Respiration and Motility in Starfish Spermatozoa at Various Times in the Breeding Season

In the spermatozoa of Asterias amurensis , patterns of changes in the respiratory rate and motility following dilution of dry sperm in sea water varied among batches and were classified into three types. The type I spermatozoa were immotile and exhibited quite low respiratory rate. Ethylenediamine tetraacetic acid made the type I spermatozoa motile and elevated their respiratory rate. The type II spermatozoa were also immotile and the respiratory rate remained quite low for about 5 min after the dilution. Thereafter, they spontaneously became motile and the respiratory rate increased. The type III spermatozoa became motile upon their dilution and exhibited high respiratory rate. Differences in the motility and respiratory rate of spermatozoa among batches probably result from degree of their maturation. In moving spermatozoa, the ADP and AMP levels increased at the expense of ATP. 2,4-Dinitrophenol elevated the respiratory rate only in immotile spermatozoa, which showed a high ATP level and quite low ADP level, but did not made them motile. Oligomycin inhibited the respiration of both motile and immotile spermatozoa. Probably, the respiratory rate is made low by a shortage of ADP in immotile spermatozoa and is enhanced by ADP production due to the initiation of their movement.     

Trout sperm motility. The transient movement of trout sperm is related to changes in the concentration of ATP following the activation of the flagellar movement

For freshwater fish the motile period of sperm is extremely brief, even after a dilution in isotonic media. This result is in contrast to most other animals (ranging from invertebrates to mammals), in which sperm are generally motile for at least several hours. We have analyzed the reasons for the brevity of this movement by studying the relationships between the metabolism of trout sperm and the activation of their motility upon dilution. Sperm motility was not initiated when the dilution medium contained an elevated concentration of potassium (20-40 mM), but dilution in an isotonic solution of sodium chloride triggered an immediate activation of motility, and sperm swam vigorously. Motility of sperm decreased rapidly and 15 s after dilution sperm were moving slowly in small circles. Sperm became abruptly immotile at 20-30 s and flagella straightened. When millimolar concentrations of Ca2+ were also present in the dilution medium, movement did not stop abruptly, flagella kept beating and stopped only after 1-2 min. When sperm remained immotile they retained a high concentration of ATP. The activation of motility induced a rapid decrease of ATP. In the absence of calcium, and after the cessation of motility, ATP increased slowly back to its original concentration. In the presence of millimolar concentrations of calcium the concentration of ATP decreased to a very low level and remained low thereafter. The progressive decrease of the flagellar beat frequency, that had been observed during the period of trout sperm movement, might be related to the rapid exhaustion of intraflagellar ATP. Motility could be reinduced in sperm that had recovered high concentrations of ATP, demonstrating the functional integrity of the motile apparatus even after flagellar arrest. In conclusion we suggest that the maximum duration of trout sperm motility, at most 2 min (as a consequence of a depletion of ATP during the movement), is due to a low mitochondrial oxidative phosphorylation capacity.     

Progression of flagellar stages during artificially delayed motility initiation in sea urchin sperm

Transition from immotile to motile flagella may involve a series of states, in which some of regulatory mechanisms underlying normal flagellar movement are working with others being still suppressed. To address ourselves to the study of starting transients of flagella, we analyzed flagellar movement of sea urchin sperm whose motility initiation had been retarded in an experimental solution, so that we could capture the instance at which individual spermatozoa began their flagellar beating. Initially straight and immotile flagella began to shiver at low amplitude, then propagated exclusively the principal bend (P bend), and finally started stable flagellar beating. The site of generation of the P bend in the P-bend propagating stage varied in position in the basal region up to 10 microm from the base, indicating that the ability of autonomous bend generation is not exclusively possessed by the very basal region but can be unmasked throughout a wider region when the reverse bend (R bend) is suppressed. The rate of change in the shear angle, the curvature of the R bend and the frequency and regularity of beating substantially increased upon transition from P-bend propagating to full-beating, while the propagation velocity of bends remained unchanged. These findings indicate that artificially delayed motility initiation may accompany sequential modification of the motile system and that mechanisms underlying flagellar motility can be analyzed separately under experimentally retarded conditions.     

