Effects of Potassium and Osmolality on Spermatozoan Motility of Salmonid Fishes

We have previously shown that in freshwater and marine fishes, sperm motility is suppressed by the osmolality of the seminal plasma (300 mosmol kg⁻¹) in the sperm duct, and that motility is induced by the decrease or increase in osmolality of the environment when they are released into water at spawning (Morisawa & Suzuki, 1980; Morisawa et al. 1983). In rainbow trout, a high concentration of potassium is contained in the seminal plasma and sperm motility is inhibited by potassium chloride (Schlenk & Kahmann, 1938; Benau & Terner, 1980; van der Horst, Dott & Foster, 1980). It has recently been shown that there is a high level of potassium in the seminal plasma of another salmonid fish, chum salmon (Morisawa, Hirano & Suzuki, 1979) and that potassium regulates sperm motility in this species also (Morisawa & Suzuki, 1980).

In the present study, we have examined the effects of osmolality and of potassium and other alkaline metals on sperm motility in three salmonid species.

Rainbow trout (Salmo gairdneri), the land-locked masu salmon (Oncorhynchus masou), the char (Salvelinus leucomaenis) and the ayu (Plecoglossus altivelis) were obtained from a commercial source. Chum salmon (Oncorhynchus keta) was captured at Otsuchi river, Iwate prefecture. The semen of rainbow trout, masu salmon and ayu was taken directly from the sperm duct by opening the abdomen. Seminal plasma was then obtained by centrifuging the semen at 10000rev. min⁻¹ for 10min. Blood was collected from the caudal vessel with a hypodermic syringe, and centrifuged at 10 000 rev. min⁻¹ for 15 min to obtain the plasma. The seminal plasma and blood plasma were immediately frozen at −20 °C. Sodium, potassium, calcium and magnesium concentrations were analysed by atomic absorption spectrophotometer (Hitachi 203), and chloride concentration by Buchler digital chloridemeter. Osmolality of blood and seminal plasma were measured, omitting the freezing step, using a melting point Knauer osmometer.

The semen, which was collected by pressing the abdomen of the fishes, was taken up into a 1μl Drummond micropipette and diluted in 0·1 ml of a buffered medium (1:100 dilution), then placed on a glass slide without cover at room temperature. The duration of sperm motility and number of moving spermatozoa were observed under the light microscope. Since the duration of sperm motility was very short, it is reasonable to consider that the temperature of the sperm suspension on the glass slide was constant for each measurement. The medium was as used in the preceding study (Morisawa et al. 1983) and was buffered with Hepes-NaOH at pH 7·7.

All chemicals were reagent grade, and the water was deionized and glass distilled. Specimens for electron microscopy were fixed with 1% glutaraldehyde buffered with Hepes-NaOH at pH 7·7 and 1% osmium tetroxide buffered with phosphate buffer at pH 7·7, and were embedded in epoxy resin. They were examined using a JEOL-100B electron microscope.

As shown in Table 1, sodium concentration was slightly higher in blood plasma than in seminal plasma in rainbow trout, whereas it was about the same in masu salmon and ayu. Potassium concentration was higher in seminal plasma than in blood plasma, by about ten times in rainbow trout and five times in masu salmon and ayu. Calcium and magnesium were at similar concentrations in seminal plasma and blood plasma in all three species. Chloride had a slightly lower concentration in seminal plasma than in blood plasma for the ayu, but was at similar concentration in the other two species. Osmolality of the seminal plasma (270-300 mosmol kg⁻¹) was isotonic to the blood plasma in the three species.

As shown in Fig. 1, maximum duration of sperm motility (spermatozoa always exhibited vigorous and progressive movement) in rainbow trout (20 s, Fig. 1A) and masu salmon (15–25 s, Fig. 1B) was obtained at a sodium concentration between 0 and 150 mmol kg⁻¹ (300 mosmol kg⁻¹). The duration of sperm motility and number of moving spermatozoa decreased with increasing sodium chloride concentrations, and sperm became immotile at 225 mmolkg⁻¹ (450mosmolkg⁻¹) for rainbow trout and at 250 mmol kg⁻¹ (500 mosmol kg⁻¹) for masu salmon.

In the nonelectrolyte, mannitol, maximum duration of motility was obtained between 0 and 225 mmol kg⁻¹ (225 mosmol kg⁻¹) in rainbow trout, and between 0 and 280mmolkg⁻¹ (280mosmolkg⁻¹) in masu salmon. The duration and number of moving spermatozoa decreased as mannitol concentration was increased, and reached zero at 340 mmol kg⁻¹ (340 mosmol kg⁻¹) in rainbow trout, and 450 mmol kg⁻¹ (450 mosmol kg⁻¹) in masu salmon.

In the char (data not shown) maximum duration of sperm motility (around 30 s) ivas obtained at a sodium chloride concentration between 0 and 150 mmol kg⁻¹ (300 mosmol kg⁻¹) and the duration of motility and number of moving spermatozoa decreased with increasing sodium chloride concentration. Sperm became immotile at 275 mmol kg⁻¹ (550 mosmol kg⁻¹). In mannitol solutions, spermatozoa were motile at concentrations between 0 and 390 mmol kg⁻¹ (390 mosmol kg⁻¹).

Sperm of rainbow trout, masu salmon (Fig. 1) and char (data not shown) were immotile in media containing potassium chloride concentrations between 1 and 300 mmol kg⁻¹ (600 mosmol kg⁻¹).

