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REVIEW |
CNRS, Univ. of Paris VI, P&M Curie, UMR 7009, Marine Station, 06230 Villefranche sur mer, France1 Biology Department, University of Bergen, Thormøhlensgate 55, Bergen 5020, Norway2 Ifremer, ARN, 29840 Argenton, France3 Ifremer, LALR, 34250 Palavas, France4 CNRS, UMR 7144, Marine Station, Place Georges Teissier, BP 74, 29682 Roscoff, France and5 Laboratory of Ichthyology, National Museum Natural History, Rue Cuvier, 75231 Paris, France
Correspondence should be addressed to J Cosson; Email: cosson{at}obs-vlfr.fr
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Abstract |
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Introduction |
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Most knowledge on sperm movement developed by simple flagella comes from studies on the classical model of sea urchin spermatozoa (Gibbons 1981) and on mammals for more structurally complex sperm cells. Nevertheless, some characteristics of fish sperm show original features: motility duration (Billard 1978, Billard & Cosson 1988, 1992), motility initiation (Morisawa 1985, 1994, Cosson et al. 1995a), or motility pattern (Boitono & Omoto 1992, Cosson et al. 1997). Most studies were carried out in fresh water fish species; there is less information concerning marine sperm characteristics. In the latter, motility activation occurs immediately after contact with SW, a high osmolarity medium compared with seminal fluid (SF). The initial velocity is very high at activation, but motility duration lasts for periods ranging only 40 s to 20 min as an energetic consequence of the high velocity.
Marine fish spermatozoa present unique features with which to study the specific aspects of sperm movement: (1) Brood fish are easily available for a part of the year in farmed species. (2) Fish sperm is easy to collect and save for short periods; marine fish sperm are easy to cryopreserve. (3) Sperm of fishes with external fertilization is immotile in the SF, and transfer to a swimming competent medium fully triggers motility, scores of which are presently used for selection of males, or for evaluation of cryopreservation results. A correlation between sperm motility and ability to fertilize the eggs has been established in many marine fish species. (4) Fish sperm cells are homogenous; all spermatozoa can be activated at the same time and then swim with very similar characteristics at a certain time point post-activation, an advantage for biochemists. (5) In many fish species, the flagellum is 50–60 µm long with a ribbon shape (presence of fins) instead of cylindrical; thus, the flagellum appears brighter by dark-field microscopy, allowing clear visualization of wave shapes. Attenuation (so-called dampening) of waves gradually invades the whole length of the flagellum during motility. At the last period before full stop, the waves are restricted to one-third of the flagellum combined with a drastic decrease of flagellar beat frequency (BF) leading to a decrease in translation efficiency (Cosson et al. 1997). (6) In several fish species, spermatozoa follow linear tracks; the flagellar bending is symmetrical, probably because of the absence of Ca2+ sensitivity of the axoneme.
Despite all these original features, few detailed studies on marine fish spermatozoa flagellar motility behavior have been conducted. In the present paper, we aim to gather the present knowledge on sperm movement characteristics of marine fish, with special emphasis on their high velocity capacities. A relationship between ionic effects, osmolality, and transience/abortion of motility is established. Using this indicator of the local and temporal ionic concentrations, we propose a model in which, following a transfer from SF to SW, the extracellular osmolality makes the intracellular ionic concentration of sperm evolve rapidly during the course of the motility phase; as a consequence the flagellar axoneme immediately activates but becomes gradually exposed to an increasing intracellular ionic environment preventing the development of distal waves and further leading to their full arrest. For general information on fish sperm, the reader is advised to consult the following review papers (Stoss 1983, Billard et al. 1994, 1995, Inaba 2003, Alavi & Cosson 2005, 2006, Lahnsteiner & Patzner 2007) and a book, Fish Spermatology (Alavi et al. 2007).
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The SF osmolality prevents sperm motility in the genital tract |
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In marine fish, the SF osmolality is much lower than that of SW and low enough to prevent motility (Table 1). In addition, the sperm cells concentration in the semen is usually high (see Table 1) which thus contributes to sperm immotility by local exhaustion of O2 and generation of high CO2 concentration in milt. In some species, spermatozoa have the ability to swim in seminal SF, sometimes only transiently. Motility also occurs when SF is slightly diluted with SW, which may accidentally happen at sperm collection. In turbot sperm, a 10% dilution of SF is enough to allow full motility. Contamination by urine may also accidentally happen when collecting sperm (Perchec et al. 1995a, 1995b, Perchec-Poupard et al. 1998): in turbot (Dreanno et al. 1998), such urine contamination leads to deleterious effects to spermatozoa.