Sodium-coupled motility in a swimming cyanobacterium.

The energetics of motility in Synechococcus strain WH8113 were studied to understand the unique nonflagellar swimming of this cyanobacterium. There was a specific sodium requirement for motility such that cells were immotile below 10 mM external sodium and cell speed increased with increasing sodium levels above 10 mM to a maximum of about 15 microns/s at 150 to 250 mM sodium. The sodium motive force increased similarly with increasing external sodium from -120 to -165 mV, but other energetic parameters including proton motive force, electrical potential, the proton diffusion gradient, and the sodium diffusion gradient did not show such a correlation. Over a range of external sodium concentrations, cell speed was greater in alkaline environments than in neutral or acidic environments. Monensin and carbonyl cyanide m-chlorophenylhydrazone inhibited motility and affected components of sodium motive force but did not affect ATP levels. Cells were motile when incubated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and arsenate, which decreased cellular ATP to about 2% of control values. The results of this investigation are consistent with the conclusion that the direct source of energy for Synechococcus motility is a sodium motive force and that below a threshold of about -100 mV, cells are immotile.     

Motility of rat spermatozoa at the site of fertilization

This study was undertaken to examine the factors that may affect the numbers and motility patterns of spermatozoa at the site of fertilization. The contents of the oviductal ampullae of previously mated cycling or superovulated immature rats were examined microscopically. We determined whether spermatozoa were free or associated with cells and whether they exhibited hyperactivated motility, forward progressive motility, or were immotile. These data were correlated with the percentage of fertilized eggs. In addition, the beat pattern of hyperactivated spermatozoa was characterized by using high-speed video microscopy. At the time when half of the eggs were fertilized, ampullae of cycling rats contained an average of less than one motile spermatozoon per ampulla. Most of these motile spermatozoa were hyperactivated. About half of these were free in the ampulla and about half were in the cumulus or zona pellucida. Hyperactivated spermatozoa displayed a nonprogressive whiplash wave form with a high amplitude recovery stroke similar to that described in hamster and guinea pig spermatozoa capacitated in vitro. In addition to motile spermatozoa, we counted about three immotile spermatozoa for each motile spermatozoon. In superovulated, immature female rats, we found about ten times as many spermatozoa in each category as in cycling rats. From our observations, it is clear that very few spermatozoa reach the ampulla of the oviduct. Furthermore our observations suggest that in cycling rats progressively swimming spermatozoa may become hyperactivated shortly after entering the ampulla of the oviduct. They probably enter the cumulus mass within a short time or become immotile.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the motility of maturing rat spermatozoa by computer-aided objective measurement.

A computer-aided sperm analysis system was optimized for objective assessment of the movement characteristics of mature and immature rat spermatozoa by testing different settings. Measurements of straight line velocity of individual motile cells were validated by manual tracking with a digitizer. Better agreement between the two methods and better performance in distinguishing between mature and immature spermatozoa was obtained by reducing the tracking rate to increase the time of analysis. However, numbers of motile and immotile cells could not be determined accurately. Manual counting of videotaped images revealed no significant differences in percentage motility of spermatozoa from five epididymal regions. Caput spermatozoa were characterized by low straight-line (VSL) and averaged-path (VAP) velocities and low path straightness (STR), whereas mature cells displayed high VSL, VAP and STR. An increase in curvilinear velocity on maturation was less obvious. Spermatozoa in the proximal corpus epididymidis were heterogeneous in their acquisition of motility maturation and the uniformity of movement pattern achieved in the distal corpus and proximal cauda regions tended to decrease again in the distal cauda epididymidis. Such objective measurements of motility patterns will facilitate studies on the regulation of motility development upon sperm maturation.     

