sperm motility in fish

Cell Biology International 30 (2006) 1e14www.elsevier.com/locate/cellbi

Review

Sperm motility in fishes. (II) Effects of ionsand osmolality: A review

Sayyed Mohammad Hadi Alavi a, Jacky Cosson b,*

a Department of Fisheries and Environmental Sciences, Faculty of Natural Resources,

University of Tehran, P.O. Box: 31585-4314, Karaj, Iranb Laboratory of Developmental Biology, UMR 7009 CNRS, University Pierre et Marie Curie,

F-06230 Villefranche-sur-Mer, France

Received 24 November 2004; revised 27 May 2005; accepted 8 June 2005

Abstract

The spermatozoa of most fish species are immotile in the testis and seminal plasma. Therefore, motility is induced after the spermatozoa arereleased into the aqueous environment during natural reproduction or into the diluent during artificial reproduction. There are clear relationshipsbetween seminal plasma composition and osmolality and the duration of fish sperm motility. Various parameters such as ion concentrations (KC,NaC, and Ca2C), osmotic pressure, pH, temperature and dilution rate affect motility. In the present paper, we review the roles of these ions onsperm motility in Salmonidae, Cyprinidae, Acipenseridae and marine fishes, and their relationship with seminal plasma composition.

Results in the literature show that:

1. KC is a key ion controlling sperm motility in Salmonidae and Acipenseridae in combination with osmotic pressure; this control is moresimple in other fish species: sperm motility is prevented when the osmotic pressure is high (Cyprinidae) or low (marine fishes) comparedto that of the seminal fluid.

2. Cations (mostly divalent, such as Ca2C) are antagonistic with the inhibitory effect of KC on sperm motility.3. In many species, Ca2C influx and KC or NaC efflux through specific ionic channels change the membrane potential and eventually lead to

an increase in cAMP concentration in the cell, which constitutes the initiation signal for sperm motility in Salmonidae.4. Media that are hyper- and hypo-osmotic relative to seminal fluid trigger sperm motility in marine and freshwater fishes, respectively.5. The motility of fish spermatozoa is controlled through their sensitivity to osmolality and ion concentrations. This phenomenon is related to

ionic channel activities in the membrane and governs the motility mechanisms of axonemes.� 2005 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Keywords: Calcium (Ca2C); Cyprinids; Motility initiation; Osmotic pressure; Potassium (KC); Salmonids; Sodium (NaC); Sperm motility; Spermatozoa; Sturgeon

1. Introduction

Fish spermatozoa are immotile in the seminal tract (Stoss,1983), in contrast to the situation in reptiles or mammals(Krasznai et al., 1995). The osmolality and composition ofthe seminal plasma usually prevent sperm motility in fish

* Corresponding author.

E-mail address: [email protected] (J. Cosson).

1065-6995/$ - see front matter � 2005 International Federation for Cell Biolog

doi:10.1016/j.cellbi.2005.06.004

sperm ducts (Billard, 1986). The seminal fluid not only immo-bilizes the spermatozoa, but also protects them (see Methodsin Cosson et al., 1997). This is the case, for example, in thecommon carp, Cyprinus carpio (Toth et al., 1995) and rainbowtrout, Oncorhynchus mykiss (Billard, 1986).

During natural reproduction, fish spermatozoa are renderedmotile after discharge into the aqueous environment (in ovip-arous species) or the female genital tract (in viviparous andovoviviparous species) (Stoss, 1983; Billard, 1986; Billardand Cosson, 1990). In the environment encountered by

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2 S.M.H. Alavi, J. Cosson / Cell Biology International 30 (2006) 1e14

spermatozoa after release in natural conditions or after dilutionin artificial fertilization media, definite ion concentrations(NaC, KC, Ca2C, Mg2C, etc.), osmolality and pH are crucial.As these conditions depolarize the cell membrane, they mayaffect the capacity of sperm tails for flagellar motility andmay stimulate this motility (Morisawa and Suzuki, 1980;Morisawa et al., 1983).

Among the above-mentioned factors, two major ones(which are eventually additive) control sperm motility and al-low it to be initiated. These key factors are KC concentrationin salmonids (Scheuring, 1925; Schlenk and Kahmann, 1937;Baynes et al., 1981; Billard, 1983; Stoss, 1983) and sturgeons(Gallis et al., 1991; Cosson and Linhart, 1996; Toth et al.,1997; Ciereszko et al., 2000; Alavi and Cosson, 2005a; Alaviet al., 2004a,b, in press), and osmolality in cyprinids (Morisawaet al., 1983; Stoss, 1983; Billard, 1986; Perchec et al., 1995a,b)and marine fishes (Morisawa and Suzuki, 1980; Oda andMorisawa, 1993; Gwo et al., 1993; Takai and Morisawa,1995; Krasznai et al., 2003a,b).

Nevertheless, it is clear that intracellular parameters such asATP and cAMP concentrations (Christen et al., 1987; Mori-sawa and Ishida, 1987; Cosson et al., 1986; Perchec et al.,1995a,b), concentrations of ions, especially Ca2C (Morisawa,1985; Cosson et al., 1989) and pH (Krasznai et al., 1995;Lahnsteiner et al., 1996), and extracellular factors such as tem-perature (Billard, 1986; Williot et al., 2000; Alavi and Cosson,2005a) and pH (Billard, 1986; Cosson and Linhart, 1996;Alavi and Cosson, 2005), affect the capacity for and durationof mobility in fish spermatozoa. To increase the efficiency ofartificial fertilization, the composition of sperm diluents isvery important and must be adjusted to mimic the species-spe-cific composition of the seminal plasma (Alavi et al., 2004a,b,in press; Rurangwa et al., 2004). In addition, it is necessary topay attention to other factors such as diluent temperature andabsence of heavy metal pollutants (Chyb et al., 2000; Epleret al., 2000; Kime et al., 1996).

The aim of the present review is to discuss the effects ofions (KC, Ca2C and NaC) and osmotic pressure as factorsstimulating sperm motility in three major groups of fishes,Cyprinidae, Salmonidae and Acipenseridae, by comparisonwith marine fish species. Available data on the ionic composi-tion and osmolality of semen are summarized.

2. Ionic composition of semen

Semen (or milt) is defined as spermatozoa plus seminalplasma. Seminal plasma (or fluid) has a unique composition:some components support the spermatozoa, while othersreflect the functions of the reproductive system and the sper-matozoa (Ciereszko et al., 2000). Studies on semen character-istics are necessary to understand the basic biochemicalprocesses that occur in sperm motility and during fertilization(Linhart et al., 1991; Coward et al., 2002; Ingermann et al.,2002; Itoh et al., 2003; Kowalski et al., 2003; Wojtczaket al., 2003), to evaluate the reproductive abilities of differentfish species (Billard, 1986; Alavi and Cosson, 2005a,b;Coward et al., 2002; Rurangwa et al., 2004), and to improve

methods for short- and long-term storage of fish semen (Piroset al., 2002). In this regard, less information is available onmarine fishes and Acipenseridae than Cyprinidae andSalmonidae.

