Effect of sodium and potassium concentrations and pH on the maintenance of motility of rabbit and rat epididymal

spermatozoa Pholpramool and G. Chaturapanich

C.

Department of Physiology, Faculty of Science, Mahidol University, Rama VI Rd, Bangkok 4, Thailand

Summary. Spermatozoa were collected from the caput and cauda epididymidis of rabbits and rats and diluted in Hank's solution containing BSA, with various concentrations of Na+ and K+. Ionic strength and osmolarity were kept constant. Motility was assessed at various intervals during incubation at 25\s=deg\C. In the pH range 7\m=.\05\p=n-\7\m=.\20,the motility of rabbit spermatozoa was not affected by changes in the ratio of K+ to Na+. Similarly, the motility of rat cauda spermatozoa was not altered but that of caput spermatozoa was slightly depressed by a high K+/Na+ ratio. In the pH range 5\m=.\45\p=n-\5\m=.\85,rabbit cauda and caput spermatozoa had much greater motility in media with a high K+/Na+ ratio. The reverse result was obtained for the rat. These findings indicate that the motility of epididymal spermatozoa is influenced by external Na+ and K+ concentrations and that this phenomenon is pH-dependent.

Introduction

Epididymal spermatozoa

non-motile in their own fluids but become intensely active upon (Blandau & Rumery, 1964; Gaddum, 1968; Turner & Howards, 1978). Ejaculated spermatozoa, however, are motile in undiluted semen and their motility is strongly influenced by various ions (Blackshaw & Emmens, 1951; Blackshaw, 1953; Bredderman & Foote, 1971; McGrady, Nelson & Ireland, 1974; Nelson, 1975; Harrison, Dott & Foster, 1978). Although many studies of the effects of various salt solutions on sperm motility have been made on ejaculated spermatozoa, we know of only one such study on epididymal spermatozoa; Turner & Howards (1978) examined the initiation, but not the maintenance, of motility of rat epididymal spermatozoa. Micropuncture studies have shown that the fluid in the cauda epididymidis of rats (Levine & Marsh, 1971; Turner, Hartmann & Howards, 1977) and hamsters (Jessee & Howards, 1976) contains extremely high K+/Na+ ratios (2-7-3-2 for rats; 1-5-3-9 for hamsters). Relatively high K+/Na+ ratios have also been found in the fluid of the distal epididymis'in rabbits (Jones & Glover, 1973), bulls and boars (Crabo, 1965). Although accumulating evidence suggests that sperm maturation is dependent on the epididymal environment (Orgebin-Crist, Danzo & Cooper, 1976) the significance of a high K+/Na+ ratio in the epididymal fluid on the motility and fertilizing capacity of epididymal spermatozoa is unknown. The present investigation is therefore a study of the motility of epididymal spermatozoa in media containing different concentration ratios of K+/Na+ at different pH. are

dilution with salt solutions

Materials and Methods Adult male New Zealand White rabbits, weighing 2-1-2-9 kg, and albino rats, weighing 209341 g, were anaesthetized with sodium pentobarbitone (35 mg/kg, i.v.) and ether, respectively. Either the right or left epididymis was quickly removed from the animal, dissected free from its fat pad and blotted on filter paper. Spermatozoa were obtained by puncturing the cauda or the caput epididymidis with a 21-gauge hypodermic needle. The emerging epididymal content was then collected in a microcapülary pipette. This fluid (1-2 pi) was added to 1-2 ml normal or modified Hank's solution (Table 1) to make a sperm concentration of 0-1-1-6 IO6 cells/ml for incubation in a small vial at room temperature (25 ± 0-3°C) for 10, 30, 60 or 80 and 120 or 130 min. After each incubation period, a small sample of fluid (10-20 pi) was removed for motility determination in a haemocytometer. Using phase-contrast microscopy, all motile spermatozoa were counted and their pattern of movement was arbitrarily classified into forward progressive movement, circular movement or weak flageUar beating with no progression. Sperm motility was scored in terms of 'percentage motility' (no. motile/total no. of spermatozoa) and 'percentage forward motility' (no. of spermatozoa with forward progressive movement/total no. of spermatozoa). Paired observations were used to compare the effects of various diluents. Table 1.