Cilia-driven fluid flow as an epigenetic cue for otolith biomineralization on sensory hair cells of the inner ear

Ciliary motility is necessary for many developmental and physiological processes in animals. In zebrafish, motile cilia are thought to be required for the deposition of otoliths, which comprise crystals of protein and calcium carbonate, on hair cells of the inner ear. The identity of the motile cilia and their role in otolith biogenesis, however, remain controversial. Here, we show that the ear vesicle differentiates numerous motile cilia, the spatial distribution of which changes as a function of the expression pattern of the ciliogenic gene foxj1b. By contrast, the hair cells develop immotile kinocilia that serve as static tethers for otolith crystallization. In ears devoid of all cilia, otoliths can form but they are of irregular shapes and sizes and appear to attach instead to the hair cell apical membranes. Moreover, overproduction of motile cilia also disrupts otolith deposition through sustained agitation of the precursor particles. Therefore, the correct spatial and temporal distribution of the motile cilia is crucial for proper otolith formation. Our findings support the view that the hair cells express a binding factor for the otolith precursors, while the motile cilia ensure that the precursors do not sediment prematurely and are efficiently directed towards the hair cells. We also provide evidence that the kinocilia are modified motile cilia that depend on Foxj1b for their differentiation. We propose that in hair cells, a Foxj1b-dependent motile ciliogenic program is altered by the proneural Atoh proteins to promote the differentiation of immotile kinocilia.     

Identification and regulation of a molecular module for bleb-based cell motility

Single-cell migration is a key process in development, homeostasis, and disease. Nevertheless, the control over basic cellular mechanisms directing cells into motile behavior in?vivo is largely unknown. Here, we report on the identification of a minimal set of parameters the regulation of which confers proper morphology and cell motility. Zebrafish primordial germ cells rendered immotile by knockdown of Dead end, a negative regulator of miRNA function, were used as a platform for identifying processes restoring motility. We have defined myosin contractility, cell adhesion, and cortex properties as factors whose proper regulation is sufficient for restoring cell migration of this cell type. Tight control over the level of these cellular features, achieved through a balance between miRNA-430 function and the action of the RNA-binding protein Dead end, effectively transforms immotile primordial germ cells into polarized cells that actively migrate relative to cells in their environment.     

鱼类精子活力研究进展

鱼类精子在精巢和精浆中一般不活动,只有当精子被排到体外并被外界环境的溶液稀释后才能活动．鱼类精子活力受渗透压、离子、ｐＨ 值、温度及ＣＯ２ 等因子的调节和影响, 不同的鱼类其精子活力有不同的调节方式;外界因子对鱼类精子活力的影响, 是通过影响ｃＡＭＰ－ＡＴＰ－Ｍｇ２＋ 系统来影响鞭毛的活动而实现的． 精子活力的评价指标主要有：精子激活后的运动时间、精子激活比例、精子运动速度及精子鞭毛摆动频率等． 大多数鱼类的精子,其活动能力是在生殖管道中获得的．     

Surface-motility induction,attraction and hitchhiking between bacterial species promote dispersal on solid surfaces

The ability to move on solid surfaces provides ecological advantages for bacteria, yet many bacterial species lack this trait. We found that Xanthomonas spp. overcome this limitation by making use of proficient motile bacteria in their vicinity. Using X. perforans and Paenibacillus vortex as models, we show that X. perforans induces surface motility, attracts proficient motile bacteria and ‘rides'' them for dispersal. In addition, X. perforans was able to restore surface motility of strains that lost this mode of motility under multiple growth cycles in the lab. The described interaction occurred both on agar plates and tomato leaves and was observed between several xanthomonads and motile bacterial species. Thus, suggesting that this motility induction and hitchhiking strategy might be widespread and ecologically important. This study provides an example as to how bacteria can rely on the abilities of their neighboring species for their own benefit, signifying the importance of a communal organization for fitness.     

Speculations on the evolution of 9+2 organelles and the role of central pair microtubules

Motility generated by 9+2 organelles, variably called cilia or flagella, evolved before divergence from the last common ancestor of extant eukaryotes. In order to understand better how motility in these organelles is regulated, evolutionary steps that led to the present 9+2 morphology are considered. In addition, recent advances in our knowledge of flagellar assembly, together with heightened appreciation of the widespread role of cilia in sensory processes, suggest that these organelles may have served multiple roles in early eukaryotic cells. In addition to their function as undulating motility organelles, we speculate that protocilia were the primary determinants of cell polarity and directed motility in early eukaryotes, and that they provided the first defined membrane domain for localization of receptors that allowed cells to respond tactically to environmental cues. Initially, motility associated with these protocilia may have been gliding motility rather than the more complex bend propagation. Once these protocilia became functional motile organelles for beating, we believe that addition of an asymmetric central apparatus, capable of transducing signals to dynein motors and altering beat parameters, provided refined directional control in response to tactic signals. This paper presents hypothesized steps in this evolutionary process, and examples to support these hypotheses.     