The osmolality and ionic composition of semen have beenstudied in cyprinids (Billard and Cosson, 1992; Billard et al.,1995a,b; Cruea, 1969; Kruger et al., 1984; Lahnsteiner et al.,1996; Linhart et al., 1991, 2003a,b,c), salmonids (Bayneset al., 1981; Billard and Cosson, 1990, 1992; Billard et al.,1995b; Linhart et al., 1991; Morisawa et al., 1979; Scott andBaynes, 1980) and sturgeons (Gallis et al., 1991; Mims,1991; Toth et al., 1997; Alavi and Cosson, 2005a,b; Linhartet al., 2003a,b,c). Data about the ionic composition of Cypri-nidae, Salmonidae, Acipenseridae and marine fish semen aresummarized in Table 1. The literature reveals wide intra-and inter-species variability in the ionic composition of fishseminal plasma and sperm, suggesting significant intra- andinter-specific differences in testicular secretion (Billardet al., 1995b) and implying differences in the precise mecha-nism of motility initiation. The epithelial cells of the spermduct have a secretory role and may fluidize goldfish semen(Yamazaki and Donaldson, 1968). From the data in Table 1the following observations can be made:

1. The NaC, KC, and Cl� ions predominate in seminalplasma.

2. The ionic composition may change during the reproduc-tive season.

3. The Cl�, NaC, and KC content is lower in sturgeons thanin cyprinids and salmonids.

4. The NaC/KC ratio is lower in cyprinids than in acipenser-ids and salmonids.

Depending on their concentrations, most of these ions areinvolved in regulating sperm motility either by contributingto the intracellular ionic composition or by their osmolality(Billard and Cosson, 1992; Scott and Baynes, 1980). Thecorrelation between the seminal plasma composition andsperm motility has been investigated in only a few species.Lahnsteiner et al. (1996) reported a correlation between spermmotility and seminal fluid composition in Alburnus alburnus,a cyprinid, and suggested that this correlation might indicatewhich components of the seminal plasma influence sperm mo-tility. These authors concluded that the NaC and KC levelshave statistically significant positive and negative relation-ships, respectively, with the percentages of motilespermatozoa.

Hwang and Idler (1969) postulated a correlation betweenthe seminal plasma NaC/KC ratio and sperm fertility in theAtlantic salmon, Salmo salar. The clear differences in NaC/KC ratios between sturgeons, cyprinids and salmonids couldexplain why sperm remain motile for longer in sturgeonsthan in the other two groups. There is also a clear differencein the intracellular Ca2C/KC ratio between S. salar and Cteno-pharyngodon idella, perhaps explaining why sperm remainmotile for longer in carps than salmonids. However, the reasonwhy sturgeon spermatozoa remain motile for longer than that

Table 1

Ionic c

Family (mmol l�1) Author(s) and remarks

Cyprin Kruger et al., 1984; 1Winter, 2Early spring

and 3Late spring; Ca2C in mg 100 ml�1

Clemens and Grant, 1965

0.04 Morisawa et al., 1983

Plouidy and Billard, 1982

Emri et al., 1998; 1Warm-adapted,2Cold-adapted

Krasznai et al., 2003b1Quiescent sperm and 2300 s after activation

Gosh, 1985

Lahnsteiner et al., 1996

0.1 Linhart et al., 2003a,b,c

1.2 Billard et al., 1995a,b

Salmon 0.12 Glogowski et al., 2000

Lahnsteiner et al., 1996

Holtz et al., 1977

Schlenk and Kahmann, 1938

0.10 Morisawa et al., 1979

Hwang and Idler, 1969

Cruea, 1969

3.6 Billard et al., 1995a,b

Acipen Gallis et al., 1991

0.021 Toth et al., 1997; 11993 and 21994

0.072

g l�1 Mims, 1991; Linhart et al., 2003a,b,c

0.03 Alavi et al., 2004a,b, in press

0.03

0.04

Marine Hayakawa and Munehara, 1998

5.2 Dreanno et al., 1998

0.28 Ciereszko et al., 2002

3S.M

.H.Alavi,

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ontents of the seminal plasma and sperm of Cyprinidae, Salmonidae, and Acipenseridae, according to the literature

Species KC (mmol l�1) NaC (mmol l�1) Ca2C (mmol l�1) Cl� (mmol l�1) Mg2C

idae Cyprinus carpio 78.87G 3.721 71.25G 3.651 10.69G 2.711 110.62G 8.811

73.01G 3.462 59.00G 3.162 11.54G 0.732 96.25G 3.582

77.56G 4.573 58.12G 5.273 8.47G 2.933 102.12G 7.253

67.8 94 12.5 0.02

82.4G 3.3 75G 3.2 2G 0.18 0.8G

43.5 51.3 0.7 0.27

60G 71

58G 82

87G 161 63G 101

64G 112 83G 122

63G 61 78G 51 43.5G 4.51

20G 52 18G 72 73G 92

Ctenopharyngodon idella 35.1 81.1 1.0 1.6

2.1 35.7 2.6 1.3

Alburnus alburnus 40.6G 5.2 66.6G 12.2

Tinca tinca 1.93G 0.6 18.40G 1.3 0.60G 0.2 0.45G

Cyprinids 39e78 94e107 0.3e12.5 0.02e

idae Oncorhynchus mykiss 30.4G 4.5 122G 14.2 1.10G 0.26 0.85G25.7G 4.1 159.8G 30.8 1.2G 0.3

25.3 104 1.4 135 1.1

25.8 107 2.6 0.8

20 133 130

Oncorhynchus keta 66.1G 4.90 142G 1.7 2.2G 0.10 134G 1.33 1.6G

Salmo salar 22 103 1.3 0.9

76.2 36.5 0.03 0.8

Salmo clarki 38.6 107 0.3 156 1.5

Salmonids 20e66 103e140 0.3e2.6 0.8e

seridae Acipenser baeri 2.5G 0.3 28G 0.7

Acipenser fulvecsens 5.78G 0.491 25.6G 2.81 0.16G 0.051 5.41G 2.791 0.21G6.97G 1.422 31.8G 7.02 013G 0.022 2.31G 1.282 0.22G

Polyodon spathula 97.3 mg l�1 500 mg l�1 7.8 mg l�1 55 m

Acipenser persicus 6.92G 0.881 62.44G 6.82 0.79G 0.03 21.11G 5.41 0.52G

5.77G 0.51 50.25G 4.83 0.80G 0.04 9.04G 1.82 0.48G7.47G 0.92 52.50G 5.86 0.77G 0.05 12.33G 3.55 0.51G

fishes Hemilepidotus gilberti 20.4G 2.60 162.0G 12.1 0.97G 0.16 2.52G 0.21

Psetta maxima 4.50G 0.31 150G 7 4.76G 0.40 30.8GPetromyzon marinus 10.5G 2.6 106.3G 12.8 0.57G 0.08 1.24G


of salmonid and carp is not entirely clear at present. An addi-tional explanation could come from structural features of sper-matozoa, such as the fact that the volume and number ofmitochondria is larger in sturgeons and paddlefish than in cyp-rinids and salmonids.