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The sperm immotility in SF suffers a few exceptions: in sharks (dogfish, Triakis scyllia), spermatozoa are immotile in the testis but become progressively motile in the spermiduct during their descent in the epididymal duct; nevertheless ejaculated sperm are immotile while motility is fully triggered only at contact with SW (Minamikawa & Morisawa 1996).
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Transfer from SF into SW triggers full motility |
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Tolerance of motility toward osmolality depends on species: motility is initiated at osmolality (in mOsm/l) of 300 or above in turbot (Chauvaud et al. 1995, Dreanno et al. 1999c); 400 in sea bass (Villani & Catena 1991, Dreanno et al. 1999b); and 333–645 in tilapia, Sarotherodon melanotheron (Linhart et al. 1999) but, according to recent results (Legendre et al. 2008), 300–970 for fishes reared in SW (450–1600 for fishes reared in twice the salinity of SW), and 480 in Atlantic croaker (Vizziano et al. 1995). In halibut, NaCl solutions with osmolalities 350–1200 mOsm/l also permit motility (Billard et al. 1993). At 300 mOsm/l, a lower percentage of turbot spermatozoa are activated (70% compared with 90% in control at 1100 mOsm/l) but the velocity is same as in control. In sea bass, activation also occurs at osmolality lower than SW; at 630 mOsm/l, there is no change of initial velocity but at 40 s, flagella produce only oscillations without resulting in any efficient forward displacement. SF separated from turbot milt (316±1.5 mOsm/l) is permissive to motility in many samples, but CO2 concentration is the main factor preventing motility in SF with total CO2 concentration of 8.97±1.53 mmol/l or 8.66±1.48 meq/l HCO3 (at a pH of 7.58±0.03), pCO2 being 6.74±1.15 (Dreanno 1998).
In sea bass, solutions of 150–300 mOsm/l reversibly prevent motility but full activation occurs at 1100 mOsm/l (Fauvel et al. 1998, Dreanno et al. 1999b). In cod, sperm motility does not activate in a twofold dilution of SW by fresh water; therefore, this solution can be used as a short-term diluent. The motility is activated by transfer in artificial SW solutions with osmolalities from 700 to 1550 mOsm/l. Sperm is not active in SF but when the SF is diluted 1:4 by SW, full flagellar activation occurs (Cosson et al. 2008b). In hake, a diluent made of SW:DW in a 1:4 ratio does not activate motility, but allows further full activation by undiluted SW (Cosson et al. 2008a).
A general rule for sperm of marine species is that the osmolality gradient must be positive between outside and inside sperm cells to trigger motility. Although a brief and transient activation is observed, when the amplitude of the difference of osmotic pressure (OP) is too low; it consists of stochastic initiation of a few flagellar waves during brief time period (about 1 s) in any individual spermatozoon intercalated with very long resting periods. Similar process probably exists in cases where semen is contaminated by low quantities of SW; motility is transiently but not fully activated, therefore such sperm do not exhaust their ATP content as discussed later in detail in this review. Such transient activation already demonstrates the reversibility of the activation process but this reversibility can also be appreciated in the more direct experiments that follow (Cosson et al. 2008b). Sperm cells were diluted in an immobilizing solution and injected through a tiny micropipette (20 µm diameter and connected to a syringe providing low pressure) into a 100 µl drop of SW settled in between glass slide and cover slip. By observation with a microscope focused on the boundary between the SW and the injected immobilization solution (IS), each individual spermatozoon can be seen alternatively active or inactive depending, whether it is in contact with SW or IS respectively. This shows that activation and inactivation periods follow each other, which demonstrates both full reversibility and immediacy of the triggering/inactivating mechanism. Using a similar experimental design (Fig. 1), one can deliver a gentle but local flow of SW through a tiny micropipette (2 µm opening) to any portion of the flagellar length of a sperm cell, the latter being stuck by its head to a second holder micropipette (3–4 µm opening) itself immersed in a drop of IS. This way, local activation and inactivation can be visualized successively at any site located along the flagellum.