A role for phosphorylation of glycogen synthase kinase-3alpha in bovine sperm motility regulation

The long-term goal of our work is to understand biochemical mechanisms underlying sperm motility and fertility. In a recent study we showed that tyrosine phosphorylation of a 55-kDa protein varied in direct proportion to motility. Tyrosine phosphorylation of the protein was low in immotile compared to motile epididymal sperm. Inhibition or stimulation of motility by high calcium levels or cAMP, respectively, results in a corresponding decrease or increase in tyrosine phosphorylation of the 55-kDa protein. Here we report purification and identification of this motility-associated protein. Soluble extracts from bovine caudal epididymal sperm were subjected to DEAE-cellulose, Affi-Gel blue, and cellulose phosphate chromatography. Tyrosine phosphate immunoreactive fractions contained glycogen synthase kinase-3 (GSK-3) activity, suggesting a possible correspondence between these proteins. This suggestion was verified by Western blot analyses following one-dimensional and two-dimensional gel electrophoresis of the purified protein using monoclonal and affinity-purified polyclonal antibodies against the catalytic amino-terminus and carboxy-terminus regions of GSK-3. Further confirmation of the identity of these proteins came from Western blot analysis using antibodies specific to the tyrosine phosphorylated GSK-3. Using this antibody, we also showed that GSK-3 tyrosine phosphorylation was high in motile compared to immotile sperm. Immunocytochemistry revealed that GSK-3 is present in the flagellum and the anterior portion of the sperm head. These data suggest that GSK-3, regulated by phosphorylation, could be a key element underlying motility initiation in the epididymis and regulation of mature sperm function.     

Single-Molecule Tracking of Inositol Trisphosphate Receptors Reveals Different Motilities and Distributions

Puffs are local Ca²⁺ signals that arise by Ca²⁺ liberation from the endoplasmic reticulum through the concerted opening of tightly clustered inositol trisphosphate receptors/channels (IP₃Rs). The locations of puff sites observed by Ca²⁺ imaging remain static over several minutes, whereas fluorescence recovery after photobleaching (FRAP) experiments employing overexpression of fluorescently tagged IP₃Rs have shown that the majority of IP₃Rs are freely motile. To address this discrepancy, we applied single-molecule imaging to locate and track type 1 IP₃Rs tagged with a photoswitchable fluorescent protein and expressed in COS-7 cells. We found that ∼70% of the IP₃R1 molecules were freely motile, undergoing random walk motility with an apparent diffusion coefficient of ∼0.095 μm s⁻¹, whereas the remaining molecules were essentially immotile. A fraction of the immotile IP₃Rs were organized in clusters, with dimensions (a few hundred nanometers across) comparable to those previously estimated for the IP₃R clusters underlying functional puff sites. No short-term (seconds) changes in overall motility or in clustering of immotile IP₃Rs were apparent following activation of IP₃/Ca²⁺ signaling. We conclude that stable clusters of small numbers of immotile IP₃Rs may underlie local Ca²⁺ release sites, whereas the more numerous motile IP₃Rs appear to be functionally silent.     

Deduction of a model for sperm storage in the oviduct of the domestic fowl (Gallus domesticus)