The ionic concentrations of C. carpio and of O. mykiss sem-inal plasma depend on the spawning period (Tables 1 and 2).This is related to frequent contamination by urine during strip-ping (Perchec-Poupard et al., 1998), phagocytosis of sperm inthe testis during the degeneration stage of spermatogenesis,thinning of the semen (Morisawa et al., 1979), and differentenvironmental conditions in the spawning seasons (Redondo-Muller et al., 1991).

3. Effects of ions on sperm motility in Salmonidae,Cyprinidae and Acipenseridae

General information is available in reviews by Billard(1986), Billard and Cosson (1992), Suquet et al. (1994),Billard et al. (1995a,b), Cosson et al. (1999), Darszon et al.(1996) and Fraser (1995). There has been extensive researchon the effects of ions on the initiation and duration of spermmotility, aimed at prolonging the duration of sperm motilityand improving reproduction in Salmonidae (Morisawa andSuzuki, 1980; Billard, 1983; Billard and Cosson, 1988, 1992;Billard et al., 1987; Cosson et al., 1989; Gatti et al., 1990;Boitano and Omoto, 1991, 1992; Kho et al., 2001; Lahnsteineret al., 1998), Cyprinidae (Morisawa et al., 1983; Krasznaiet al., 1995, 2000, 2003a,b; Cosson et al., 1991a,b; Christet al., 1996; Lahnsteiner et al., 1996; Perchec-Poupard et al.,1997; Emri et al., 1998; Linhart et al., 2003a,b,c), Acipenser-idae (Gallis et al., 1991; Toth et al., 1997; Linhart et al., 1995,2002; Cosson and Linhart, 1996; Mims, 1991; Alavi et al.,2004a,b, in press; Alavi and Cosson, 2005a,b) and marinefish species (Hines and Yashov, 1971; Oda and Morisawa,1993; Billard et al., 1993; Lahnsteiner et al., 1997; Gwo,2000; Ciereszko et al., 2002; Litvak and Trippel, 1998; Inabaet al., 2003; Elofsson et al., 2003; Krasznai et al., 2003a,b; Leeet al., 1992).

4. Effect of potassium (KC)

4.1. In salmonids

Scheuring (1925) was the first to observe that rainbow troutspermatozoa were immobilized after dilution with solutionscontaining high potassium concentrations. Schlenk and

Table 2

The ionic content and osmotic pressure in the seminal plasma of rainbow trout,

O. mykiss, in relation to spermiation time (Munikittrick and Moccia, 1987)

Time after start of

spermiation (months)

KC

(mM l�1)

NaC

(mM l�1)

Cl�

(mM l�1)

Osmolality

(mmol/kg)

1e3 12.4e18.8 54e91.9 53.7e86.3 138.1e210.3

2e4 9.4e14.2 42.8e63 39.6e65 159.1e79.4

3e5 7.4e9.8 37.4e44.3 35e48.1 106.4e120.9

Kahmann (1938) directly associated the immotility of troutspermatozoa with the high concentration of KC ions in theseminal fluid. Kusa (1950) found that a high potassium con-centration inhibits both motility and fertilizability in chumsalmon sperm, Oncorhynchus keta. Benau and Terner (1980)showed that dilution of KC or its removal from the seminalplasma by dialysis induces motility in salmonid spermatozoa.KC at 20e40 mM (Morisawa, 1985; Billard et al., 1987;Cosson et al., 1989) completely suppresses sperm motility inthe rainbow trout (Morisawa et al., 1983; Billard et al.,1987). Dilution media containing KC concentrations that areelevated in relation to the ionic composition and osmotic pres-sure of the seminal plasma eliminate the initiation of spermmotility in salmonids (Billard et al., 1995a; Kho et al.,2001). However, dilution in isotonic NaCl triggers immediateactivation of motility (Billard et al., 1987; Gatti et al., 1990).

Comparing the results obtained on induction of spermmotility (see above) and ionic composition (Table 1) suggeststhe hypothesis: ‘‘the inhibition of motility in salmonids ismainly due to KC ions’’. In other words, membrane hyperpo-larization caused directly by transmembrane KC efflux is thefirst trigger for initiating sperm motility in salmonid fishes(Billard et al., 1987; Kho et al., 2001). The discrepancies inthe literature could be explained by other variables such aspH and osmotic pressure (Petit et al., 1973; Baynes et al.,1981). However, Stoss et al. (1977) found that pH and osmoticpressure had no marked effects on trout sperm motility.Because motility is not inhibited in physiological solutions,such as ovarian fluid, containing more than 1 mM KC (Hwangand Idler, 1969; Stoss et al., 1977), interaction with other com-ponents must occur.

Scheuring (1925) reported that ions such as NaC, Ca2C andMg2C counter the inhibitory action of KC, and bivalent cati-ons are more effective than NaC. Baynes et al. (1981) drewthe same conclusion concerning KC control of trout spermmovement and pointed to an antagonism between potassiumand calcium ions. Several results confirm that divalent cationsusually antagonize KC inhibition and are more effective thanmonovalent cations. Billard et al. (1987) studied the interac-tion between KC and Ca2C concentrations in media and per-centage sperm motility in rainbow trout. Their results showedthat in 2 mM, 20 mM and 40 mM KC, sperm motility is in-hibited when combined with Ca2C concentrations lower than10�6, 10�3 and 10�2 mM, respectively (Fig. 1). More recentstudies have shown that the extent of inhibition of sperm mo-tility by KC changes during the spawning season (Table 2): atthe beginning and end of the spermiation period, a high per-centage of sperm become motile even in the presence ofhigh KC concentrations (40 and 80 mM; see Table 3) (Billardand Cosson, 1992). The differences seem to be due to altera-tions in semen KC concentration, which are related to changesin sperm sensitivity to potassium. Total KC and Ca2C concen-trations in the seminal fluid show no apparent correlation withvariations in the levels of these ions or the insensitivity of troutspermatozoa to KC (Billard and Cosson, 1992).

