Induction of Potential for Sperm Motility by Bicarbonate and pH In Rainbow Trout and Chum Salmon

In mammals, it is well known that spermatozoa acquire the potential for motility as they leave the testis and pass along the epididymis. In rainbow trout and chum salmon, we recently demonstrated that spermatozoa obtained from the sperm duct are motile on dilution in a potassium-free medium; however, spermatozoa from the testis are immotile or less motile in the same medium (Morisawa & Morisawa, 1986). From these results it is clear that spermatozoa acquire the potential for motility during the transition from the testis to the sperm duct in salmonid fishes. However, it is still unknown which factor is indispensable for the acquisition of sperm motility not only in these species but also in mammals. The simpler structure of the reproductive organ in salmonid fish in comparison with that of mammals offers a fascinating opportunity to investigate this phenomenon.

Mature male rainbow trout, Salmo gairdneri, obtained from Yamanashi Prefectural Fisheries Experiment Station, Yamanashi Prefecture, were kept in the laboratory tank without food and used within a week. Chum salmon, Oncorhynchus keta, were caught in Otsuchi Bay and Otsuchi River, Iwate Prefecture, in the breeding season (November to December), kept in a seawater tank and in a freshwater tank, respectively, and used within 2 days after capture. The fish in Otsuchi Bay reach Otsuchi River within 12 h (see Morisawa & Morisawa, 1986). Spermatozoa in the testis of fish caught in the bay were mature (histochemical observation). The sperm duct was ligated into four portions (I-IV, see Fig. 1) as described previously (Morisawa & Morisawa, 1986). The surface of the testis or each portion of the sperm duct was cut with scissors and extruded semen was collected with a pipette. Semen was preserved in a small tube with a tight cap, placed on ice and used immediately.

Spermatocrit value was determined with a haematocrit meter after centrifugation of the semen in a haematocrit tube at 10 000g for 10 min at room temperature.

For analysis of ion concentration and osmolality, semen was centrifuged at 2000g for 10 min and the resulting supernatant was then recentrifuged at 7000g for 10min at 4°C. Sodium, potassium, calcium and magnesium concentrations were measured with an atomic absorption spectrophotometer (Hitachi 180-50) and osmolality with an osmometer (Halbmicro-osmometer, Knauer). Chloride concentration was measured with a chloride meter (digital chloridometer, Buehler). The total amount of inorganic carbon in the seminal plasma, which was obtained by centrifugation of semen at 10000g for 10min, was measured with a carbon analyser (Model 524C, O. I. Corporation). In the equipment, total inorganic carbon in the seminal plasma was converted to free CO₂ by reaction with dilute phosphoric acid and measured by a non-dispersive infrared analyser. Sodium bicarbonate solution was used as a standard, since the form HCO₃⁻ predominates over the form CO₂ + CO₃²⁻ in the pH range 7·0-8·6 (Skirrow, 1975).

The reproduction organs of rainbow trout and chum salmon were removed from the abdomen just after the fish had been killed by a blow on the head. A small incision was immediately made on the surface of the sperm duct and testis, a combination pH electrode with a diameter of 5 mm (GS-195C, Toa) or 1·2 mm (MI-410, Microelectrode) was inserted into semen running out of the incision, and the pH value was measured. Each measurement was completed within about 3 min and all measurements for one fish were completed within 30min.

One volume of semen of rainbow trout or chum salmon obtained from the testis was mixed with two volumes of the artificial seminal plasma (ASP) (in mmol 1^-1: NaCl, 100; KC1, 40 for rainbow trout, 70 for chum salmon; CaCl₂, 3; MgCl₂, 1·5; Tris buffer, 50) at various pH values, with or without HCO₃⁻, at room temperature (20°C for rainbow trout, 17°C for chum salmon) and incubated. At an appropriate time, 0·1 μl of semen was obtained in a Drummond micropipette, diluted in 100 μ1 of the K⁺-free 100 mmol 1^-1 NaCl solution in which spermatozoa that have acquired the potential for motility actually exhibit motility (see Morisawa, Suzuki & Morisawa, 1983) and sperm motility was observed as described before (Morisawa & Morisawa, 1986). After incubation, the pH value was remeasured for verification.