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An ambiguity is encountered in the case of the sperm of the wolffish (Anarhichas lupus), which is motile on stripping and remains as such for several days; motility is restricted to a 200–500 mOsm/l osmolality range. It has been suggested that motility is adapted to the local environment of the egg, which in this species presents osmolality lower than the surrounding SW (Kime & Tveiten 2002) which could be related to the fact that fertilization seems to be close to ‘internal’ in this species (Pavlov 1994, Pavlov et al. 1997).
Concerning pH, it is of low influence on motility and therefore pH is considered as a critical factor controlling motility. Values of pH in SF of several species are presented in Table 1.
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Immediately after activation, marine fish sperm swim with very high efficiency |
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These high velocity values are a consequence of the high flagellar BF ranging 50–70 Hz depending on species. Such high BF values are reminiscent of the so-called ‘hyperactivation’ process occurring to mammalian spermatozoa in the vicinity of ova, which consists of change in activity. However, in the case of fish, spermatozoa present much less chaotic movement characteristics, i.e. much more propulsive in a straightforward manner, probably corresponding to a different need and function.
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The motility period is limited to minute range duration for marine fish's sperm |
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The total duration of motility can be estimated either by visual microscopic observation of movement until full cessation of activity or by extrapolation to zero of the curve representing the percentage of motility versus time obtained by Computer Assisted Sperm Analysis (CASA; Cosson 2007a). The latter estimation is easiest in case of a linear decrease but more difficult in case of exponential or sigmoidal decrease. Table 2 gives values for the two types of estimation. In contrast to the motility durations described in Table 2, eel spermatozoa can swim for more than 20 min with little change in their motility characteristics (Gibbons et al. 1985). The same is true in conger spermatozoa (Cosson et al. 2008b).
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1 min for L. yokohamae and L. herzensteini, 2 min for Verasper moseri, 5 min for Pecten maximus, and 7–10 min for Vaejovis variegatus, to an hour in cod (Trippel & Morgan 1994) at 2 °C in the latter case. In hagfish, it was observed that sperm motility can last for periods up to 10 min (Morisawa 1995). Motility duration is also limited by damage that appears during the motility period. This damage appears in SW during the motility phase; it can be observed at high microscopical magnification. Either cytoplasmic blebs emerge anywhere along flagellar length, eventually impairing the propagation of waves (f to i in Fig. 2a) or a curling process (i in Fig. 2a) may develop at the flagellar tip, which obviously shortens the efficient part for wave propagation along the flagellum. Such curling is reminiscent of the naturally occurring curling (helicoidal) observed in intact eel flagella (Gibbons et al. 1985). Such blebs or curling damages usually result from local membrane defects engendered by osmotic stress and they are usually reversible by reversing the osmolality of the surrounding solution to correct the values (Perchec et al. 1996). Such damages may occur when milt happens to be contaminated by urine at collection by stripping (Dreanno et al. 1998, Perchec et al. 1998).
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Most motility parameters decrease during the motility period |
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Additional methods, designed to describe the details of wave parameters, were initially developed to study the sperm flagella of invertebrates (Brokaw 2004) and were adapted to marine fish spermatozoa (Cosson 2004, 2007a). In sperm cells from marine fishes, the initiation of the flagella waves occurs just at the junction between head and tail, therefore wave propagation occurs from head to flagellar tip, leading to opposite forward movement of the spermatozoon, head first (Cosson 2007b). The wave's velocity along the flagellum ranges from two to three times the translational velocity of the spermatozoon itself.
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Wave shape changes during the motility period |
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Flagellar waves are almost planar, i.e., each sine wave is ‘flat’ and the successive waves are coplanar. The waves are also most of the time coplanar with the plane of observation, which is the focal plane of the objective lens and which usually coincides optically with the glass slide plane as well as that of the cover slip. An exception to wave's flatness is found in eel spermatozoa in which waves are of corkscrew shape, i.e., helicoidal (Gibbons et al. 1985, Wooley 1997, 1998). Even though eel spermatozoa swim with very high BF, 95 Hz, their original wave pattern of helicoidal shape is poorly efficient in terms of forward velocity. They mainly present a rolling motion at 19 Hz, and flagella develop a 3D bending, recently detailed by Wooley (1998).