In conclusion, (1) high potassium concentration is a majorinhibitor of sperm motility in salmonids, but the extent of

5S.M.H. Alavi, J. Cosson / Cell Biology International 30 (2006) 1e14

inhibition depends on the sensitivity of the spermatozoa toKC, which varies through the reproductive season. (2)A decrease in environmental KC concentration causes KC

efflux through specific membrane channels, leading to mem-brane hyperpolarization and the initiation of sperm motilityin salmonids.

4.2. In cyprinids

Since the concentration of KC in the seminal plasma(82.4 mM;Morisawa et al., 1983) is higher than that in the sper-matozoa (60.5 mM; Balkay et al., 1997), the sperm plasmamembrane is markedly depolarized in semen. Nevertheless,the KC concentration difference across the plasma membraneis not enough to account for the amplitude of the membrane po-tential (Krasznai et al., 2000). Even if the high seminal plasmaKC concentration is a major inhibitor of sperm motility in theduct, the change of external osmolality seems likely to be thefirst trigger of sperm motility initiation, preceding KC efflux(Fig. 2; see Section 8). Krasznai et al. (1995, 2000) found thatpotassium channels affect sperm motility and regulate hypo-osmotic shock induced in the common carp. KC ions also in-crease sperm velocity and motility in carp (Morisawa et al.,1983; Billard and Cosson, 1992). These results essentiallyshow that carp spermatozoa are less KC sensitive than thoseof trout.

Fig. 1. The inhibitory action of Ca2C on rainbow trout spermatozoa in the

presence of various KC concentrations, according to Billard et al. (1987). A

competitive effect between the two ion species is clearly apparent.

Table 3

Motility of trout spermatozoa in solutions containing various concentrations of

KC and Ca2C (Billard and Cosson, 1992)

KC (mM) Ca2C (mM)

0.1 1 2 3 10 20 40

2 C C C C C C C

20 � � � C/� C C C40 � � � � C/� C C

60 � � � � � � C/�C, Nearly all spermatozoa motile;C/�, about half of the spermatozoa motile;

�, no motility. In all cases the final solution was adjusted to 125 mM NaCl,

20 mM TriseHCl, pH 9.0.

In the presence of KC channel inhibitors, flagellar motionis attenuated or completely abolished, while NaC channel oranion channel inhibitors do not affect carp sperm motility.When using semen of poor quality, Cosson et al. (1991a,b) ob-served that the potential for motility in common carp sperma-tozoa recovered after incubation in KC-rich media, in whichthe spermatozoa are immotile. This improved potential wasobserved in solutions containing 50e200 mM KCl (orNaCl), conditions that led to a complete recovery of spermmotility (Redondo-Muller et al., 1991).

The potent effects of KC ions were also investigated in de-membranated flagella; axonemal motility was found to be di-rectly controlled by the ion concentration (Linhart et al.,2003a,b,c). It is also clear that the KC concentrations in di-luents used for cryopreservation strongly influence the poten-tial motility of carp spermatozoa (Linhart and Cosson, 1997).

4.3. In sturgeons and paddlefish

There is less information about the effect of KCon themotil-ity of sturgeon (acipenseridae) and paddlefish (polyodontidae)

Fig. 2. Suggested cell-signaling pathways for the mechanism of initiation of

carp sperm motility, according to Krasznai et al. (2000). Change in osmolality

of the external milieu would lead to membrane polarization changes mainly

because of variation in the internal KC concentration combined with variation

in Ca2C concentration; cAMP/phosphorylation signaling would not be

necessary.


spermatozoa. The potential mobility of spermatozoa from theSiberian sturgeon, Acipenser baeri (Gallis et al., 1991), thepaddlefish, Polyodon spathula (Cosson and Linhart, 1996),and the Lake sturgeon, Acipenser fulvescens (Toth et al.,1997), are attenuated by increasing the KC concentrationin the media. Gallis et al. (1991) reported that KC drasticallyinhibited motility at 0.1 mM, while a concentration of0.05 mM had no effect. Paddlefish spermatozoa are sensitiveto very low KC concentrations (Cosson and Linhart, 1996).These authors observed that the percentage of motile sperma-tozoa decreased rapidly by 10% after 10 s, and to 0% after1 min, in a solution containing 1 mM KCl. Toth et al.(1997) studied the effect on A. fulvescens spermatozoa ofadding KC to a swimming medium composed of 50 mMTris/glycine buffer, pH 9.0. They showed that activationwas prevented with an apparent EC50 of 0.5 mM potassium.

To date, the mechanism regulating mobility in sturgeon andpaddlefish spermatozoa has not been fully identified, but itpresents quite striking similarities with that in salmonid sper-matozoa. A recent study by Alavi et al. (2004a) showed thatthe potassium concentration in Acipenser persicus seminalplasma was 6.92G 0.88 mM. Extracellular KC concentrationsover 2 mM were also inhibitory (Alavi et al., 2004b). Thesefindings indicate that seminal plasma KC is a major inhibitorof sperm motility in A. persicus.

As in carp spermatozoa, KC has potent effects on demem-branated sturgeon flagella: axonemal motility is controlled bythe KC ionic concentration (Linhart et al., 2003a,b,c) and thesame applies to the paddlefish (Cosson and Linhart, 1996):these results may help to better understand the KC regulatorymechanism.

5. Effects of calcium (Ca2C)

5.1. In salmonids

As mentioned above, the inhibition of sperm motility bymillimolar concentrations of KC can be overcome by increasedexternal Ca2C concentration. Preliminary results indicate thatthe intracellular Ca2C concentration increases when motilityis initiated (Billard et al., 1989; Cosson et al., 1989; Boitanoand Omoto, 1991). Christen et al. (1987), Billard et al.(1995a) and Cosson et al. (1989) reported that external Ca2C

ions are necessary for initiating motility. These observations(Cosson et al., 1991a,b) led to the following conclusions:

1. A chelator of calcium ions (EGTA) decreases the freeCa2C concentration in a sperm suspension to 10�9 M,completely preventing the activation of motility; but motil-ity can be fully restored by adding millimolar concentra-tions of Ca2C.

2. Prevention of Ca2C entry into the cell by a voltage-depen-dent calcium channel blocker (desmethoxyverapamil)completely inhibits motility.

3. The increase of intracellular free Ca2C is produced bya flux of external Ca2C into the cell rather than by a mobi-lization of internal Ca2C stores.

4. The increase in free intracellular Ca2C varies from 30 nMin quiescent cells (before swimming) to 180 nM in cellsafter the cessation of movement (after swimming).

Gonadotropin and steroid hormones are involved in thematuration and motility of sperm in various fish species(Billard et al., 1995a; Nagahama, 1994; Yaron, 1995). In mam-malian sperm, progesterone stimulates Ca2C influx via non-genomic receptors located on the sperm head (Blackmoreand Lattanzio, 1991; Blackmore et al., 1990, 1991), possiblyby changes in Ca2C membrane permeability (Shivaji andJagannadhan, 1992) and by induction of acrosome reaction(Blackmore et al., 1990, 1991) and hyperactivation (Suarezand Pollard, 1990).