To obtain a sufficient volume for measurement in rainbow trout, semen collected from portions I and II (the anterior portion of the sperm duct; AP) was combined, as was semen from portions III and IV (the posterior portion of the sperm duct; PP) (Fig. 1A). The amount of semen in the PP in three fish was more than five times that in the AP and the amount of seminal plasma in the PP was eight times more than in the AP. The ratios of the volumes of seminal plasma to semen were about 0·4 in the AP and 0·65 in the PP. These facts suggest that seminal thinning may occur by secretion of fluid during the passage of spermatozoa through the sperm duct from the anterior to the posterior portion.

The degree of thinning of the semen was precisely measured by its spermatocrit value (Fig. 1B). The spermatocrit value of the semen from testis was 80%. This value decreased to 65% in portion I, 50% in portion II, and reached 30% in portions III and IV. There were mutually significant differences in spermatocrit values among the testis (T), and portions I, II and III or IV of the sperm duct (P<001).

Ion concentrations and osmolality of seminal plasma from various portions of the sperm duct are presented in Table 1. Values for all constituents and osmolality decreased as the semen passed down the duct; however, except for magnesium, none of the differences were significant. Values in portion IV are similar to those reported by Morisawa et al. 1983.

The amount of total inorganic carbon in the seminal plasma increased as the semen moved from the testis to the sperm duct in both rainbow trout (P < 0·02) and chum salmon (P < 0·00l) (Table 2). The level in the testis of chum salmon caught in the bay, in which spermatozoa were morphologically ripe, was similar to that in the testis of rainbow trout and was much lower than for fish caught in the river. The level in the sperm duct was 1·6 times higher than that in the testis of fish caught in the river.

The pH values of semen and seminal plasma increased significantly from the testis to the sperm duct (P < 0·001) in both rainbow trout (Table 3A) and chum salmon (Table 3B). The value of semen from the testis in chum salmon caught in the bay was lower than for fish caught in the river.

Spermatozoa from the testis of both rainbow trout and chum salmon were immotile on suspension in potassium-free 100 mmol 1^-1 NaCl solution after incubation in ASP without HCO₃⁻. When HCOa⁻ (20 mmol 1^-1 for rainbow trout, 40 mmol 1^-1 for chum salmon) was included in the incubation medium (ASP), spermatozoa acquired the capacity for motility: spermatozoa were motile when suspended in potassium-free 100 mmoll^-1 NaCl solution (Fig. 2). Only 10min incubation was required for trout, but 60 min was needed for salmon. In rainbow trout, values of pH after incubation were 8·18 and 8·08 in the experiments with and without addition of HCO₃^_, respectively. If the ASP contained a lower concentration of HCO₃⁻, the acquisition of sperm motility occurred slowly (data not shown).

If testis spermatozoa of rainbow trout were incubated in ASP containing HCO₃⁻ at pH 7·8 for Ih, the capacity for sperm motility developed in 30% of spermatozoa (Fig. 3). When the pH was 8·18, it developed in 70% of spermatozoa. A pH over 8·5 was less effective, judged by the proportion of motile spermatozoa.

We recently demonstrated that spermatozoa of salmonid fish acquire the potential for motility during their passage through the sperm duct (Morisawa & Morisawa, 1986). In the present study, we showed that the volume of semen included in each portion of the sperm duct varies in each fish. This suggests that physiological conditions in the male reproductive organ vary in individuals. Despite this variation, the volume of semen in the posterior portion of the sperm duct was always larger than that in the anterior portion. The increase of seminal volume may be a result of an increase in seminal plasma, since the spermatocrit value decreased to one-third of that in the testis along the sperm duct towards the posterior portion (Fig. 1B). Seminal storage may also be possible, since the relative increase in the volume of seminal plasma is greater than the decrease in spermatocrit value. Increase in water in the sperm duct may be under the hormonal control of the pituitary gland (Clemens & Grant, 1965). The space surrounding a spermatozoon must increase. However, most testis spermatozoa that were diluted in ASP and incubated did not acquire the potential for motility (Fig. 2). Therefore, the increase of the surrounding space per se can scarcely be considered as a factor in the induction of aquisition of sperm motility.

