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.     

Bacterial chemotaxis: a field in motion

Many different types of studies are being combined to provide an increasingly detailed picture of the bacterial chemotaxis system. The structures of periplasmic receptors and a cytoplasmic response regulator, along with structures of domains of a membrane receptor, a receptor-modifying enzyme and a cytoplasmic histidine kinase, have been determined. These structures provide a basis for other work which is likely to open up new structural avenues.     

Changes in human sperm motion during capacitation in vitro

Spermatozoa from 10 fertile donors and from 10 patients with infertile marriages were washed and centrifuged (time zero, T0), and incubated in vitro in capacitation media for 6 h (T6), or 24 h (T24). At each time individual spermatozoa were classified as being morphologically normal or abnormal, and their movement characteristics were determined using high-speed videomicrography. Zona-free hamster oocytes were added to the T24 sperm suspensions. At all times, morphologically normal spermatozoa from donors and patients swam faster and had greater rolling frequency, flagellar beat frequency and amplitude than did abnormally shaped cells. Morphologically normal spermatozoa from donors exhibited a significant change in their movement pattern at T6. This change, which resembles hyperactivation in other species, was characterized by higher values of amplitude of lateral head displacement, and lower values of linearity, beat frequency and flagellar curvature ratio. In contrast, normal spermatozoa from patients showed only a decrease in straight line velocity at T6, with no other significant changes in movement characteristics. No changes in sperm movement could be demonstrated for the abnormal cells in either group of subjects. In sperm suspensions from donors and patients examined at T24, sperm vigour declined regardless of the morphological type. Spermatozoa from all 10 donors were able to penetrate the zona-free hamster oocytes, but spermatozoa from 5 of the 10 patients failed to penetrate oocytes. Correlations between hamster oocyte penetration and indicators of sperm vigour were demonstrated only for spermatozoa of patients.     

Lack of species-specificity in mammalian sperm chemotaxis

Attraction of spermatozoa by way of chemotaxis to substances secreted from the egg or its surrounding cells has been demonstrated in marine species, amphibians, and mammals. This process is species- or family-specific in marine invertebrates: a chemoattractant for one marine species is usually not recognized by another species or by a member of another family. It is not known whether this selectivity is also the rule in other phyla. Furthermore, it is not at all obvious that such selectivity would be advantageous to species with internal fertilization. Here, using a directionality-based assay for chemotaxis, we studied in vitro the chemotactic response of human and rabbit spermatozoa to human, rabbit, and bovine egg-related factors. We found that spermatozoa from each of the two sources responded similarly well to egg-related factors obtained from any of the three species examined. These results indicate lack of chemotaxis-related, species specificity between these species, suggesting that their sperm chemoattractants are common or very similar. The findings further suggest that mammals do not rely on species specificity of sperm chemotaxis for avoidance of interspecies fertilization.     

Particulate adenylate cyclase plays a key role in human sperm olfactory receptor-mediated chemotaxis

Human sperm chemotaxis is a critical component of the fertilization process, but the molecular basis for this behavior remains unclear. Recent evidence shows that chemotactic responses depend on activation of the sperm olfactory receptor, hOR17-4. Certain floral scents, including bourgeonal, activate hOR17-4, trigger pronounced Ca(2+) fluxes, and evoke chemotaxis. Here, we provide evidence that hOR17-4 activation is coupled to a cAMP-mediated signaling cascade. Multidimensional protein identification technology was used to identify potential components of a G-protein-coupled cAMP transduction pathway in human sperm. These products included various membrane-associated adenylate cyclase (mAC) isoforms and the G(olf)-subunit. Using immunocytochemistry, specific mAC isoforms were localized to particular cell regions. Whereas mAC III occurred in the sperm head and midpiece, mAC VIII was distributed predominantly in the flagellum. In contrast, G(olf) was found mostly in the flagellum and midpiece. The observed spatial distribution patterns largely correspond to the spatiotemporal character of hOR17-4-induced Ca(2+) changes. Behavioral and Ca(2+) signaling responses of human sperm to bourgeonal were bioassayed in the presence, or absence, of the adenylate cyclase antagonist SQ22536. This specific agent inhibits particulate AC, but not soluble AC, activation. Upon incubation with SQ22536, cells ceased to exhibit Ca(2+) signaling, chemotaxis, and hyperactivation (faster swim speed and flagellar beat rate) in response to bourgeonal. Particulate AC is therefore required for induction of hOR17-4-mediated human sperm behavior and represents a promising target for future design of contraceptive drugs.     