In trout, the ATP concentration decreases rapidly during thefirst 30 s after initiation of sperm motility in an activation me-dium (AM) containing 125 mM NaCl, 20e30 mM TriseHCl,pH 9.0, then slowly increases to reach its original levels15 min after dilution in the absence of Ca2C. When the spermwere diluted in AM containing 10 mM Ca2C, the fall in ATPconcentration was greater and lasted 1e2 min after the initia-tion of sperm motility, and the recovery of ATP was totallysuppressed (Christen et al., 1987).

Moreover, the ability of the Ca2C channel blocker, desme-thoxyverapamil, to prevent both the initiation of motility andthe rise of internal free Ca2C suggests that the latter is gener-ated by an influx of external Ca2C and not by mobilization ofinternal stores. Also, experiments show that 80% of the in-crease of internal Ca2C takes place within 30 s. Ca2C influxmight be implicated in the initiation of flagellar beating, con-stituting an intermediate step between the decrease of externalKC and the increase of sperm adenylate cyclase activity(Morisawa, 1985).

Recent research by Tanimoto et al. (1994) and Kho et al.(2001) showed that L-type Ca2C and T-type channel blockersinhibited the initiation of sperm motility in the rainbow trout.These findings suggest that the influx of external Ca2C consti-tutes an additional transmembrane process distinct from KC

efflux, and that the Ca2C entering the sperm cytoplasm partic-ipates in the activation of certain enzymes or other proteins.New data reported by Kho et al. (2001) showed that calmod-ulin, or regulatory compounds interfering with adenylyl cy-clase (Bookbinder et al., 1990) or with cAMP productionand degradation (Tash and Means, 1983), may be prerequisitesfor the initiation of sperm motility in salmonids.

High concentrations of a calmodulin inhibitor suppressedsperm motility, membrane hyperpolarization and cAMP syn-thesis in live rainbow trout sperm. Several studies indicatea key role for cAMP in the phosphorylation of specific pro-tein subunits involved in the triggering of axonemal motilityin trout sperm (Itoh et al., 2003). Nevertheless, cAMP in-creases slowly, especially at low temperatures, and peaksmuch later, after 100% of the spermatozoa have begun tomove (Cosson et al., 1995). In addition, in some under- orover-mature sperm samples, the requirement of cAMP for ac-tivation could be bypassed. Therefore, cAMP is certainly in-volved in the regulation of trout sperm axonemal movement,


but it is dispensable in some conditions, possibly related tomodulation by Ca2C in the environment (Cosson et al.,1995).

5.2. In cyprinids

Extracellular Ca2C is a prerequisite for the initiation of livesperm motility in cyprinids (Krasznai et al., 2000). Spermato-zoa motility was observed after 30 s when 10�4 M NaCl wasadded to the swimming solution (Krasznai et al., 2000).Also, when spermatozoa were demembranated with TritonX-100, they exhibited high motility in 10�6 and 10�5 MCa2C. Verapamil used as a Ca2C channel blocker inhibitedthe motility of mature carp spermatozoa prediluted and incu-bated in physiological solution (140 mM NaCl, 10 mM KCl,1 mM CaCl2, and 20 mM HEPES, pH 8.5), and completelyeliminated the increase in intracellular Ca2C (Krasznaiet al., 2000). The intracellular Ca2C concentration in carpspermatozoa increased from 48G 3 nM to 78G 3 nM afteractivation of motility in a hypotonic solution containing0.1e1 mM Ca2C.

The hypo-osmotic shock results in hyperpolarization of thecell membrane (Krasznai et al., 1995), which re-activates thecalcium channels (Krasznai et al., 2000). The intracellular cal-cium concentration did not change in the absence of externalCa2C, but subsequent addition of Ca2C caused an increaseand triggered sperm motility. Krasznai et al. (2000) also sug-gested that influx of extracellular Ca2C through specific chan-nels leads to induction of Ca2C release from stores (Krasznaiet al., 2000) and initiates sperm motility through the calmod-ulin system (Krasznai et al., 2000, 2003a,b). Several specificCa2C channel blockers (verapamil, flunarizine and the cono-toxin family) prevent the increase of intracellular Ca2C inthe common carp, and the initiation of sperm motility is sub-sequently suppressed (Krasznai et al., 2003b).

On the other hand, Ca2C increase itself does not cause theinitiation of sperm motility, suggesting that the entry ofextracellular Ca2C is a critical event (Krasznai et al.,2003b). Krasznai et al. (2003a,b) exploited the observationsof Perchec-Poupard et al. (1997) on the effect of gadolinium,a stretch-activated ion channel blocker, on sperm motility inthe common carp. Gadolinium inhibits carp sperm activationand the effect is dose- and incubation time-dependent(Krasznai et al., 2003a,b). It significantly decreases the spermmembrane fluidity and also abolishes the fluidizing effect ofhypotonic treatment. It seems possible that the membranestructural changes caused by gadolinium alter the water per-meability of the membrane and therefore its osmoticproperties.

Studies on the ‘‘revival’’ mechanism showed that carp sper-matozoa do not require external calcium ions for a second mo-tility phase, in contrast to trout spermatozoa (Billard et al.,1995b; Perchec et al., 1995a,b, Perchec-Poupard et al., 1997).Such a second motility phase, obtained by transient incubationof spermatozoa in a non-swimming medium that allows respi-ration, leads to recovery not only of the capacity for movementbut also of fertilizing ability (Cosson et al., unpublished).

5.3. In sturgeons

The effect of Ca2C on motility has not been so intensivelystudied in sturgeons. Ca2C at 100 mM reversed the KC inhib-itory effect, as in salmonid sperm, and EGTA abolished theCa2C effect (Cosson et al., 1999). Recently, different concen-trations of CaSO4 in 20 mM TriseHCl, pH 8.0, at 1:50 dilu-tion were tested on A. persicus (Alavi et al., 2004b).The percentage of motile spermatozoa and the duration of mo-tility of sperm were maximal in 3 mM CaSO4. A rapid de-crease in the percentage of motile spermatozoa wasobserved in solutions containing 5 mM CaSO4 or more. Theseobservations corroborate the sensitivity of sturgeon spermato-zoa to Ca2C ions (Alavi et al., 2004a,b, in press); this was con-firmed by the use of demembranated spermatozoa (Cossonet al., 1999; Linhart et al., 2003a,b,c).

