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Julien Berro Michael M. Wallace F. Methods in Enzymology Volume Volume Methods in Cell Biology, Volume 86 Volume Kinase Inhibitors Methods and Protocols. Another type of wave distortion is also observed, when turbot or sea bass sperm are exposed to pollutants such as mercury derivatives see in Fig. Another characteristic of beating flagella is the ability to develop either symmetric or asymmetric ways of beating: waves follow each other and successive waves are called direct and reverse waves Gibbons When both are of equal curvature, symmetrical movement is developed and forces the sperm cells to describe linear tracks.
In case of unequal curvature, movement becomes consequently circular and sperm cells describe circles of corresponding diameter Brokaw a. This is observed in sea bass sperm flagella, in vivo as well as in vitro Dreanno et al. During the motility period, the wave pattern of fish spermatozoa rapidly evolves Fig. The distal part of the partially beating flagellum appears straight, rigid, and devoid of any propagating wave Fig. The fully developed waves are initiated and propagated in the proximal segment of the flagellum over a distance covering one-third to one-fourth of the total length Fig.
Flagellar wave dampening also occurs in invertebrate spermatozoa and was described more extensively by Tombes et al.
In turbot spermatozoa, the wave dampening is also induced in vitro on demembranated flagella by the non-adequate ionic strength Fig. Dampening occurs because waves persist preferentially in the portion of the flagellum close to the head. The observations of wave dampening obtained in vivo and in vitro on turbot sperm flagella Fig.
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Figure 4 Download Figure Download figure as PowerPoint slide Wave shape of turbot sperm observed in vivo and in vitro. Upper panel: a—d just after the triggering of motility, fully developed waves occupy most of the length of the flagellum; their amplitude is constant and their frequency remains high; e—g at the middle of the motility phase, the frequency drops while the wave propagation becomes restricted to the proximal part of the flagellum and the very tip of the flagellum is in rigor; h—k waves develop only in a very short portion close to the head; and l no wave remains and the whole flagellum stops beating; it adopts a rigor aspect.
Bottom right: schematic of the progression of the wave shape in fish sperm flagella during the swimming period in vivo upper part of the panel and in vitro lower part of the panel. The rigidification process following dampening observed for turbot spermatozoa by Chauvaud et al. Published results show that all the elements necessary for the functioning of a phosphocreatine PCr shuttle are present in turbot as well as in trout spermatozoa Saudrais et al. This shuttle would allow a more homogenous distribution of ATP along the flagellum.
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However, as emphasized above, dampening also occurs in vitro in demembranated spermatozoa, where no such shuttle can be active. In the case of turbot sperm, a similar sequence of wave dampening leading to full stiffening can also be precociously induced by CO 2 application Dreanno et al. The BF represents the number of waves generated every second; the BF is directly in proportion to the activity of dyneins and therefore of the rate of ATP hydrolysis.
In the six species described in this paper, the BF shows high values at initiation of motility, but decreases rapidly as a function of time. Fish sperm advantageously exhibits a high homogeneity of movement in the sperm population at a given time point; that is, the successive images of one single sperm cell are representative of the majority of the population but this is only true when considering any defined time point after activation.
This is mainly obtained by the use of a double dilution procedure first dilution of milt in a non-swimming solution followed by a second dilution in the SM , which allows homogeneity and synchrony in the motility initiation for the whole population of sperm cells. Within a very brief period, i. These characteristics are similar to those of the sea urchin's sperm flagellum, which is commonly used as a model for sperm movement studies Gibbons In the sea urchin, such behavior is constantly exhibited for very long periods of time, i.
The wave dampening features mentioned for turbot sperm as example occurs in all teleosts fish spermatozoa so far studied Cosson et al. After a first period post-activation, fish sperm show a decrease not only in BF, in the case of Oncorhynchus mykiss sperm Cosson et al. After a first period post-activation, fish sperm show a decrease not only in BF but also in WA in the distal portion of the flagellum Cosson et al. The flagellar beat efficiency is a measurement of the propulsive efficiency or swimming performance; it represents a combination between the BF and the WA.
This process is accelerated as the time progresses within the movement period, because waves travel in a more and more restricted part of the proximal flagellum, while a longer and longer distal part becomes inactive and straight. This local paralysis may be paralleled by a curling process similar to that also observed in carp sperm Perchec et al.
Nevertheless, both the rigidification and the curling are reversible processes; this reversal is obtained when sperm cells are transferred from SM high OP back to IM low OP. The reversibility process is related to both reconstitution of energy stores and internal ionic concentration. A subsequent transfer to SW needs to be applied after a delay in IM, during which cells reload their ATP and their ionic levels to a normal value compatible with full motility Dreanno , Cosson et al.
This delay probably involves both mitochondrial respiratory activity and ion-pumping activities, the latter being also ATP dependent. Energy stores are allowed to reconstitute during the incubation in an opposite OP situation sustaining no motilility and this allows a second motility sequence to be triggered through a new transfer in SW.
