INDUCTION OF POTENTIAL FOR SPERM MOTILITY BY BICARBONATE AND ph IN RAINBOW TROUT AND CHUM SALMON

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1 J. exp. Biol. 136, (1988) 13 Printed in Great Britain The Company of Biologists Limned 1988 INDUCTION OF POTENTIAL FOR SPERM MOTILITY BY BICARBONATE AND ph IN RAINBOW TROUT AND CHUM SALMON BY SACHIKO MORISAWA Biological Laboratory, St Marianna University, School of Medicine, Sugao, Miyamae-ku, Kawasaki 213, Japan AND MASAAKI MORISAWA* Laboratory of Physiology, Ocean Research Institute, University of Tokyo, Minamidai, Nakano-ku, Tokyo 164, Japan Accepted 4 January 1988 Summary Spermatozoa of rainbow trout and chum salmon, which have no potential for motility in the testis, acquire that potential in the sperm duct. This paper demonstrates that there is little difference between the levels of sodium, potassium, calcium, magnesium, chloride and osmolality of the seminal plasma in the testis and in the sperm duct. However, the bicarbonate concentration of the seminal plasma and the ph value of semen were higher in the sperm duct than in the testis. When immotile spermatozoa obtained from the testis were incubated in artificial seminal plasma with a high ph and containing HCO 3 ", spermatozoa became motile within 1 h. These results suggest that spermatozoa of salmonid fish acquire the potential for motility as a result of the increase in seminal bicarbonate concentration and ph that occurs as spermatozoa pass from the testis to the sperm duct. Introduction 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 * Present address: Misaki Marine Biological Station, Faculty of Science, University of Tokyo, 1024, Koajiro, Misaki-cho, Miura-shi , Japan. Key words: sperm motility, HCO 3 ~, ph, trout, salmon.

2 14 S. MORIS AW A AND M. MORIS AW A 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. Materials and methods Materials 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. Analysis of spermatocrit value, ion concentration and osmolality Spermatocrit value was determined with a haematocrit meter after centrifugation of the semen in a haematocrit tube at g for lomin 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 lomin at 4 C. Sodium, potassium, calcium and magnesium concentrations were measured with an atomic absorption spectrophotometer (Hitachi ) and osmolality with an osmometer (Halbmicro-osmometer, Knauer). Chloride concentration was measured with a chloride meter (digital chloridometer, Buchler). 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 CO 2 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 HCO 3 ~ predominates over the form CO 2 + CO 2 3 " in the ph range (Skirrow, 1975). Measurement of ph value of semen 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

3 Acquisition of sperm motility by HCO3 and ph 15 (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. Incubation of spermatozoa 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 mmoll" 1 : NaCl, 100; KC1, 40 for rainbow trout, 70 for chum salmon; CaCl 2, 3; MgCl 2, 1-5; Tris buffer, 50) at various ph values, with or without HCO 3 ~, at room temperature (20 C for rainbow trout, 17 C for chum salmon) and incubated. At an appropriate time, 0-1 jul of semen was obtained in a Drummond micropipette, diluted in 100/il of the K + -free loommoll" 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. Results Semen and seminal volume and spermatocrit value 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. IB). 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 Ion concentrations and osmolality of the seminal plasma 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

4 16 S. MORISAWA AND M. MORISAWA B [T, rli rh rh III IV Fig. 1. (A) Diagram of the male reproductive organ of salmonid fish, illustrating zones of the sperm duct. (B) Spermatocrit values of the semen from the testis and sperm duct of rainbow trout. T, testis; I, II, III and IV, portions of the sperm duct (see Materials and methods); AP and PP, anterior and posterior portion of the sperm duct, respectively. N=8, vertical bars indicate means ± S.E. Amount of total inorganic carbon in the seminal plasma 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<002) and chum salmon (P< 0-001) (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 offish caught in the river.

5 Table 1. Inorganic electrolytes and osmolality of seminal plasma from various portions of the sperm duct in rainbow trout ^ Portion of Na+ K + Ca 2+ Mg 2+ Cl" Osmolality <* sperm duct (mmoir 1 ) (mmolp 1 ) (mmolp 1 ) (mmolp 1 ) (mmolp 1 ) (mosmolkg"') 3i I.II(AP) ± 3-30 (6) 37-3 ±0-65 (6) 3-26 ±0-16 (6) 1-48 ±0-10 (6) 153 ±4-1 (3) 335 ±7-3 (4) III ±5-95 (7) 33-3 ±2-38 (7) 2-86 ±0-28 (7) 1-11 ±0-07 (7) 149 ±2-9 (4) 333 ±2-9 (4)». IV ±5-87 (7) 32-5 ±2-14 (7) 2-69 ±0-27 (7) 1-02 ±0-05 (7) 143 ±4-6 (4) 316 ±11-2 (4) ^ Numbers of experiments are indicated in parentheses. V Values are means ± S.E. 15; n R o 3 a a.