A more detailed analysis of the exact shape of the flagellum during motion can be obtained by determining the local curvature of the flagellum and plotting the curvature versus the distance on the flagellum (Cosson 2004).
The flatness of waves is not perfect, but waves slightly deviate from the plane; this was shown in sperm flagella of several species including marine fishes (Cosson et al. 2003). Such slight distortion is in the shape of alternating helical segments and is designed in such a way that each linear segment intercalated in between two successive curvatures generates a thrust that is not coplanar with the main beating plane: as a result, while swimming, sperm cells have the tendency to be pushed by their flagella toward surfaces, and then to remain swimming in the vicinity of these surfaces. This presents an advantage for observers because sperm cells remain in focus very close to the glass slide plane (usually the focal plane of the microscope). But the drawback is that a majority of spermatozoa swim in the vicinity of these surfaces, which lead to a bias when counting the moving spermatozoa by automatic techniques such as CASA. Such an ability to swim in the vicinity of the egg surface can be a biological advantage for fertilization efficiency. Swimming close to a surface frequently leads to the adhesion of sperm to glass; in order to make sure that spermatozoa can swim freely, prevention of sticking to glass surfaces can be ensured by the addition of BSA (at 0.1–0.5%) or Pluronic F-127 (at 0.1% from Sigma).
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. 5).
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The symmetry of flagellar waves and their dampening usually evolves during the motility period |
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During the motility period, the wave pattern of fish spermatozoa rapidly evolves (Fig. 2) successively from a fully beating pattern, where waves propagate along the whole length of the flagellum, to a partially beating pattern, where waves occupy only the portion of the flagellum proximal to the head, finally leading to the full absence of any beating wave (Cosson et al. 1997, 1999). The blocking process occurs to the distal part of the flagella at a precise time post-activation (20–30 s or later depending on species). The distal part of the partially beating flagellum appears straight, rigid, and devoid of any propagating wave (Fig. 2). 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. 4). Such a decrease of the wave amplitude (WA) along the flagellum from the proximal to distal part is called ‘wave dampening’. Flagellar wave dampening also occurs in invertebrate spermatozoa and was described more extensively by Tombes et al. (1987) in sea urchin flagella. In turbot spermatozoa, the wave dampening is also induced in vitro on demembranated flagella by the non-adequate ionic strength (Fig. 4): by contrast, ionic concentration shows little effect on trypsin-induced microtubule sliding (an in vitro measurement of dynein activity), BF, and proximal WA. In turbot sperm, this dampening is not related to Ca2+ induction of beat asymmetry as no sensitivity to this ion has been observed. 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. 4) show traits common to sperm of most other fish species (Cosson et al. 1997, 1999).
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Flagellar BF and efficiency both decrease during the motility period |
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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.e., less than 3–5 s, the minimal period of time required to achieve the second dilution and mixing, motility is initiated for almost 100% of the spermatozoa. This initial swimming period, which lasts 5–60 s depending on the species, is characterized by the high BF (up to 70 Hz) of fully developed waves proceeding throughout the whole flagellar length with an almost constant amplitude (Figs 2 and 4). 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 1981). In the sea urchin, such behavior is constantly exhibited for very long periods of time, i.e., hours, with BF of 45 Hz. The wave dampening features mentioned for turbot sperm as example occurs in all teleosts fish spermatozoa so far studied (Cosson et al. 1997, 1999). 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. 1985, 1991), for Acipenser baeri sperm (Cosson et al. 1995b) and for Scophthalmus maximus (Chauvaud et al. 1995) but also in WA in the distal portion of the flagellum (Cosson et al. 1999) as clearly demonstrated in turbot (Fig. 2).
