidence of sperm selection at the gametic level. We mixed the sperm of sibling and non‐sibling males, and observed a fertilisation bias towards the sperm of non‐sibling males. The number of motile sperm and sperm swimming performance did not differ between competitors among the replicate assays. Therefore, our result can only be ascribed to egg‐driven sperm selection against related sperm. We conclude that the expression or secretion of gametic proteins could provide the molecular basis for this mechanism of cryptic female choice.     

Chemoresponsiveness to cAMP and Folic Acid during Growth, Development, and Dedifferentiation in Dictyostelium discoideum

Chemoresponsiveness to cAMP and to folic acid are monitored in growing, developing, and dedifferentiating amebae of the cellular slime mold Dictyostelium discoideum . Two semiquantitative assays are employed, one measuring the directed movement of cells up a gradient of chemoattractant ('chemotaxis' assay) and the other measuring the outward spreading of cells in response to a chemical stimulant distributed equally throughout the substratum ('spreading' assay). Vegetative amebae possess relatively insignificant levels of chemotactic responsiveness to cAMP. Six h after the initiation of development, at approximately the same time as the onset of aggregation, cells rapidly acquire chemotactic responsiveness to cAMP. During 'erasure', a dedifferentiation induced by resuspending aggregating cells in fresh nutrient medium, chemotactic responsiveness to cAMP is lost just after the erasure event. By the same chemotactic assay, it is demonstrated that vegetative amebae possess a significant level of chemotactic responsiveness to folic acid. Two h after the initiation of development, cells completely lose chemotactic responsiveness to folic acid. During erasure, cells reacquire chemotactic responsiveness to folic acid at approximately the same time that they lose responsiveness to cAMP.
Dramatically different results are obtained by the spreading assay. When cells lose chemotactic responsiveness to folic acid early in development and when erasing cells lose chemotactic responsiveness to cAMP, they retain the spreading response to the two stimulants, respectively. The different results obtained for chemoreception employing the two assays are discussed in terms of molecular mechanisms, and a testable hypothesis is proposed for the possible roles of chemoresponsiveness and erasure in late morphogenesis.     

Chemoresponsiveness to cAMP and folic acid during growth, development, and dedifferentiation in Dictyostelium discoideum

Chemoresponsiveness to cAMP and to folic acid are monitored in growing, developing, and dedifferentiating amebae of the cellular slime mold Dictyostelium discoideum. Two semiquantitative assays are employed, one measuring the directed movement of cells up a gradient of chemoattractant ('chemotaxis' assay) and the other measuring the outward spreading of cells in response to a chemical stimulant distributed equally throughout the substratum ('spreading' assay). Vegetative amebae possess relatively insignificant levels of chemotactic responsiveness to cAMP. Six h after the initiation of development, at approximately the same time as the onset of aggregation, cells rapidly acquire chemotactic responsiveness to cAMP. During 'erasure', a dedifferentiation induced by resuspending aggregating cells in fresh nutrient medium, chemotactic responsiveness to cAMP is lost just after the erasure event. By the same chemotactic assay, it is demonstrated that vegetative amebae possess a significant level of chemotactic responsiveness to folic acid. Two h after the initiation of development, cells completely lose chemotactic responsiveness to folic acid. During erasure, cells reacquire chemotactic responsiveness to folic acid at approximately the same time that they lose responsiveness to cAMP. Dramatically different results are obtained by the spreading assay. When cells lose chemotactic responsiveness to folic acid early in development and when erasing cells lose chemotactic responsiveness to cAMP, they retain the spreading response to the two stimulants, respectively. The different results obtained for chemoreception employing the two assays are discussed in terms of molecular mechanisms, and a testable hypothesis is proposed for the possible roles of chemoresponsiveness and erasure in late morphogenesis.     

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.     

Rhizobium meliloti swims by unidirectional, intermittent rotation of right-handed flagellar helices.