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Sperm flagella exhibit a new full wave pattern with normal waves developed and high beat efficiency. The hypothesis of such a restoration of the initial energy store has been confirmed by the direct measurement of the ATP concentration see below , which shows a low value at the end of the first motility phase compared with that obtained after regeneration; this was observed for the sperm of S. Complementary information leading to the understanding if this specific in vivo behavior comes from experiment using demembranated sperm models; wave parameters of permeabilized models were measured in the presence of a variable concentration of ions.
Below we detail these results on the in vitro sperm models of two species turbot and sea bass , because they are crucially important to better understand the two main features specific to fish sperm: the mechanism of activation and the briefness of motility, and finally give rise to a common model allowing an explanation of both features. In vitro wave patterns are shown in Fig.
Briefly, when increasing ionic concentration, waves activity parameters vary successively from zero to an optimal value, then decrease more and more down to zero again at much higher ionic concentrations. By contrast, media containing glucose from 10 to mM but no KAc did not allow any flagellar motility. It is concluded that the effects observed in vitro are not due to a direct sensitivity of axonemes toward osmolality. In order to identify which element of the axonemal machinery is affected by ionic strength, experiments of microtubule sliding were conducted.
Nevertheless, our in vivo observations show that in some cases, only distal portions of the flagellum can be active Cosson et al. In similar assays using demembranated turbot spermatozoa, in vitro reactivation media made of KCl, K propionate, and NaCl gave equal results but K acetate was preferred because sperm motility was more stable, as already stated for sea urchin spermatozoa in such media Gibbons et al.
In turbot, other ions were also tested, such as NaHCO 3 at concentrations from 2. A common feature is the decrease in sperm ATP content during the motility period. Presently, most studies in marine fishes concern turbot Dreanno et al. In sea bass, ATP values of 1. Dreanno et al. In turbot, the ATP content is dependent on an aging phenomenon related to the maturity period Suquet et al.
Nuclear magnetic resonance studies on turbot spermatozoa Dreanno et al. The presence of creatine kinase was described in turbot spermatozoa and therefore a PCr shuttle is probably present in turbot as well as in trout spermatozoa Saudrais , Saudrais et al. In hake sperm, preliminary results Groison et al. In other species such as cod and tuna, no information is available. In eel spermatozoa, the dynein ATPase is located in axonemal inner arms only Baccetti et al. The respiration rate of marine fish sperm is boosted at activation.
This was measured in few species because of the briefness of the motility period relative to the time period needed to obtain this respiration rate using an oxygen electrode.
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In cod, respiration at rest is of 1. The effect of respiratory inhibitors are detailed in Dreanno et al. Marine fish spermatozoa are able to sustain a second motility period after a certain period of rest, provided previously activated spermatozoa remain metabolically active. In turbot, the revival of spermatozoa rendered immotile by a first incubation in SW can be obtained by allowing these cells to settle in an artificial SF Cosson After a subsequent transfer into SW, spermatozoa reinitiate motility and swim similarly to the first activation in SW. This second transfer needs to be applied after a delay, during which cells reload their ATP level Dreanno , Cosson et al.
This delay probably involves both mitochondrial respiratory activity and ion-pumping activities, the latter being both ATP and motility dependent. Even though one generally believes intuitively that sperm is attracted by egg, there are very few examples of the demonstration of such phenomenon, so-called chemotaxis: this has been well established in sea urchins, several jelly fishes, and ascidians Bohmer et al. In fish, the only clear demonstration is in the pacific herring Clupea pallasi ; spermatozoa are not active when delivered at spawning in SW but only when they happen to reach the egg chorion, more precisely the vicinity of the micropyle Yanagimachi , Yanagimachi et al.
Physicotaxis is necessary for physical guidance of sperm cells on the surface of eggs and eventually toward the micropyle. The first aspect of physicotaxis is the tendency for spermatozoa to swim on any surface, including the surface of an egg, which at this scale appears large and flat. This ability to swim in the close vicinity of surfaces is due to a slight deviation of the beating plane of sperm flagella, which generates a thrust of small amplitude out of the main beat plane and leads to a chiral shape in two successive helices inverted relative to each other Cosson et al.
These chances are further improved by the presence of guidance grooves located on the surface of some eggs. In order to meet the egg, sperm should come into its vicinity, therefore the total averaged distance covered called D by sperm cells is a key factor. D can be calculated from the change in velocity with time by use of the formula:.
Integral calculation needs first a curve fitting of the plot of velocity versus time after activation. In theory, this calculation must take into account the linearity index weighted according to the percentage of motility decrease. As tracks are usually not linear but circular, the efficient distance covered is even lower than that predicted by calculation. A gross estimation of the distance covered by a spermatozoon is 2.
Compared with many other oviparous animal species, the egg size of marine fishes is relatively small but definitely big when compared with the spermatozoon size. The egg diameter is in mm 1. Comparison of egg size relative to the distance covered by a spermatozoon leads to similar values; therefore, any marine fish spermatozoon should be delivered in the close proximity of the ovocyte in order to reach its micropyle. This may explain a double reproductive strategy for marine fishes in order to accomplish the reproductive task: 1 a very large excess of sperm cells relative to one egg and 2 a local delivery of sperm resulting from a close proximity between the two spawners, male and female.