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. 1999; Fig. 4). The flagellar beat efficiency is a measurement of the propulsive efficiency or swimming performance; it represents a combination between the BF and the WA. The combination of decrease in BF and WA leads to a faster decrease of the swimming performance (P), because P=BFxWA. 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 is clearly illustrated in turbot spermatozoa where the distal straight segment occupies 60–80% of the length or even the total length by the end of the translational motility period. This local paralysis may be paralleled by a curling process similar to that also observed in carp sperm (Perchec et al. 1996): the appearance of such a distal loop represents an additional contribution to the rapid slow down of sperm cells; it is followed by a full arrest. 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 1998, Cosson et al. 2008b). 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. 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. maximus (Perchec et al. 1993, Suquet et al. 1994, Chauvaud et al. 1995) similarly to observations in fresh water species such as O. mykiss (Christen et al. 1987, Billard & Cosson 1992), or Cyprinus carpio (P A second activation of motility can also be induced in spermatozoa from Ictalurus punctatus (Guest et al. 1976).
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The regulation of axonemal motility by ionic concentration can be observed in vitro |
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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. 4: the WA, the wavelength as well as portion of the flagellum where waves fully develop are all controlled by the ionic concentration of the RM. 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. As in vivo motility can be triggered in no electrolyte solutions with osmolality higher than 300 mOsm/kg, a potent effect of osmolality on axonemal machinery was tested. When glucose, sucrose, or mannitol were added at up to 500 mM to the reactivation solution (containing 75 mM KAc as major ions) neither distal blockage nor perturbation of the wave shape or the frequency were observed. By contrast, media containing glucose from 10 to 500 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. When KAc concentration was varied from 25 to 250 mM, little effects of ionic strength were observed neither on the portion of the flagellum where sliding occurred (distal versus proximal) nor on the rate of sliding (Cosson et al. 2008b); dynein itself would not be the direct target of ionic effects. The same is concluded for the CO2 effects, either in turbot (this paper) or in sea urchin sperm (Brokaw & Simonick 1976).
The variations of BF of axonemes reactivated in the presence of various ATP–Mg2+ concentrations indicate that the Km for ATP and the corresponding Vm are unchanged when KAc concentration varies from 25 to 250 mM. These results could tend to show that the axonemal component preferentially blocked by the increasing ionic strength could be some dynein subspecies located in the distal portion of the axoneme, as observed in Chlamydomonas flagella (Piperno & Ramanis 1991). Nevertheless, our in vivo observations show that in some cases, only distal portions of the flagellum can be active (Cosson et al. 2008b) and do not favor such a hypothesis.
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. 1982, Gibbons & Gibbons 1983). These assays also showed that K+ ions, even at high concentrations, are not inhibitors of the reactivated movement. The absence of inhibitory effects of K+ also holds for trout (Saudrais et al. 1998) and carp demembranated sperm models (Cosson & Gagnon 1988) and therefore cannot explain the K+ blocking effect observed in vivo in trout. In turbot, other ions were also tested, such as NaHCO3 at concentrations from 2.5 to 50 mM. When combined with the optimum reactivation medium containing 50–75 mM KAc, no effect was observed with 1–5 mM NaHCO3, but a progressive blockage of the axoneme in its the distal part was shown when higher NaHCO3 concentrations were used. These results confirm an effect of the ionic strength on distal blockage of the axoneme as NaHCO3 contributes more efficiently to the ionic strength (3 meq/molecule). The pH was controlled and it was little affected by the addition of NaHCO3. The results obtained in vitro with NaHCO3 will be discussed later for comparison with in vivo effects of CO2.
The results of the in vitro experiments with sea bass demembranated flagella show effects of Ca2+ ions combined with effects of ionic strength on the circularity of tracks, as seen in many species (Brokaw 1991a).
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The energy available in marine fish spermatozoa is rapidly exhausted |
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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. 1999c) and sea bass. In sea bass, ATP values of 1.22 µmol/mg protein (Zilli et al. 2004) were observed. It is difficult to correlate with the previously published results of 90 nmol/109 spermatozoa by Dreanno et al. (1999b) due to the difference of unit. Dreanno et al. (1997) and Zilli et al. (2004) have proposed to use ATP as a sperm quality marker for sperm used for cryoconservation.
In turbot, the ATP content is dependent on an aging phenomenon related to the maturity period (Suquet et al. 1997, Dreanno et al. 1999a). Usually, ATP content is around 200 nmol/109 spermatozoa (Dreanno et al. 1999b). In turbot, inhibitors of respiration (KCN) or ATP synthesis (oligomycin) have little effects on the internal ATP concentration.