Biased excitable networks: how cells direct motion in response to gradients

The actin cytoskeleton in motile cells has many of the hallmarks of an excitable medium, including the presence of propagating waves. This excitable behavior can account for the spontaneous migration of cells. A number of reports have suggested that the chemoattractant-mediated signaling can bias excitability, thus providing a means by which cell motility can be directed. In this review, we discuss some of these observations and theories proposed to explain them. We also suggest a mechanism for cell polarity that can be incorporated into the existing framework.     

Prediction of human sperm penetrating ability using computerized motion parameters

The sperm penetration assay (SPA) is used to assess male fertilizing potential but it is tedious and costly. Computer analysis could replace the need for the SPA in some cases, if computerized sperm motility parameters are highly predictive of SPA performance. The objective of this study was to determine whether computerized motility parameters from fresh semen samples could be used to predict SPA performance. Computer automated semen analysis (CASA; CellSoft, Cryo Resources) was used to quantitate sperm concentration (CONC), percent motility (MOT), curvilinear velocity (VEL), linearity of swimming trajectory (LIN), mean amplitude of lateral head displacement (ALH), and beat/cross frequency (B/CF). The SPA was performed using either Biggers, Whitten, and Whittingham's medium (BWW) or TEST-yolk buffer (TYB). Patients were divided into three groups depending on SPA performance: group 1, BWW-treated, 0% versus greater than 0% penetration; group 2, TYB-treated, 0% versus greater than 0% penetration; and group 3, TYB-treated, less than 20% versus less than or equal to 20% penetration. SPA performance was highly correlated with CONC, MOT, VEL, and B/CF. CONC, MOT, VEL, and B/CF were significantly higher for patients who penetrated in the SPA than for those who failed to penetrate. Discriminant function analysis (DFA) successfully classified 76% of all patients treated with TYB (group 2) who penetrated and 86% of nonpenetrators based on their computerized motility parameters. For group 2 DFA predicted that 93 men would penetrate in the SPA with TYB. Of these, 90 (97%) successfully penetrated at least one egg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermal fluctuations biased for directional motion in molecular motors

Recently developed single molecule measurements have demonstrated that the mechanisms for numerous protein functions involve thermal fluctuation, or Brownian motion. Protein interactions bias the random thermal noise in a manner such that the protein can perform its given functions. This phenomenon has been observed in molecular motor unidirectional movement where Brownian motion is used to preferentially bind the motor heads in one direction causing directional motility. This is analogous to that used by proteins in which spontaneous structural fluctuations are used to switch function. Seeing that two very different systems implement similar mechanisms suggests there exists a general scheme applied by diverse proteins that exploits thermal fluctuations in order to achieve their respective functions.     

Ciliary contractile model applied to sperm flagellar motion

The contractile model developed previously to describe ciliary motions was applied to the case of invertebrate sperm flagella. The model could describe waveforms of these flagella as a function of the flagellar frequency and as a function of the external viscosity. Rapid spontaneous transients from rest to motion were also correctly described by the model. The calculations showed that the time course of the internal, active, moments during a cycle is only weakly displayed in the time course of the transverse motion of the flagella. This is apparently due to nonlinearities which suppress higher harmonics in the frequency spectrum of the active moments. The local curvature of the flagella was found to follow the time course of the internal, active moments directly. It is concluded that to derive the time course of the active contractile moments from the transverse motion, data on the flagellar motion with a position accuracy of 0·1 μm and with a time resolution of 1 msec are required. For curvature data an accuracy of 50 cm^?1 and a time resolution of 2·5 msec would suffice.     