The mechanism of sperm storage in the fowl oviduct has remained a mystery since the 1960s, when sperm storage tubules (SST) were discovered between the shell gland and vagina. Previously, it was known that only motile sperm could ascend the vagina and enter these tubules. However, the means by which sperm resided therein was not clear. Research with computer-assisted sperm motion analysis has demonstrated that 1) seminal plasma glutamate acts as a motility agonist via N-methyl-d-aspartate receptors; 2) motility depends on extracellular Ca2+ and Na+; 3) straight-line velocity is a variable with a skewed distribution; 4) sperm cell trajectory is a function of straight-line velocity; and 5) specific inhibition of phospholipase A2 renders sperm immotile. An additional experiment demonstrated that Ca2+ acts as a second messenger and thereby modulates the content of long-chain acylcarnitine within sperm. Therefore, it is proposed that 1) the release of endogenous fatty acids fuels sperm as they ascend the vagina; (2) on entering the SST, motile sperm maintain position against a fluid current generated by SST epithelial cells; 3) resident sperm metabolize exogenous fatty acids released from lipid-laden epithelial cells; (4) motile sperm emerge from the SST when their velocity declines to a threshold at which retrograde movement begins; and 5) the skewed distribution of straight-line velocity accounts for the exponential pattern of sperm emergence from the SST. In summary, sperm residence within and emergence from the SST are phenomena most likely explicable in terms of sperm cell motility.     

Inactivation of Campylobacter jejuni flagellin genes by homologous recombination demonstrates that flaA but not flaB is required for invasion.

The role of the Campylobacter jejuni flagella in adhesion to, and penetration into, eukaryotic cells was investigated. We used homologous recombination to inactivate the two flagellin genes flaA and flaB of C. jejuni, respectively. Mutants in which flaB but not flaA is inactivated remain motile. In contrast a defective flaA gene leads to immotile bacteria. Invasion studies showed that mutants without motile flagella have lost their potential to adhere to, and penetrate into, human intestinal cells in vitro. Invasive properties could be partially restored by centrifugation of the mutants onto the tissue culture cells, indicating that motility is a major, but not the only, factor involved in invasion.     

Redistribution of cytosolic FGFR1 after induced migration of urothelial cells in culture

The distribution of cytosolic fibroblast growth factor receptor 1 (FGFR1) was studied in correlation to cell migration in urothelial cell line g/G. Cell motility was analysed with a new method using consecutive series of photographs of cells relocated on CELLocate coverslips and with image analysis software. The results confirmed that FGF1 stimulated cell motility only when cells were grown on collagen I coating. During the transition from sessile to motile cell phenotype a complete redistribution of cytosolic FGFR1 was revealed. In sessile cells, FGFR1 had a filamentous distribution and its location matched cytokeratin 7. In cells of the migrating phenotype, the distribution of FGFR1 was diffuse, mainly located in cytosol. Our data reveal that the location of cytosolic FGFR1 depends on the motile characteristics of the cell. The results also indicate that attachment of cells to collagen I is crucial for the induction of urothelial cell motility with FGF1.     

Formation,collective motion,and merging of macroscopic bacterial aggregates

Chemotactic bacteria form emergent spatial patterns of variable cell density within cultures that are initially spatially uniform. These patterns are the result of chemical gradients that are created from the directed movement and metabolic activity of billions of cells. A recent study on pattern formation in wild bacterial isolates has revealed unique collective behaviors of the bacteria Enterobacter cloacae. As in other bacterial species, Enterobacter cloacae form macroscopic aggregates. Once formed, these bacterial clusters can migrate several millimeters, sometimes resulting in the merging of two or more clusters. To better understand these phenomena, we examine the formation and dynamics of thousands of bacterial clusters that form within a 22 cm square culture dish filled with soft agar over two days. At the macroscale, the aggregates display spatial order at short length scales, and the migration of cell clusters is superdiffusive, with a merging acceleration that is correlated with aggregate size. At the microscale, aggregates are composed of immotile cells surrounded by low density regions of motile cells. The collective movement of the aggregates is the result of an asymmetric flux of bacteria at the boundary. An agent-based model is developed to examine how these phenomena are the result of both chemotactic movement and a change in motility at high cell density. These results identify and characterize a new mechanism for collective bacterial motility driven by a transient, density-dependent change in motility.     

Selection of nonmotile Tetrahymena with Ficoll underlayers.