The 5 to 10 peritrichously inserted complex flagella of Rhizobium meliloti MVII-1 were found to form right-handed flagellar bundles. Bacteria swam at speeds up to 60 microns/s, their random three-dimensional walk consisting of straight runs and quick directional changes (turns) without the vigorous angular motion (tumbling) seen in swimming Escherichia coli cells. Observations of R. meliloti cells tethered by a single flagellar filament revealed that flagellar rotation was exclusively clockwise, interrupted by very brief stops (shorter than 0.1 s), typically every 1 to 2 s. Swimming bacteria responded to chemotactic stimuli by extending their runs, and tethered bacteria responded by prolonged intervals of clockwise rotation. Moreover, the motility tracks of a generally nonchemotactic ("smooth") mutant consisted of long runs without sharp turns, and tethered mutant cells showed continuous clockwise rotation without detectable stops. These observations suggested that the runs of swimming cells correspond to clockwise flagellar rotation, and the turns correspond to the brief rotation stops. We propose that single rotating flagella (depending on their insertion point on the rod-shaped bacterial surface) can reorient a swimming cell whenever the majority of flagellar motors stop.     

Chemotaxis is required for virulence and competitive fitness of the bacterial wilt pathogen Ralstonia solanacearum

Ralstonia solanacearum, a soilborne plant pathogen of considerable economic importance, invades host plant roots from the soil. Qualitative and quantitative chemotaxis assays revealed that this bacterium is specifically attracted to diverse amino acids and organic acids, and especially to root exudates from the host plant tomato. Exudates from rice, a nonhost plant, were less attractive. Eight different strains from this heterogeneous species complex varied significantly in their attraction to a panel of carbohydrate stimuli, raising the possibility that chemotactic responses may be differentially selected traits that confer adaptation to various hosts or ecological conditions. Previous studies found that an aflagellate mutant lacking swimming motility is significantly reduced in virulence, but the role of directed motility mediated by the chemotaxis system was not known. Two site-directed R. solanacearum mutants lacking either CheA or CheW, which are core chemotaxis signal transduction proteins, were completely nonchemotactic but retained normal swimming motility. In biologically realistic soil soak virulence assays on tomato plants, both nonchemotactic mutants had significantly reduced virulence indistinguishable from that of a nonmotile mutant, demonstrating that directed motility, not simply random motion, is required for full virulence. In contrast, nontactic strains were as virulent as the wild-type strain was when bacteria were introduced directly into the plant stem through a cut petiole, indicating that taxis makes its contribution to virulence in the early stages of host invasion and colonization. When inoculated individually by soaking the soil, both nontactic mutants reached the same population sizes as the wild type did in the stems of tomato plants just beginning to wilt. However, when tomato plants were coinoculated with a 1:1 mixture of a nontactic mutant and its wild-type parent, the wild-type strain outcompeted both nontactic mutants by 100-fold. Together, these results indicate that chemotaxis is an important trait for virulence and pathogenic fitness in this plant pathogen.     

Sex cells in changing environments: can organisms adjust the physiological function of gametes to different temperatures?

Over the past decade, hundreds of studies have examined the abilities of whole organisms to modify their physiology and behaviour in response to environmental temperature changes; despite this, virtually nothing is known about the ability of sex cells to adjust to different temperature conditions. In fact, a recent meta‐analysis based on studies of 309 species and 112 physiological and ecological traits found no studies examining the influence of temperature on gamete function. Because sex cells play a critical role in the adaptation and persistence of species, this represents a severe oversight in physiological studies of thermal adaptation. Our study examines whether sex cells can respond phenotypically to variation in the thermal environment that is experienced by the whole‐organism. Specifically, we studied the thermal dependence of sperm swimming and the critical thermal limits of sperm cells in males of the poeciliid fish, Gambusia holbrooki. This species is well known for its ability to modify physiological function and maintains burst and sustained swimming performance and mating ability across a wide range of thermal conditions. In contrast, we found that sperm cells from male G. holbrooki did not adjust their physiological function as predicted by adaptive models. After acclimation of adult males to cool or warm temperatures, we found that the critical thermal limits of sperm function remained unchanged, as did the effect of temperature on sperm swimming performance. However, warm‐acclimated fish had sperm with higher swimming speeds across all temperatures. The absence of phenotypic changes in the critical thermal limits of sperm or thermal dependence of sperm swimming performance is surprising given that whole‐organism traits in G. holbrooki generally show improved performance after exposure to novel environments. As such an inability to thermally adjust gamete function may be widespread among other organisms, we urge biologists to investigate the generality of this result.     