In mammals, it is well known that the composition and concentration of ions and other components, such as protein and sugar, change markedly in the seminal fluid, as spermatozoa move down from the testis to the cauda epididymis, and these changes are thought to play important roles in the acquisition of sperm motility (Hoskins, Brandt & Acott, 1978; Howards, Lechene & Vigersky, 1979). In rainbow trout, however, there were no significant changes in concentrations of Na⁺, K⁺, Ca²⁺ or Cl⁻ or in osmolality between the anterior portion of the sperm duct and the posterior portion (Table 1). Although there was some difference in Mg²⁺ concentration, its role in the acquisition of sperm motility is not known.

However, both the amount of total inorganic carbon (Table 2) and the pH level (Table 3) were significantly higher in the seminal plasma obtained from the sperm duct than in that from the testis in both rainbow trout and chum salmon. Almost all inorganic carbon is in the form of HCO₃⁻ at the high pH in the sperm duct (7·8-8·15), whereas at the pH value in the testis (below 7·5) the percentage of inorganic carbon in the form of HCO₃⁻ decreases with decreasing pH (Skirrow, 1975). From these facts, it is clear that the difference in HCO₃⁻ concentrations between the testis and the sperm duct must be larger than that suggested in Table 2. In salmonid fish, the concentration of HCO₃⁻ and the pH level in the environment surrounding spermatozoa increase as they move from the testis to the sperm duct.

A previous report showed that spermatozoa obtained from the testis acquire motility following incubation in seminal plasma obtained from the sperm duct in rainbow trout (Morisawa & Morisawa, 1986). As shown in Tables 2 and 3A, the seminal plasma must contain 8–10 mmol 1⁻¹ HCO₃⁻ and the pH must be 7·9-8·0. Thus, the testis spermatozoa must be exposed to a higher pH and [HCO₃⁻] in the sperm duct than in the testis. In the experiments described here, when the testis spermatozoa were incubated in artificial seminal plasma (ASP) containing HCO₃^–, they acquired the potential for motility (Figs 2A, 3). The testis spermatozoa of chum salmon also acquired the potential for motility with incubation in ASP containing HCO₃⁻ (Fig. 2B). Thus, ASP that contained a similar concentration of HCO₃⁻ to the natural seminal plasma and was of similar pH bestowed the potential for sperm motility. Motility was induced within a pH range of 8-8·5, but diminished at higher levels (Fig. 3). These results suggest that appropriate pH and [HCO₃⁻] in the seminal plasma combine to give spermatozoa the ability to acquire motility.

In our preliminary experiment, cyclic AMP levels in the testis spermatozoa increased gradually during their incubation with HCO₃⁻. Hoskins et al. (1978) reported that the gradual increase of cyclic AMP levels in bull spermatozoa during their passage through the epididymis causes the acquisition of motility. Thus, it seems probable that increased cyclic AMP levels in spermatozoa during incubation with HCO₃⁻ may cause the acquisition of sperm motility in salmonid fish. It seems possible that the cyclic AMP level in the testis spermatozoa may be too low to initiate motility and that accumulation of intraspermatozoan cyclic AMP to the basal level is necessary for the induction of sperm motility.

We thank J. P. Barron, Associate Professor, St Marianna University, School of Medicine, for reading the manuscript, and the director and staff of the Oshino Branch of Yamanashi Prefectural Fisheries Experiment Station, Yamanashi Prefecture, and the Otsuchi Marine Research Center, Ocean Research Institute, University of Tokyo, for supplying materials.