6. Effects of sodium (NaC)

6.1. In salmonids

When NaCl is added to the seminal plasma, motility is in-duced. Schlenk and Kahmann (1938) observed motility in me-dia combining NaC and KC, provided the NaC/KC ratio was16/1 or higher. This was confirmed by Scheuring (1925), Stosset al. (1977), Baynes et al. (1981) and Christen et al. (1987),who found, in contrast, that sperm motility was activated by ad-dition of 125 mM NaCl to solutions containing 125 mM KCl,and that in such conditions the ATP concentration decreasedvery rapidly. These authors also observed that the concentra-tion of ATP returned to its original level 15 min after dilution,and that sperm diluted in swimming solution with 10 mM cal-cium became motile again for 1e2 min, then stopped (the socalled ‘‘second motility phase’’ or ‘‘revival’’, see above).

Marshall et al. (1993) studied hormonal mechanisms ofsperm motility in brook trout. They reported that gonadotropindirectly induced KC secretion into the seminal fluid and there-absorption of NaC. As a consequence of this active iontransport, the seminal fluid has a low NaC concentration anda high KC content, which keeps the sperm quiescent until itis released into the external water.

6.2. In cyprinids

The activation of sperm motility is followed by the alkali-zation of the intracellular medium in the carp (Billard, 1988;Krasznai et al., 1995; Perchec et al., 1995a,b; Alavi andCosson, 2005a). Fast intracellular alkalization is most proba-bly caused by activation of the NaC/HC exchanger (Tronet al., 1990; Marian et al., 1993). Recent studies show that so-dium channel inhibitors do not affect the motility of carp sper-matozoa (Krasznai et al., 1995). However, similar activationwas observed when carp spermatozoa were incubated in vari-ous NaCl concentrations. In a semen sample in which nearlyall the spermatozoa were immotile, 90% became capable ofmovement after 100 min incubation at 0 �C in 125 mM


NaCl (Redondo-Muller et al., 1991). However, higher NaClconcentrations appeared less favorable and prolonged incuba-tions did not increase the percentage of motile spermatozoa. Itis unclear whether a high NaCl and KCl solution improves themotility of poor quality carp semen. It is interesting to notethat, after such reactivation, sperm motility is not pH depen-dent (Cosson et al., 1991a,b; Redondo-Muller et al., 1991).

6.3. In sturgeons

The effect of NaC on sturgeon sperm motility has been stud-ied by Gallis et al. (1991) in A. baeri and by Toth et al. (1997) inA. fulvescens. In A. baeri, sodium ions in a concentration rangesimilar to that of seminal plasma (20 mM) had no effect onsperm motility (Gallis et al., 1991). Fig. 3 shows the effect ofNaC added to the activation solution (containing Tris/glycinebuffer) on the duration of Lake Sturgeon sperm motility andthe percentage of motile sperm. The latter parameter remainedunchanged after 5 min in an activation solution containing10 mM NaC (Table 4); the maximum duration of motilitywas observed in this solution (Fig. 3) (Toth et al., 1997). Thepercentage of motile cells and the period of sperm motilitywere maximized in a 25 mM NaC activation solution in A. per-sicus (Alavi et al., 2004b). No motility was initiated in an acti-vation solution containing more than 25 mM NaC (Alavi et al.,2004a,b, in press). These findings are in agreement with the re-sults of Gallis et al. (1991) and Toth et al. (1997), who claimedthat sturgeon spermatozoa are sensitive to high extracellularNaC concentrations. In this respect, sturgeon spermatozoa con-trast with cyprinid and salmonid spermatozoa, which have lowor zero sensitivity to extracellular NaC.

7. Effects of ions on sperm motility in marine fishes

In contrast to Cyprinidae and Salmonidae, the literature onthe effect of ions on sperm motility in marine fishes is fairlylimited (Linhart et al., 1999; Gwo, 2000; Vines et al., 2002;Cosson, 2004). Nevertheless, studies on turbot spermatozoa

Fig. 3. Effect of NaC concentration of the swimming solution on the duration

of motility in sturgeon (Acipenser fulvescens) spermatozoa, according to Toth

et al. (1997). The optimal duration is observed around 10 mM NaC.

have yielded information (Chauvaud et al., 1995; Suquetet al., 1994; Inaba et al., 2003). Increased intracellular Ca2C

and KC have been proposed as triggers of sperm motility ini-tiation in marine fishes (Yanagimachi et al., 1992; Oda andMorisawa, 1993; Takai and Morisawa, 1995; Krasznai et al.,2003a,b; Vines et al., 2002). Linhart et al. (1999) showedthat NaCl solutions without CaCl2 did not activate sea-watertilapia spermatozoa, but the addition of 1.5e30 mM CaCl2(with 0e934 mOsmol kg�1) led to full activation.

These findings suggest that, at least in some species: (1)sperm motility is controlled by extracellular Ca2C ions com-bined with NaC solution (e.g. tilapia) as well as osmotic pres-sure; and (2) the presence of Ca2C allows motility to occur athigh osmotic pressure. Krasznai et al. (2003a,b) reported thatpuffer fish spermatozoa were immotile in a seminal plasma-like solution with or without Ca2C, but motility was activatedin either Ca2C-free or Ca2C-containing artificial sea-water. In-tracellular Ca2C depletion inhibited sperm motility. Thesefindings indicate that extracellular Ca2C is not required for ini-tiation of motility, in contrast to intracellular Ca2C. This is in-consistent with the results of Vines et al. (2002) in herring,showing that extracellular calcium is a prerequisite for the ini-tiation of sperm motility.

Recent findings by Krasznai et al. (2003a,b) show that in-creased intracellular Ca2C alone is not sufficient to initiatesperm motility, and that hyper-osmotic shock is required;this induces Ca2C release from internal stores. These authorsalso observed that (1) neither Ca2C nor KC channel blockersinfluence sperm motility and (2) gadolinium decreases spermmotility in the puffer fish. The inference is that interaction be-tween calmodulin and Ca2C from internal stores plays a keyrole in the hyper-osmolality triggered initiation of sperm mo-tility in the puffer fish, Takifugu niphobles. Vines et al. (2002)reported that a sperm motility initiation factor in the herring,an egg chorion ligand, acts by regulating ionic channel activ-ities, specifically the voltage-gated calcium channel and theNaC/Ca2C exchanger, causing membrane hyperpolarization.The effect of NaCl and KCl on sperm motility in the sea lam-prey was studied by Ciereszko et al. (2002). Their resultsshowed that (1) motility was inhibited by concentrations ofNaCl and KCl higher than 40 mM just after initiation of move-ment, and (2) 2 min after activation, NaCl and KCl concentra-tions over 20 mM were inhibitory.

Nevertheless, other ions such as Mg2C, KC and Mn2C,with or without NaCl, did not activate spermatozoa in tilapia,a sea-water fish (Linhart et al., 1999). There is a need for stud-ies on sperm cell signaling to elucidate the mechanism of ini-tiation of sperm motility involving osmolality.