The adenylate energy charge (AEC), a measurement of the percentage of energy present in ATP and ADP relative to adenylate compounds (Atkinson 1968), decreases from 90–95% at activation by SW to 50% at 1 min post-activation (Dreanno et al. 1999c).
Nuclear magnetic resonance studies on turbot spermatozoa (Dreanno et al. 1999c, 2000) show that other energetic compounds such as creatine phosphate contribute to the energetic balance during motility. 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 1996, Saudrais et al. 1998) allowing a more homogenous distribution of ATP along the flagellum. This shuttle involves ATP/ADP and creatine/PCr is similar to that described in sea urchin sperm (Tombes et al. 1987). In turbot, the presence of CO2 also affects the internal ATP level.
In hake sperm, preliminary results (Groison et al. 2007) indicate AEC initial value of 0.71 before motility activation, with a large individual variability from 0.17 to 0.96. In other species such as cod and tuna, no information is available.
In turbot, the ATP concentration in vivo can be calculated from the ATP content per cell: assuming a volume of 16x10–9 µl per sperm cell (Christen et al. 1987) supposedly constant during motility, the results of Dreanno et al. (1999c) lead to 6 mM ATP and 2 mM ADP initial concentration; in arrested spermatozoa, the ATP drops down to 1.5 mM. The Km of ATP for the dynein ATPase is about 150 µM and plateaus at 80 Hz (extrapolation of fmax; Cosson et al. 2008b). In halibut demembranated sperm flagella, Km ATP is 170 µM with fmax 51 Hz (Billard et al. 1993). In eel, Km ATP is 150 µM with fmax 83 Hz (Gibbons et al. 1985). In eel spermatozoa, the dynein ATPase is located in axonemal inner arms only (Baccetti et al. 1979), outer arms being absent. In turbot, at the end of the motility period, the intraflagellar ATP concentration drops down spectacularly but it is still high enough to sustain motility, while ADP (2 mM) is not high enough to fully inhibit the dynein activity by competition with ATP.
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. In turbot, this is possible because motility lasts long enough: initial respiration ranges 35 nmol O2 per min per 109 spermatozoa but at transfer in SW, it reaches 135 (same units) and then decreases to 40 at 2 min (Dreanno et al. 1999c). In cod, respiration at rest is of 1.5–3 (same units, Robitaille et al. 1987). The effect of respiratory inhibitors are detailed in Dreanno et al. (1999c): FCCP or KCN does not affect respiration of swimming spermatozoa; oligomycin is of low inhibitory effect; and KCN, NaN3, or NaHCO3 does not affect motility or flagellar BF.
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 2004). 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 1998, Cosson et al. 2008b). This delay probably involves both mitochondrial respiratory activity and ion-pumping activities, the latter being both ATP and motility dependent.
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The ultimate task for sperm is to meet an egg: how to attract sperm and guide it to micropyle |
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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. 2003). The consequence is that spermatozoa at surfaces mostly follow two dimensional tracks instead of three dimensional ones when swimming in space in the absence of any surface (for example in turbot; J Cosson & E Corset, unpublished observations), following the egg surface highly increases the chances for any spermatozoon to reach the micropyle. These chances are further improved by the presence of guidance grooves located on the surface of some eggs. Such guidance tracks physically converge toward the micropylar funnel, resulting in fine into a significant increase of sperm concentration in the close vicinity of the micropyle (estimated to 10 µm range in turbot eggs; Cosson et al. 2008b). This was also documented in some fresh water species such as Puntius conchonius (Amanze & Iyengar 1990) and medaka (Iwamatsu et al. 1993).
In addition to the delivery of DNA information through the egg micropyle, the ultimate task of the fish spermatozoon would be to induce a factor responsible for Ca2+ waves in the eggs, a necessary signal for egg activation (Coward et al. 2003).
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The ability of spermatozoa to fertilize eggs: limitations to the fertilization process |
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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. From the above formula, one can also predict that when velocity decreases logarithmically with time (the most common case), 80–90% of the distance is covered by sperm during the first half of the motility period (D1/2).