6 18 S. MORISAWA AND M. MORISAWA Table 2. Amount of total inorganic carbon in seminal plasma from the testis and sperm duct in rainbow trout and chum salmon Total inorganic carbon Testis Sperm duct (mmoir 1 ) (mmoir 1 ) Rainbow trout 4-9 ± 0-86 (5) 9-2 ± 0-99 (6) Chum salmon caught in the bay 5-8 ± 0-68 (4) no semen caught in the river 14-7 ±2-99 (3) 23-5 ±1-15 (3) Values are means ± S.E. Number of fish is indicated in parentheses. For explanation of the units see Materials and methods. ph in the testis and sperm duct 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. Effect of bicarbonate on sperm motility Spermatozoa from the testis of both rainbow trout and chum salmon were immotile on suspension in potassium-free 100mmoir 1 NaCl solution after incubation in ASP without HCO 3 ~. When HCO 3 " (20mmoir 1 for rainbow trout, ^mmoll" 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 loommou" 1 NaCl solution (Fig. 2). Only lomin Table 3. ph values of semen and seminal plasma in the testis and sperm duct in rainbow trout A Testis Sperm duct Semen 7-29 ± 0-04 (12) 7-81 ± 0-07 (15) Seminal plasma 7-48 ± 0-06 (7) 7-99 ± 0-06 (10) Number of fish is indicated in parentheses. B ph values of semen in the testis and sperm duct in chum salmon Fish Testis Sperm duct Caught in the bay 7-38 ±0-02 no semen Caught in the river (N=3) Values are means ± S.E ± ±0-07

7 Acquisition of sperm motility by HCO 3 and ph A 100 B 50 I Tune (min) Fig. 2. Effect of bicarbonate on induction of sperm motility in salmonid fish. Testis spermatozoa were incubated in artificial seminal plasma (ASP) containing 20mmol I" 1 (for rainbow trout) or ^mmolp 1 (for chum salmon) HCO 3 ~ ( ) and in ASP without addition of bicarbonate (O) at ph8-2. (A) Rainbow trout, N = 4 for ASP containing HCO 3 " and N=3 for ASP; (B) chum salmon, N = 4. Vertical bars indicate means ± S.E. 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 HCO 3 ~, respectively. If the ASP contained a lower concentration of HCO3", the acquisition of sperm motility occurred slowly (data not shown). Effect of ph on sperm motility If testis spermatozoa of rainbow trout were incubated in ASP containing HCO3- at ph7-8 for lh, 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. Discussion 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

8 20 S. MORIS A WA AND M. MORIS AW A 100 o Fig. 3. Effect of ph on the induction of sperm motility with ( ) or without (O) addition of HCO3~ in rainbow trout. Motility was assessed after incubation for 60 min. Numbers of fish are indicated. Bars indicate means ± S.E. ph posterior portion (Fig. IB). 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 +, Ca 2+ 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 Mg 2+ 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 HCO 3 ~ at the high ph in the sperm duct ( ), whereas at the ph value in the testis (below 7-5) the percentage of inorganic carbon in the form of HCO 3 " decreases with decreasing ph (Skirrow, 1975). From these facts, it is clear that the difference in HCO 3 ~ concentrations between the testis and the sperm duct must be larger than that suggested in

9 Acquisition of sperm motility by HCO 3 and ph 21 Table 2. In salmonid fish, the concentration of HCO 3 ~ 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 S-lOmmoll" 1 HCO 3 ~ and the ph must be Thus, the testis spermatozoa must be exposed to a higher ph and [HCO 3 ~] 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 HCO 3 ~, 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 HCO 3 ~ (Fig. 2B). Thus, ASP that contained a similar concentration of HCO 3 ~ 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 [HCO 3 ~] 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 HCO 3 ~. 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 HCO 3 ~ 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. Barren, 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. References CLEMENS, H. P. & GRANT, F. B. (1965). The seminal thinning response of carp {Cyprinus carpio) and rainbow trout (Salmo gairdnerii) after injections of pituitary extracts. Copeia 2, HOSKINS, D. D., BRANDT, H. & ACOTT, T. S. (1978). Initiation of sperm motility in the mammalian epididymis. Fedn Proc. Fedn Am. Socs exp. Biol. 37, HOWARDS, S., LECHENE, C. & VIGERSKY, R. (1979). The fluid environment of the maturing spermatozoon. In The Spermatozoon (ed. D. W. Fawcett & J. M. Bedford), pp Baltimore, Munich: Urban & Schwarzenberg. MORJSAWA, M., SUZUKI, K. & MORISAWA, S. (1983). Effects of potassium and osmolality on spermatozoan motility of salmonid fishes. J. exp. Biol. 107,

10 22 S. MORIS AWA AND M. MORIS AW A MORISAWA, S. & MORISAWA, M. (1986). Acquisition of potential for motility in rainbow trout and chum salmon. J. exp. Biol. 126, SKIRROW, G. (1975). The dissolved gases- carbon dioxide. In Chemical Oceanography, vol. 2, 2nd edn (ed. J. P. Riley & G. Skirrow), pp London, New York, San Francisco: Academic Press.