As shown in Fig. 2, the duration of motility of rainbow trout sperm in 100 mmol kg⁻¹ sodium chloride was unaffected by the presence of potassium chloride at concentrations between 0·03 and l-5mmolkg⁻¹. Sperm motility was completely suppressed at potassium chloride concentrations over 3·0 mmol kg⁻¹. When the medium contained rubidium chloride in addition to 100 mmol kg⁻¹ sodium chloride, the duration of motility and number of moving spermatozoa decreased with rubidium concentrations over 0·3 mmol kg⁻¹, and motility ceased at 3·0 mmol kg⁻¹.In contrast, lithium chloride did not inhibit sperm motility in 100 mmol kg⁻¹ sodium chloride, at concentrations between 0 and 25 mmol kg⁻¹. Almost all spermatozoa exhibited vigorous and progressive movement. Neither lithium chloride nor sodium chloride alone inhibited sperm motility at concentrations up to 100 mmolkg⁻¹ (Fig. 3).

Potassium and rubidium chloride at concentrations of 100 mmol kg⁻¹ completely suppressed sperm motility. Substitution of the sodium chloride by Tris chloride or sodium acetate had little effect on sperm motility.

The concentration of potassium required for inhibition of sperm motility of rainbow trout depended on sodium concentration (Fig. 4). Potassium suppressed sperm motility at concentrations between 6·0 and 6·5 mmol kg⁻¹ in the presence of 150 mmol kg⁻¹ sodium. At lower sodium concentrations, less potassium was needed to suppress motility. Only 0·25–0·5 mmol kg⁻¹ potassium was needed to suppress sperm motility in the absence of sodium chloride.

Duration of sperm motility in 100 mmol kg⁻¹ sodium chloride solution was unaffected by the presence of calcium or magnesium at concentrations between 0 and 16 mmol kg⁻¹, as shown for chum salmon (Fig. 5).

Rainbow trout spermatozoa in the semen had a bullet-shaped head, a midpiece which contained several mitochondria, and a tail. The plasma membrane tightly covered the head, midpiece and tail (Fig. 6A). When semen was diluted and the spermatozoa were allowed to swim in 100 mmol kg⁻¹ sodium chloride solution for 90s, the plasma membrane of the tail became swollen and the space between the plasma membrane and axoneme became considerably wider (Fig. 6B). When semen was diluted in the buffer solution, the head maintained the original shape although the electron-sparse region increased. The plasma membrane became swollen, membranes fused with each other, and the envelope enclosed several axonemes (Fig. 6C). These morphological alterations are identical to those occurring in fresh water (Billard, 1978).

Spermatozoa of the salmonid fishes, rainbow trout, masu salmon and char, were motile in sodium chloride and mannitol solutions hypertonic to the seminal plasma (Fig. 1), in agreement with previous observation on chum salmon (Morisawa & Suzuki, 1980). Spermatozoa of rainbow trout, for example, were able to move at sodium chloride concentrations up to at least 225 mmol kg⁻¹ (450 mosmol kg⁻¹) or at mannitol concentrations up to 340mmol kg⁻¹ (340 mosmol kg⁻¹). This suggests that the osmolality of the seminal plasma is not the factor inhibiting sperm motility in the sperm duct of salmonid fishes, and that sodium chloride may slightly stimulate motility. This contrasts with the situation in freshwater cyprinid fishes, where sperm motility is inhibited at the osmolality of the seminal plasma, and where sodium chloride and mannitol have similar effects on motility (Morisawa & Suzuki, 1980; Morisawa et al. 1983).

Potassium chloride suppressed sperm motility as found previously for rainbow ipout (Schlenk & Kahmann, 1938; Benau & Tener, 1980; van der Horst et al. 1980) and chum salmon (Morisawa & Suzuki, 1980). Even in the presence of 100mmol kg⁻¹ sodium chloride, 3 mmol kg⁻¹ potassium chloride could inhibit motility. This effect could not be due to chloride, since up to 225 mmol kg⁻¹ sodium chloride, and removal of sodium or chloride, have no effect. Thus potassium, which is a major constituent of the seminal plasma, is the factor inhibiting sperm motility in the sperm duct, and sodium as well as chloride can maintain sperm motility.

In freshwater cyprinid fishes, potassium accelerates sperm motility (Morisawa et al. 1983). Another difference between cyprinid and salmonid spermatozoa is the resistance to hypotonicity even though both groups of teleosts release spermatozoa into fresh water at spawning. As shown in the previous paper (Morisawa et al. 1983), in hypotonic solutions, sperm motility is reduced and sperm structure is disrupted in cyprinid fish; spermatozoa of carp become completely amorphous in fresh water. In rainbow trout, sperm head chromatin and flagellar axoneme (9+2 structure) retain the original shape even in fresh water (Fig. 6; see also Billard, 1978), and sperm lifespan is almost the same in fresh water and in isotonic solution. Salmonid fishes are thought to belong to a primitive group of teleosts. They migrate in the ocean for several years and transfer to fresh water to reproduce. Freshwater cyprinid fishes live all their life in fresh water. Thus it seems probable that the difference of potassium effect on sperm motility and of resistance to hypotonicity between Cyprinidae and Salmonidae is related to the course of adaptation and evolution of teleosts.

Rubidium also inhibited motility of rainbow trout sperm. In contrast, lithium and sodium had no inhibiting effect. Indeed, an increase in sodium concentration reduced the sensitivity to potassium (Fig. 4). Detailed experiments using the potassiumsodium system to regulate sperm motility may provide further insight into the events which occur at the plasma membrane during the initiation of sperm motility of salmonid fishes.

The authors wish to thank Jean Gilder, Stazione Zoológica di Napoli, for her help in preparing the manuscript and Yabe Hatchery, Yamanashi Prefecture and Tokyo for supplying the materials. This work was supported in part by grant in aid from Ministry of Education Japan to M. M. We would like to thank M. Di Genova for drawing.