A gross estimation of the distance covered by a spermatozoon is 2.3 mm in sea bass (Dreanno et al. 1999b), 12 mm in turbot (Chauvaud et al. 1995), 14 mm in cod (this paper), 11 mm in hake (this paper), 10 mm in tuna, and 9 mm in halibut (Billard et al. 1993). Trippel & Neilson (1992) estimated an initial velocity of 2 mm/min for turbot, which leads to a total distance of 4–6 mm after integration during the motility period.
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) 1.02–1.39 (Carillo et al. 1995) and 1.16–1.89 in sea bass (Kjorsvik & Holmefjord 1995), 1.2–1.8 (Miller et al. 1995) in cod, 3.00–3.80 in halibut (Kjorsvik & Holmefjord 1995), 1.06±0.10 (AL Groison, M Suquet & J Cosson, unpublished observations) in hake, 1.4–3.5 (Ehrenbaum 1905) or 1.01–1.07 in turbot, and 1.0–1.2 in red tuna (Doumenge 1999).
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. 1999). 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. (2001) about cod in Murua & Motos (2006) about hake and more generally in Turner (1993).
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Sperm movement specificities in some species: effects of Ca 2+ and CO2 |
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Flagella can change their beating pattern (asymmetric or symmetric) in response to Ca2+ concentration perceived by the axoneme (Brokaw 1991a), which contains several calcium binding proteins: calmodulin in the spokes (Yang et al. 2001), centrin (caltractin) at the attachment point of the I2 and I3 inner dynein heavy chains (dynein regulatory complex; LeDizet & Piperno 1995), all elements by which asymmetry of flagellar beating could be controlled by Ca2+ in marine fish sperm.
The main CO2 effect is a blockage of axonemal motility both in vivo (Dreanno et al. 1995) and in vitro: this is specific to flatfishes, including turbot, due to the high concentration of carbonic anhydrase in their sperm flagella (Inaba et al. 2003). Both in vivo and in vitro, CO2 controls dynein activity through a NaHCO3 ionic effect similar to that of the other ions (Dreanno et al. 1999d). In the sea urchin’s sperm flagella, CO2 was also shown to affect wave shape without blockage, therefore probably involving a different mechanism (Brokaw & Simonick 1976, Brokaw 1977).
Additional signaling such as protein phosphorylation was shown to be involved in flagellar motility regulation (Inaba 2003) 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. 2004); similar results were observed in tilapia (Morita et al. 2006). Viviparous fish like guppy (Poecilia reticulata), a fresh water species, adopt a strategy requiring cAMP signaling, but it needs application to the spermatophore of some chaotropic chemicals that destabilize specific flagellar protein(s) (Tanaka & Oka 2005) to reach motility activation.
Flagellar shape modifications, i.e., stiffening of distal part of fish sperm flagella, could result from a regulation by hydin, a central pair protein of cilia and flagella (Lechtreck & Witman 2007).
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The osmolarity control of motility: toward a global explanatory model |
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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.e. 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 2002). By the OP effect, sperm motility in marine fishes is induced by the hyperosmotic shock of the surrounding medium (Billard et al. 1993, Chauvaud et al. 1995, Gwo 1995, Linhart et al. 1999, Krasznai et al. 2003, Cosson 2007b). Nevertheless, sperm motility is triggered in turbot and other flatfish in isoosmotic as well as in hyperosmotic media (Suquet et al. 1994) relatively to the SF because of the extra control by CO2. Motility occurs in a wide range of osmolalities, below or above that of SW (Suquet et al. 1994, Billard et al. 1995, Chauvaud et al. 1995): optimal osmolality (in mOsm/kg) is at 900–1100 in halibut (Billard et al. 1993), 300–1100 in turbot, 333–645 in tilapia (for fishes raised in SW, Linhart et al. 1999) but higher for tilapia fish raised in hypersalinity (Legendre et al. 2008), and 480 in Atlantic croaker (Vizziano et al. 1995). A general model of marine fish sperm motility control by osmolality is proposed in Fig. 6, where turbot sperm is taken as an example. It is based on results published by Suquet et al. (1994), Chauvaud et al. (1995), and Inaba et al. (2003) as well as additional in vitro result.