Composition of the incubation media Choline chloride

Solution*

NaCl(mM)

Normal Hank's solution Solution A Solution

130-9 10-0

KC1

(mM)

5-4 54-0 54-0

(mM)

K+/Na+ratio

72-3 82-3

0-05 3-06 6-88





•All the solutions also contained; 1-3 mM-CaCl2, 1-0 mM-KH2P04, 1-9 mM-Na2HP04, 1-4 mM-MgS04, 4-2 mM-NaHC03, 5-8 mM-glucose and 4% bovine serum albumin. The pH of these solutions was 5-45-5-85. When solutions with pH 7-05-7-20 were needed, 1 M-Tris buffer was added to a final concentration of 20 mM.

AU data

are

presented as mean

+ s.e.m. and

analysis.

paired Student's t test was employed in statistical

Results

Effect of varying the ratio ofK+/Na+ in the medium at neutral pH Rabbits

Caput epididymidis. At pH 7-10-7-15

a considerable number of immature spermatozoa motile in normal Hank's solution, but motUity declined progressively during incubation (Table 2). At all periods of incubation less than 10% of spermatozoa showed forward progressive movement, and most motUe spermatozoa moved in circular fashion. Simüar results were obtained when spermatozoa were incubated in media containing higher ratios of K+/Na+ were

initially

(Table 2).

Cauda epididymidis. Spermatozoa from the cauda epididymidis were initially very active in normal Hank's solution, with about 50% showing progressive movement (Table 2). Sperm motUity was maintained throughout the experiment, but at 130 min the number of forwardmoving spermatozoa slightly decreased (P < 0-05). Incubation of spermatozoa in Solutions A and had no effect on the total number of motile spermatozoa but caused a rapid reduction in the percentage of cells showing progressive motion at 80 and 130 min (Table 2).

Table 2. Effect of varying the ratio of K+/Na+ and pH in the medium on the percentage forward progressive motility (in parentheses) of rabbit epididymal spermatozoa

motility

and

Incubation time (min) 10

30

80

130

601 ± 11-6 (3-7 ± 1-6) 52-3 ± 12-4 (9-0 ± 4-5) 52-0 ± 10-2

57-1 ± 10-4 (2-6 ± 1-7) 48-7 ± 8

36-0 ± 10-0 (4-7 ± 2 20-0 ± 5 (2-0 ± 2

28-7 ±8-8 (2-4 ± 2-4)

(5-0 ±2-2)

(3-4

7 ± 1

22-4 ± 9 (0-9 ± 0

19-1 ± 7-4

26-2 ±5-3

27-0 ± 6 (0-9 ± 0 16-8 ±8 (2-7 ± 2 31-5 ± 4

12-5 ±3 (0-7 ± 0

Solution

pH

Caput epididymidis 7-10-7-15

Normal Hank's A

5 -45-5 80 ·

Normal Hank's

(1-4 ±0-9) A

19-7 ±61 (4-3 ± 2-2) 44-8 ± 4-0" (6-3 ± 2-8)

Cauda epididymidis 7-10-7-15 Normal Hank's

81-4 ±2-9

(53-3 ±7-5) A

Normal Hank's A

6-8 ±3-9

19-0 ±6 (2-8 ± 1

14-3 ±5-5 (3-3 ± 1-7)

78-3

±

+

(0)

72-6 ±4-1

2-6

(55-0 ±4-4)

(38-4 ± 6-9)

75-6 ±4 5

(32-1 ±5-3)*

81-6 ± 1-6

7-5

9-3 ±4 1

75-6 ±3-6

(60-6 ±

+

(0)

(1-0 ±

75-4 ±4-1

72-4 ± 5-4

(14-6 ±4-6)** 70-9 ±5-7

(12-4 ±5-0)***

68-9 ±3

42-9 ±6-6

(51-1 ±4-8)

(37-9 ±4

(14-0 ±4-4)

(5-5 ±4-1)

77-1 ±3-4

68-5 ±3 (32-4 + 7

44-6 ± 6-6

35-1 ± 7-1

+

2-6

78-5 ± 1 (60-3 ± 7

28-9 ±6-0

(11-4 ±5-4) 70-3

+

(6-0 ±5-7)

4-0***

(37-8 ± 10-0)*

from 6-8 experiments. different from those in normal Hank's solution at the < 0001.

are mean

61-9 ±4-7"* (26-6 ± 10-2)*

s.e.m.

significantly

0-01;***

6

7-5

(34-1 ±6-7)*

(60-3 ± 7-9)

Effect of sodium and potassium concentrations and pH on the maintenance of motility of rabbit and rat epididymal spermatozoa.

Effect of sodium and potassium concentrations and pH on the maintenance of motility of rabbit and rat epididymal spermatozoa Pholpramool and G. Chatu...
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