The mechanisms regulating the development of cilia in Tetrahymena are poorly understood but might be revealed through the study of ciliogenesis mutants. Failure to regenerate cilia after dibucaine deciliation results in continued absence of motility. Therefore, to isolate ciliogenesis mutants efficiently, methods for separating motile and nonmotile cells are essential. We examined the efficacy of Ficoll underlayers for these separations. Ciliates of T. thng type IV) were mixed with Ficoll and added as underlayers to separatory funnels containing growth medium. At 27 C most of the cells remained motile and were found in the top layer; at 37 C, there was a time-dependent increase in the number of nonmotile cells and the number of cells in the Ficoll layer. After 150 min at 37 C, most of the cells became nonmotile and were found in the Ficoll layer. Other studies indicated that at 37 C, the cells remained alive and capable of regenerating cilia when deciliated. Thus, it is clear that the Ficoll underlayer effectively separates the majority of nonmotile cells from the majority of motile cells. Evidently, however, at 37 C wild-type T. thermophila exhibit temperature-sensitive phenotypic variability with regard to motility which should be minimized when selecting for mutations affecting motility and ciliogenesis.     

The role of hair cells, cilia and ciliary motility in otolith formation in the zebrafish otic vesicle

Otoliths are biomineralised structures required for the sensation of gravity, linear acceleration and sound in the zebrafish ear. Otolith precursor particles, initially distributed throughout the otic vesicle lumen, become tethered to the tips of hair cell kinocilia (tether cilia) at the otic vesicle poles, forming two otoliths. We have used high-speed video microscopy to investigate the role of cilia and ciliary motility in otolith formation. In wild-type ears, groups of motile cilia are present at the otic vesicle poles, surrounding the immotile tether cilia. A few motile cilia are also found on the medial wall, but most cilia (92-98%) in the otic vesicle are immotile. In mutants with defective cilia (iguana) or ciliary motility (lrrc50), otoliths are frequently ectopic, untethered or fused. Nevertheless, neither cilia nor ciliary motility are absolutely required for otolith tethering: a mutant that lacks cilia completely (MZovl) is still capable of tethering otoliths at the otic vesicle poles. In embryos with attenuated Notch signalling [mindbomb mutant or Su(H) morphant], supernumerary hair cells develop and otolith precursor particles bind to the tips of all kinocilia, or bind directly to the hair cells' apical surface if cilia are absent [MZovl injected with a Su(H)1+2 morpholino]. However, if the first hair cells are missing (atoh1b morphant), otolith formation is severely disrupted and delayed. Our data support a model in which hair cells produce an otolith precursor-binding factor, normally localised to tether cell kinocilia. We also show that embryonic movement plays a minor role in the formation of normal otoliths.     

Effects of osmolality, morphology perturbations and intracellular nucleotide content during the movement of sea bass (Dicentrarchus labrax) spermatozoa.

Sea bass spermatozoa are maintained immotile in the seminal fluid, but initiate swimming for 45 s at 20 degrees C, immediately after dispersion in a hyperosmotic medium (1100 mOsm kg-1). The duration of this motile period could be extended by a reduction of the amplitude of the hyperosmotic shock. Five seconds after the initiation of motility, 94.4 +/- 1.8% of spermatozoa were motile with a swimming velocity of 141.8 +/- 1.2 microns s-1, a flagellar beat frequency of 60 Hz and a symmetric type of flagellar swimming, resulting in linear tracks. Velocity, flagellar beat frequency, percentage of motile cells and trajectory diameter decreased concomitantly throughout the swimming phase. After 30 s of motility, the flagellar beat became asymmetric, leading to circular trajectories. Ca2+ modulated the swimming pattern of demembranated spermatozoa, suggesting that the asymmetric waves produced by intact spermatozoa after 30 s of motility were induced by an accumulation of intracellular Ca2+. Moreover, increased ionic strength in the reactivation medium induced a dampening of waves in the distal portion of the flagellum and, at high values, resulted in an arrest of wave generation in demembranated spermatozoa. In non-demembranated cells, the intracellular ATP concentration fell immediately after transfer to sea water. In contrast, the AMP content increased during the same period, while the ADP content increased slightly. In addition, several morphological changes affected the mitochondria, chromatin and midpiece. These results indicate that the short swimming period of sea bass spermatozoa is controlled by energetic and cytoplasmic ionic conditions and that it is limited by osmotic stress, which induces marked changes in cell morphology.