Crisp proteins and sperm chemotaxis: discovery in amphibians and explorations in mammals

Crisp proteins appear to play multiple roles in the life history of sperm. One of these roles is to act as a sperm chemoattractant. Allurin, a 21 kDa Crisp protein rapidly released from the egg jelly of at least two frogs, X. laevis and X. tropicalis, elicits directed motility in both homospecific and heterospecific sperm. In X. tropicalis, allurin is coded for by the newly documented Crisp A gene. Recently, the observation that allurin can also elicit chemotaxis in mouse sperm raises the question of whether allurin-like proteins might act as sperm chemoattractants in mammals. Although an allurin gene has yet to be documented in mammals, Crisp proteins truncated post-translationally appear to exist in both the male and female reproductive tract of mammals.     

Factors influencing the ability to detect motion reversals in rotation simulations

Five experiments probed the conditions under which observers fail to report instantaneous reversals in the direction of motion of pixels that define the rotation of a transparent sphere or plane. Our results showed that the extent to which rotation reversals were not reported depended upon whether observers used strict or lax criteria to make their judgments, the degree of perspective present in the rotation simulations, and the percentage of pixels that actually reversed direction. Furthermore, we found failures to report rotation reversals both with stimuli whose pixels were confined to smooth surfaces and scattered within volumes. Reversal detection with planar stimuli, unlike sphere stimuli, depended upon the orientation of the stimulus at the moment of reversal. Treue et al. (1995) postulated a surface-interpolation process as the explanation for the apparent insensitivity of observers to such reversals. However, we suggest that other stages of processing (e.g. a structure-from-motion process) are required to account for these results.     

Phase variation: how to create and coordinate population diversity

Phase variation yields phenotypic heterogeneity in a clonal population as the result of one of a limited number of known molecular mechanisms. These include slipped strand mispairing, site-specific recombination and epigenetic regulation mediated by DNA methylation. Recently new regulatory variants utilizing these mechanisms have been identified, which is facilitating the identification of additional phase variation events solely from genome sequence analysis. Furthermore, it is becoming increasingly clear that in many cases phase variation control is integrated with regulatory networks and with cellular processes of a growing cell. This review focuses specifically on these recent advances in the understanding of the regulation of phase variation.     

Testing population differentiation in plant species – how important are environmental maternal effects

The maternal environment may contribute to population differentiation in offspring traits if growing conditions of mother plants are different. However, the magnitude of such environmental maternal effects compared with genetic differentiation is often not clear. We tested the importance of environmental maternal effects by comparing population differentiation in parental seed directly collected in the field and in F₁ seed grown under homogeneous conditions. The F₁ seeds were obtained by random crosses within populations. We used five populations in each of four plant species to analyse seed mass and growth chamber germination of both generations at the same time. In two species, we additionally tested offspring performance in the field. We found a significant population differentiation in all species and for nearly all measured traits. Population‐by‐generation interactions indicating environmental maternal effects were significant for germination (three species) and for seed mass (two species) but not for growth and reproduction. The significant interaction was partly due to a reduction of among‐population differentiation from the parental to the F₁ generation that can be explained by a decrease of maternal provisioning effects. However, in some species by trait combinations a change in population ranking and not a decrease of variation was responsible for significant population‐by‐generation interactions indicating environmental maternal effects beyond maternal provisioning. Fitting of seed mass as covariate was not successful in reducing environmental maternal effects on population differentiation in germination. We discuss alternative methods to account for environmental maternal effects in studies on genetic differentiation among populations.     

Motility and chemotaxis of Spirochaeta aurantia: computer-assisted motion analysis.

A computer program has been designed to study behavior in populations of Spirochaeta aurantia cells, and this program has been used to analyze changes in behavior in response to chemoattractants. Three kinds of behavior were distinguished: smooth swimming, flexing, and reversals in direction of swimming after a short pause (120 ms). Cell populations exposed to chemoattractants spent, on average, 66, 33, and 1% of the time in these modes, respectively. After the addition of a chemoattractant, behavior was modified transiently--smooth swimming increased, flexing decreased, and reversals were suppressed. After addition of D-xylose (final concentration, 10 mM), the adaptation time (the time required for the populations to return to the unmodified behavior) for S. aurantia was 1.5 to 2.0 min. A model to explain the behavior of S. aurantia and the response of cells to chemoattractants is described. This model includes a coordinating mechanism for flagellar motor operation and a motor switch synchronizing device.     