8. Osmotic pressure of seminal plasma

Table 5 shows some selected data on the osmotic pressure(or osmolality) of the seminal plasma of different fish speciesfrom the families Cyprinidae, Salmonidae and Acipenseridaeand from marine fishes. The osmotic pressure is lower in Aci-penseridae than in Cyprinidae, Salmonidae and marine fishseminal plasma. It is worth remarking that the osmotic


Table 4

Effect of concentration of added NaC on motility of Acipenser fulvescens (A.f.), Scaphirhynchus platoryncus (S.p.), and Polyodon spathula (P.s.)

Species Time

after

activation

0 mM NaC 10 mM NaC 20 mM NaC 25 mM NaC Author(s) and

remarks% Motile

sperm

Total

duration

period

% Motile

sperm

Total

duration

period

% Motile

sperm

Total

duration

period

% Motile

sperm

Total

duration

period

A.f. 0 min 56.4G 22.11 51.8G 25.51 49.8G 24.61 Toth et al., 1997;11994 and 2199387.0G 1.92 58.1G 6.6 78.5G 9.32

2 min 26.4G 23.51 45.8G 25.51 34.9G 18.51

56.5G 11.32 65.7G 3.32 14.8G 10.92

5 min 12.2G 8.811 21.0G 27.71 42.4G 18.01

36.0G 3.02 55.5G 10.82 13.3G 9.82

S.p. 10 s 90G 01 166G 11.6a 100G 0 360G 14.1 93G 3.5 205G 7.0 Linhart et al., 1995;aDistilled water andbHatchery water

90G 2.9 222G 7.6

P.s. 10 s 80G 01 110G 100a 100G 0 370G 14.1 93G 3.5

97G 8.32 177G 15.3b

pressure is higher in marine than in freshwater fish seminalplasma. Like the ionic composition, the osmotic pressurecan vary between individuals, and this is correlated with the‘‘thinning’’ (hydration) of the semen (Table 5) (Morisawaet al., 1979). Also, the variations in osmotic pressure observedin the literature might be due to hormonal induction of sper-miation outside the natural reproductive season (Redondo-Muller et al., 1991). Recent studies by Piros et al. (2002)and Alavi et al. (2004a,b, in press) indicate that the strippingfrequency has no effect on the seminal plasma osmotic pres-sure in sturgeons 12 h after the first stripping.

9. Effect of osmotic pressure on sperm motility inSalmonidae, Cyprinidae and Acipenseridae

It is obvious that ions contribute to the final osmolality ofany solution. In this section, the effect of osmotic pressure istentatively distinguished from that of specific ions; much ex-perimental evidence shows that the activation (or inactivation)of fish sperm is controlled even in ion-free solutions contain-ing sucrose or similar solutes, which directly govern the finalosmolality.

9.1. In salmonids

High osmotic pressure (400 mOsmol kg�1) inhibits spermmotility in Salmonidae, but the osmotic pressure of the semi-nal plasma (approximately 300 mOsmol kg�1) is not highenough to account for the inhibition of motility in semen(Billard and Cosson, 1992). The mechanism of motility initi-ation in salmonid spermatozoa is not completely elucidated,especially the events occurring in the intracellular medium, in-cluding ATP/cAMP regulation (Cosson et al., 1995) and regu-lation by other energetic compounds such as creatinephosphate (Saudrais et al., 1998).

9.2. In cyprinids

According to Morisawa and Suzuki (1980), hypotonic me-dia initiate the motility of spermatozoa from freshwater fishes

such as the common carp and goldfish, Carassius auratus.However, there are examples where isotonic media activatesperm motility of freshwater spawners. Although hypotonicityis a major stimulant of motility of carp spermatozoa, it is notthe only factor involved in initiating sperm motility in allfreshwater fish species. Carp sperm motility is fully initiatedin media of osmotic pressure below 150e200 mOsmol kg�1

(Plouidy and Billard, 1982; Perchec-Poupard et al., 1997),but exposure of the sperm to extreme osmotic conditions leadsto changes in morphology and capacity for movement; thisis the case for distilled water (Perchec et al., 1996) or urine(Perchec et al., 1995a,b; Perchec-Poupard et al., 1998). Insome cases, the seminal plasma osmolality is higher thannecessary for sperm activation; for example, the osmolalityof carp seminal plasma is 286 mOsmol kg�1 (Plouidy andBillard, 1982). These findings suggest that the change in exter-nal osmolality is the first trigger, preceding KC efflux in theinitiation of sperm motility in cyprinids (Fig. 2). In contrastwith trout sperm, the triggering of carp sperm motility doesnot involve cAMP-dependent phosphorylation of axonemalproteins (Cosson and Gagnon, 1988).

Statistically, there is a significant relationship betweensperm motility and osmolality in A. alburnus (Lahnsteineret al., 1996); these authors found an association between lowosmolality and weak motility. The ATP stores in carp sperma-tozoa could be re-established by preventing the residual motil-ity phase 5e10 min post-activation in a high osmotic pressuremedium (300 mOsmol kg�1) (Billard et al., 1995b). The sper-matozoa could sustain a second round of motility if the exter-nal osmolality was decreased.

9.3. In sturgeons

The motility of A. baeri spermatozoa is inhibited by highosmotic pressure (100 mOsmol kg�1 or more) (Gallis et al.,1991), but the average values of seminal plasma osmotic pres-sure are low (38G 3 mOsmol kg�1). Therefore, osmolalityseems not to be the main inhibitor of sperm motility in seminalfluid; rather, motility is controlled by the KC concentration.Nevertheless, Linhart et al. (1995) reported that the motility


Table 5

Osmolality of seminal plasma in different fish species, according to published results

Family Species Osmolality (mOsmol kg�1) Authors and remarks

Cyprinidae Carassius auratus 290G 3 Grant et al., 1969

317G 11 Morisawa, 1985

Tinca tinca 230G 82 Linhart et al., 2003a,b,c

Cyprinus carpio 286G 3 Plouidy and Billard, 1982

302G 5 Morisawa et al., 1983

346.00G 18.261 Kruger et al., 1984; 1Winter, 2Early spring

and 3Late spring290.25G 5.972

297.75G 14.203

258G 20 Redondo-Muller et al., 1991

254G 16.7 Toth et al., 1995

Alburnus alburnus 266.6G 20.1 Lahnsteiner et al., 1996

Salmonidae Salmo salar 245G 11 Aas et al., 1991

232G 13 Hwang and Idler, 1969

Oncorhynchus mykiss 322.1G 21.76 Glogowski et al., 2000

313.3G 22.3 Lahnsteiner et al., 1996

Oncorhynchus keta 332G 5.1 Morisawa et al., 1979

Acipenseridae Acipenser ruthenus 69.7G 1.81 Piros et al., 2002; 1Danube River and2Raised in cages submerged in warm water