Obviously, the fertilizing ability not only depends on the ratio of the number of active spermatozoa per egg but also of the time elapsed since motility activation Fauvel et al. In turbot, after a 3-min period of sperm swim, the number of swimmers decreases: the efficiency for fertilization decreases, reaching zero after tens of minutes. The fertilization ability of gametes is one among other traits of the reproductive biology of fishes; in this regard, specific information concerning fish mating can be found in Rakitin et al.
As seen in sea bass, tuna and tilapia Morita et al. The main CO 2 effect is a blockage of axonemal motility both in vivo Dreanno et al. Both in vivo and in vitro , CO 2 controls dynein activity through a NaHCO 3 ionic effect similar to that of the other ions Dreanno et al. Additional signaling such as protein phosphorylation was shown to be involved in flagellar motility regulation Inaba but little is known in this respect in marine fish spermatozoa.
In striped bass, flagellar activation seems to occur through phosphorylation of some specific proteins via a cAMP-independent pathway Shuyang et al. Flagellar shape modifications, i. Osmolality effects on fish motility have been studied by Morisawa and Perchec-Poupard et al. Regarding osmolality adaptation of marine fish spermatozoa it is worth to remind the presence of membrane folding which develops in most species on both sides of the flagellar membrane.
These fins flank the whole length of the flagellum, ranging to several micrometers in width, and obviously increase the ratio surface to volume of the flagellar organelle. Not only do they contribute to the efficiency of the thrust generated by waves by increasing the flagellar surface used for the friction on the surrounding medium during movement, but they also contribute to a large increase of the membrane surface when compared with a simple cylindrical axoneme. A calculation applied to turbot flagellum leads to the following: the surface of a flagellum in shape of a simple cylinder is about 34 millions square nm; this is about one-fourth compared with the surface of the same cylinder comprising fins millions square nm.
At initiation of movement of marine fish spermatozoa in SW, the first signal received by the membrane is osmotic, followed by a water flux in either direction, or provokes local membrane distortions due to osmotic constraints. A significant increase of the membrane surface due to these fin-shaped creases Cosson et al. The distortion ability of creases would lead to the blebs or coils observed on exposure to extreme osmotic situations Perchec et al. Subsuming the above remarks on marine fish sperm, we have developed the following model to explain motility activation then inhibition resulting from non-optimal internal ionic concentration, according to in vitro results.
Sudden exposure of an animal cell to an extreme and drastic osmotic environment, i. SW, causes various reactions including volume and shape changes because, in contrast to vegetal cells, they are devoid of the constraints of a polysaccharide wall Stein By the OP effect, sperm motility in marine fishes is induced by the hyperosmotic shock of the surrounding medium Billard et al. Nevertheless, sperm motility is triggered in turbot and other flatfish in isoosmotic as well as in hyperosmotic media Suquet et al. Motility occurs in a wide range of osmolalities, below or above that of SW Suquet et al.
A general model of marine fish sperm motility control by osmolality is proposed in Fig. It is based on results published by Suquet et al. Figure 6 Download Figure Download figure as PowerPoint slide Activation process and signal transduction in marine spermatozoa: general schematic of the interacting processes occurring during the motility period of a turbot spermatozoon. Seawater is of much higher osmolality compared with SF: the osmolality jump induces an osmolality reaction at the sperm membrane level; water exits the sperm cell, a process accelerated by water pumps aquaporins.
As a consequence of water exit, internal ionic concentration increases and reaches optimal values for dynein motor activity. Beating of flagella is at maximal velocity but decreases with time because of two reasons: the ionic concentration becomes too high to sustain correct dynein activity and ATP concentration declines and becomes limiting for flagellar beating.
After some period, flagellar activity stops because of these unfavorable conditions. In some cases, resistance to very low osmolality is surprisingly high; turbot sperm can sustain dilution in distilled water and resist reversibly for several minutes. Osmolality is definitely a key factor for fish gametes released in the surrounding medium. In the case of male gametes, it is worth mentioning that mechanical activation could be the second signal in response to the first osmotic signal via the stretch-activated channels SACs located in the sperm membrane.
It has been shown that a specific and reversible inhibitor of the SACs, gadolinium, is active on carp spermatozoa Krasznai et al. Mechanosensitivity is biologically important Ingber especially considering that flagella and cilia are acting as mechanosensitive detectors: the signal is transduced through gene products of the polycistic kidney disease family Pan et al. By proteomic analysis, the presence of a polycystinlike receptor was revealed in Chlamydomonas cilia Pazour et al.
In fish spermatozoa, the same situation probably occurs when sperm are put in a medium limiting the initiation of motility. Activation by SW probably involves such mechanosensitivity; at first, mechanosensitive channels are activated which themselves mechanically activate the axoneme Fig. The SACs may associate with other membrane proteins to modulate their activity Vandorpe et al.