IL-12 elispot assays to detect and enumerate IL-12 secreting cells

The cytokine IL-12 promotes Th(1)type immune responses and plays a key role in immune regulation. The complex nature of IL-12 hampered its detection without use of stimulants that might give less relevant information. To detect circulating IL-12 p40, we developed enzyme-linked immunospot (ELISPOT) assays that allow enumeration of IL-12 p40 secreting cells without prior in vitro stimulation of the cells. In parallel, intracellular staining of IL-12 p40 by flow cytometry was performed to compare the two methods. IL-12 p40 secreting cells were detected in healthy subjects at a mean number of 103+/-155 per 10(5)blood mononuclear cells (MNC). Numbers of IL-12 p40 secreting blood MNC correlated with IL-12 p40 positive blood MNC detected by flow cytometry. Bacterial endotoxins and the inflammatory cytokines TNF-alpha and IFN-gamma control IL-12 production by antigen presenting cells. Utilizing IL-12 p40 ELISPOT assays, we could confirm occurrence of elevated numbers of IL-12 p40 secreting blood MNC after stimulation with TNF-alpha, IFN-gamma, LPS, LPS+TNF-alpha or LPS+IFN-gamma, compared to cultures without stimulant. Due to its central role in inflammation and autoimmunity, IL-12 is an attractive target for immunotherapy. IL-12 p40 ELISPOT assays represent a sensitive, specific and reliable tool for investigating the role of IL-12 in both health and disease.     

Mutagen sensitivity assays in population studies

Human population monitoring studies are frequently conducted to determine if exposure to environmental mutagenic agents can cause health problems or not. In these studies, a variety of biomarkers are used to identify biological events that are predictive of health consequences. An emphasis in this report is on the use of mutagen sensitivity assays to understand health risk. The assay is based on the assumption that exposure to mutagenic chemicals or mixtures of chemicals for a long time can cause cellular changes that are expressed as mutagen sensitivity. From experience in using these assays in cancer patients and in mutagen-exposed populations, it is clear that the expression of mutagen sensitivity is based on the interactions between mutagen exposure and individual susceptibility. When studies are conducted under appropriate conditions, expression of mutagen sensitivity is suggestive of increased risk for environmental disease such as cancer.     

Number of SNPS loci needed to detect population structure

The study of the association of polymorphic genetic markers with common diseases is one of the most powerful tools in modern genetics. Interest in single nucleotide polymorphisms (SNPs) has steadily grown over the last decade. SNPs are currently the most developed markers in the human genome because they have a number of advantages over other marker types. One of the critical problems responsible for 'spurious' association findings in case-control studies is population stratification. There are many statistical approaches developed for detecting population heterogeneity. However the power to detect population structure by known methods is highly dependent on the number of loci utilised. We performed an analysis of SNPs data available in the public domain from The Single Nucleotide Consortia Ltd. (TSCL). Three populations, Afro-American, Asian and Caucasian, were compared. Estimation of the minimum number of SNPs loci necessary for detection of the population structure was performed. Two clustering approaches, distance-based and model-based, were compared. The model-based approach was superior when compared with the distance-based method. We found more than 65 random SNPs loci are required for identifying distinct geographically separated populations. Increasing the number of markers to over 100 raises the probability of correct assignment of a particular individual to an origin group to over 90%, even with conventional clustering methods.     

Strategies for sperm chemotaxis in the siphonophores and ascidians: a numerical simulation study

Chemotactic swimming behaviors of spermatozoa toward an egg have been reported in various species. The strategies underlying these behaviors, however, are poorly understood. We focused on two types of chemotaxis, one in the siphonophores and the second in the ascidians, and then proposed two models based on experimental data. Both models assumed that the radius of the path curvature of a swimming spermatozoon depends on [Ca(2+)](i), the intracellular calcium concentration. The chemotaxis in the siphonophores could be simulated in a model that assumes that [Ca(2+)](i) depends on the local concentration of the attractant in the vicinity of the spermatozoon and that a substantial time period is required for the clearance of transient high [Ca(2+)](i). In the case of ascidians, trajectories similar to those in experiments could be adequately simulated by a variant of this model that assumes that [Ca(2+)](i) depends on the time derivative of the attractant concentration. The properties of these strategies and future problems are discussed in relation to these models.