(17 �C) supplied by Dona Odra power plant

79.1G 4.82

Acipenser baeri 38G 3 Gallis et al., 1991

93.6G 7.31 Piros et al., 2002; 1First and 2Second stripping

with intervals 12 h95.7G 5.42

Acipenser persicus 82.56G 8.101 Alavi et al., 2004a,b, in press; 1First, 2Second and3Third stripping with intervals 12 h55.13G 5.542

56.50G 9.383

Marine fishes Dicentrachus labrax 400 Villani and Catena, 1991

Sparus aurata 365G 1 Chambeyron and Zohar, 1990

Petromyzon marinus 249.0G 20.0 Ciereszko et al., 2002

Oreochromis mossambicus 351.00 Linhart et al., 1999

Psetta maxima 306G 3 Suquet et al., 1993

317G 2 Dreanno et al., 1998

Hemilepidotus gilberti 328.0G 13.2 Hayakawa and Munehara, 1998

Gadus morhua 400 Hwang and Idler, 1969

414G 30 Litvak and Trippel, 1998

of shovelnose sturgeon and paddlefish spermatozoa were in-hibited by increased extracellular osmotic pressure, as ob-served in cyprinids. Their results showed that shovelnosesturgeon and paddlefish sperm were motile only in solutionsin the osmotic pressure ranges 0e100 or 0e120 mOsmol kg�1,respectively. Similarly, our recent studies show that the sper-matozoa of the Persian sturgeon, A. persicus, are motile inthe osmotic pressure range 0e100 mOsmol kg�1 (Alaviet al., 2004b). The duration of sperm motility and the percent-age of motile sperm are maximal in a sucrose solution with anosmotic pressure of 50 mOsmol kg�1.

10. Effect of osmotic pressure on sperm motility inmarine versus freshwater fish

It is clear that sperm motility is induced by hypo- and hy-per-osmotic pressure in freshwater and marine fishes, respec-tively (Billard, 1986; Billard et al., 1993; Suquet et al.,1994; Gwo, 1995; Linhart et al., 1999; Krasznai et al., 2000,2003a,b; Cosson, 2004). Nevertheless, sperm motility in theturbot is triggered in solutions isotonic with seminal fluid aswell as in hyper-osmotic media (Suquet et al., 1994). In the

case of turbot, motility in the seminal fluid is prevented byCO2, a mechanism specific to flat fish spermatozoa, whichwere shown to have a high carbonic anhydrase content (Inabaet al., 2003). Motility occurs over a wider range of osmolal-ities (below or above that of sea-water) in marine fishes thanin freshwater fishes (Chauvaud et al., 1995; Suquet et al.,1994; Billard et al., 1995a). The optimal osmolality (inmOsmol kg�1) for sperm motility is 900e1100 in halibut(Billard et al., 1995a), 300e1100 in turbot (Chauvaud et al.,1995), 333e645 in tilapia (for fish raised in sea-water; Linhartet al., 1999), and 480 in Atlantic croaker (Vizziano et al.,1995). The control of motility by osmolality is efficient inthe absence of extracellular ions as was shown by the use ofnon-ionic carbohydrate solutions (Chauvaud et al., 1995). Ageneral model of the control of fish sperm motility by osmo-lality is proposed in Fig. 4, with sea-water fish sperm as theexample, based on several published results (Chauvaudet al., 1995; Suquet et al., 1994; Inaba et al., 2003).

It is worth mentioning parenthetically that mechanical acti-vation could be the immediate response to the osmotic signal,acting via the stretch-activated channels of the sperm mem-brane. First, it was established that gadolinium, a specific


and reversible inhibitor of stretch-activated channels (SAC), isactive in carp spermatozoa (Krasznai et al., 2003b), and in otherfish spermatozoa including those of marine fish (Cosson,unpublished), but not in species other than fish (Krasznaiet al., 2003a). The SAC are mechanosensitive channels that in-crease conductivity to ions such as Ca2C or KC when mechan-ical constraints distort the membrane (Yang and Sachs, 1993).They can associate with other membrane proteins and modifytheir activity (Vandorpe et al., 1994); this could be the case forwater channels such as aquaporins, which are involved in

Fig. 4. Proposed sequence of events showing how sperm motility is controlled

by external osmolality in marine fishes (example of turbot spermatozoa sug-

gested in Cosson, 2004). The osmolality (sea-water) rises drastically at spawn-

ing when the spermatozoa move out of the seminal fluid. Osmoregulation

immediately triggers water efflux, which gives rise to local membrane distor-

tions (eventually blebs). The latter are signals for stretch-activated channels

(SAC), and such signals self-amplify by propagation from place to place along

the membrane, allowing very rapid water efflux, possibly mediated and accel-

erated by aquaporin. The result is a rapid increase of internal ionic concentra-

tion, reaching optimal values for dynein motor activity (flagella fully motile

with high velocity). After 10e60 s, because of continuing water efflux, the in-

ternal ionic concentration is further increased, gradually becoming incompat-

ible with dynein activity (slow movement of flagella). Much later, the internal

ion concentration becomes so high that it completely blocks the dynein motors

(immotile spermatozoa after several minutes). The whole process is experi-

mentally reversible.

transmembrane water transport. The putative presence ofaquaporins in fish spermatozoa is supported by the fact thatfish sperm motility is sensitive to specific inhibitors of aqua-porins such as HgCl2 at very low concentrations (Cosson,unpublished).

Taking account of these findings and of current knowledgeabout osmotic signals, the SAC have been included in themodel of the signaling pathway for fish sperm activation pro-posed in Fig. 4. According to this working hypothesis, the veryfirst signal perceived by the membrane is osmotic; water fluxin either direction would provoke local membrane distortion.Here it is worth recalling that the flagellar membrane of fishsperm presents unusual fin-shaped creases (Cosson et al.,1999), which significantly increase the membrane surfacearea (apparent membrane ‘‘excess’’ favoring water exchange)but can be easily distorted, appearing as blebs on exposure toextreme osmotic situations (Cosson et al., 2000; Perchec et al.,1996). The SAC would respond immediately to this mechani-cal signal by increasing the local permeability, therefore al-lowing ions such as Ca2C or KC to move in or out throughchannels and/or water to move rapidly through porins. Thiswould trigger an autocatalytic effect along flagellar mem-branes from place to place, which would explain why fishsperm activation is so rapid (less than 1/50th of a second ac-cording to our estimates; Cosson, unpublished).

Acknowledgements

We are grateful to Prof. R. Billard for his helpful com-ments. The authors wish to thank Mrs. F. Ramzani andMrs. M. Madadi for their assistance. The support of TehranUniversity to SMHA and of CNRS to JC is greatlyappreciated.

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