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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. 2003), and more generally in spermatozoa of several fish species including marine ones such as sea bass, turbot, and tuna (Cosson et al. 2008b), but inactive in sperm of other species apart from fishes (Krasznai et al. 2003). SACs are mechanosensitive channels that increase the membrane conductivity to ions such as Ca2+ or K+ when mechanical constraints induce distortion of this membrane (Yang & Sachs 1993). Mechanosensitivity is biologically important (Ingber 2006) 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. 2005). By proteomic analysis, the presence of a polycystin-2-like receptor was revealed in Chlamydomonas cilia (Pazour et al. 2005) and in metazoan cilia as well (Pazour & Rosenbaum 2002); in addition polycystin-2 is a ‘transient receptor potential’, a cation channel with mechanosensory properties (Nauli et al. 2003). Mechanosensitivity is also a specific property of flagellar axonemes; no longer beating cut-off pieces of axoneme that have lost coordination can be reinitiated by bending the flagellum with a microprobe (Lindemann & Rickemenspoel 1972). 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. 6).
The SACs may associate with other membrane proteins to modulate their activity (Vandorpe et al. 1994); those can be water channels (aquaporins) that are involved in the water transportation across membranes and may increase up to 1000-fold the diffusion rate of water molecules through membranes. In fish spermatozoa, the putative presence of aquaporins comes from observations where sperm motility is sensitive to low concentrations of inhibitors of aquaporins such as HgCl2 (Cosson et al. 1999, Abascal et al. 2007). In turbot sperm, the effect of HgCl2, supposedly targeted to aquaporins, is chronologically double: first, inhibiting initiation of motility and secondly, inducing a ‘twist’ of the flagellum (Fig. 5). Both aquaporins and polycystin-like receptors genes are present in a fish genome, the zebra fish.
Putting all together these features with the knowledge about the osmotic signal, we propose to involve SACs and aquaporins in the signaling pathway of the fish sperm activation (Fig. 6). This paradigm proposes several steps: the very first signal perceived by the membrane is osmotic; water exit would provoke a local membrane distortion or stretching. In this respect, the role of unusual creases shaped as fins (Cosson et al. 1999, Cosson 2007b) as discussed previously could be crucial not only in significantly increasing the membrane surface, this membrane ‘excess’ favoring water exchange, but also when distortions such as blebs appear on flagella exposed to extreme osmolalities (Perchec et al. 1996, Cosson et al. 2000). The SAC would respond immediately to this mechanical signal by increasing the local permeability, which would therefore allow ions such as Ca2+ or K+ and/or water to move rapidly in or out through channels or aquaporins. The triggering of an autocatalytic effect along flagellar membrane transmitted from place to place would explain why fish sperm activation proceeds in an extremely fast way (less than 20 ms according to our estimations). In fine, the local stretching of membranes would be the signal perceived by the axoneme because of the mechanosensitivity of this micromachine.
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Conclusions |
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The in vivo and in vitro observations of ionic strength control of axonemal activities are strengthened by observations of Billard (1978), Lahnsteiner & Patzner (1998), and Groison et al. (AL Groison, M Suquet & J Cosson, unpublished observations), which show that partly diluted SW supports longer sperm motility period than normal SW, as it leads to less harmful osmotical environment to sperm. Therefore, our in vitro observations combined with ATP measurements have lead us to a possible general schematic flow chart explaining how changes in the internal ionic concentration occurring in response to external osmolality could control fish sperm motility (Fig. 6). The response immediacy to the osmolality signal may be related to one major constraint endured by fish spermatozoa that are to obey a reproduction strategy in which a very brief period of reaction is needed to achieve the task. They exhibit a hypermotile behavior (high but brief BF) remarkably similar to the hyperactivated motility exhibited by mammalian spermatozoa in the vicinity of eggs before fertilization. The hypermotility of fish spermatozoa is demonstrated by a high velocity and a fast consumption of energy, which was stored during the spermatogenesis process. This strategy is probably dictated by another main constraint of many fishes, the short period of competence of the egg for fertilization in which the micropyle remains open only for 10–20 s after contact with SW.
As complementary information, some video files about fish sperm motility are available at http://biodev.obs-vlfr.fr/cosson/fishsperm/fishsperm.html.
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Declaration of interest |
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Funding |
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Acknowledgements |
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Received 20 November 2007
First decision 30 April 2008
Revised manuscript received 1 February 2008
Accepted 2 June 2008
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References |
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Abstract
Introduction