The rate of change in Ca(2+) concentration controls sperm chemotaxis

During chemotaxis and phototaxis, sperm, algae, marine zooplankton, and other microswimmers move on helical paths or drifting circles by rhythmically bending cell protrusions called motile cilia or flagella. Sperm of marine invertebrates navigate in a chemoattractant gradient by adjusting the flagellar waveform and, thereby, the swimming path. The waveform is periodically modulated by Ca(2+) oscillations. How Ca(2+) signals elicit steering responses and shape the path is unknown. We unveil the signal transfer between the changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) and path curvature (κ). We show that κ is modulated by the time derivative d[Ca(2+)](i)/dt rather than the absolute [Ca(2+)](i). Furthermore, simulation of swimming paths using various Ca(2+) waveforms reproduces the wealth of swimming paths observed for sperm of marine invertebrates. We propose a cellular mechanism for a chemical differentiator that computes a time derivative. The cytoskeleton of cilia, the axoneme, is highly conserved. Thus, motile ciliated cells in general might use a similar cellular computation to translate changes of [Ca(2+)](i) into motion.     

Motility response of Rhodobacter sphaeroides to chemotactic stimulation.

Tethered rotating cells of Rhodobacter sphaeroides varied widely in their stopping frequency; 45% of cells showed no stops of longer than 1 s, whereas others showed stops of up to several seconds. Individual cells alternated between stops and rotation at a fairly constant rate, without continuous variation. Addition of the chemoattractant propionate to free-swimming cells of R. sphaeroides increased the mean population swimming speed from 15 to 23 microns s-1. After correction for nonmotile cells, the percentage swimming at less than 5 microns s-1 dropped from approximately 22 to 8, whereas the percentage swimming at greater than 50 microns s-1 increased from 6 to 15. However, cells already swimming did not swim faster after propionate addition; the increase in the mean population speed after propionate addition was caused by an increase in the mean run length between stops from 25 to 101 microns. The increased run length was the result of a drop in both the stopping frequency and the length of a stop. Addition of propionate over the range of 10 microM to 1 mM decreased the stopping frequency; this decrease was almost entirely blocked by benzoate, a competitive inhibitor of propionate transport. The chemoattractants acetate and potassium had the same effect as propionate on the distribution of stopping frequency, which demonstrated that this is a general behavioral response to chemotactic stimulation. Adaptation to propionate stimulation was slow and very variable, cultures frequently showing little adaptation over 30 min. This characteristic may be the result of the lack of a highly specific chemosensory system in R. sphaeroides.     

An automated assay for quantifying the swimming behavior of Paramecium and its use to study cation responses

Paramecium tetraurelia is a ciliated protist that alters its swimming behavior in response to various stimuli. Like the sensory responses of many organisms, these responses in Paramecium show adaptation to continued stimulation. For quantitative studies of the initial response to stimulation, and of the time course of adaptation, we have developed a computerized motion analysis assay that can detect deviations from the normal swimming pattern in a population of cells. The motion of an average of ten cells was quantified during periods ranging from 15 to 60 seconds, with a time resolution of 1/15 seconds. During normal forward swimming, the maximum deviation from a straight-line path was less than 17 degrees. Path deviations above this threshold value were defined as changes in swimming direction. The percentage of total path time that cells spent deviating from forward swimming was defined as percent directional changes (PDC). This parameter was used to construct dose-response curves for the behavioral effects of various externally added cations known to induce behavioral changes and also to show the time course of adaptation to a depolarizing K+ stimulus. This assay is a valuable tool for studies of chemoeffectors or mutations that alter the swimming behavior of Paramecium and may also be applicable to other motile organisms.