MOLECULAR REPRODUCTION AND DEVELOPMENT 30:241-249 (1991)

Effect of Ethanol and Methanol on the Motility of Saccostrea commercialis Sperm and Sperm Models F.C. MOLINIA AND M.A. SWAN Department of Histology and Embryology, University of Sydney, New South Wales, Australia

ABSTRACT The organic solvents methanol and ethanol at concentrations of 2.5% and 5% (v/v), respectively, were found t o significantly (P < 0.001) decrease the radius of curvature and track velocity of S. commercialis sperm. To observe the effects of the solvent directly on the axoneme, S. commercialis sperm models were prepared by extraction with Triton X-100 and reactivation with ATP in media containing acetate anions, DTT, magnesium, and CAMP. Concentrations of 0.1% Triton X-100 demembranated sperm while 0.01% and 0.05% Triton X-100 permeabilized sperm. Sperm models were successfully produced after reactivation with 1 m M ATP. At pH 8.25, 1% (v/v) ethanol or methanol was observed to increase waveform asymmetry and significantly (P < 0.001) decrease track velocity of 0.1%Triton X-100 demembranated sperm models. Similarly 1% (v/v) ethanol increased tailwave asymmetry and decreased track velocity of 0.01% and 0.05% Triton X-100 permeabilized sperm models. Reactivated motility of 0.05%Triton X-100 permeabilized sperm models prepared at pH 7.8 were poor and improved after treatment with 7% (v/v) ethanol, which increased waveform asymmetry and doubled the track velocity of sperm. This stimulatory effect of ethanol was unchanged in the presence of the alcohol dehydrogenase inhibitor pyrazole. Concerning the precise mechanism of action of ethanol on the axoneme, we conclude that a stimulatory or inhibitory effect of ethanol is dependent on the pH of the sperm model system used. Key Words: Asymmetry, Ethanol, Methanol, Permeabilization, pH, Sperm, Track velocity

INTRODUCTION Organic solvents exert various effects on spermatozoa in vitro. Ethanol (0.05-0.5%) inhibits sperm capacitation and therefore fertilization in mammalian sperm without affecting sperm motility (Anderson et al., 1982; Salonen, 1986; Rogers e t al., 1987). Incubation of semen with concentrations of ethanol a s low as 0.01-1% (Vishwanath et al., 1987) and a s high as 5% (Ivanov, 1913), cause no observable change in the motility of intact sperm. In Triton X-100 extracted ATP-reactivated sea urchin sperm models, low concentrations of methanol and 2-propanol ( ethanol or methanol decreased the track velocity of reactivated

sperm extracted with 0.1% Triton X-100 (P < 0.001) (N = 30), as did 1%(v/v) ethanol in 0.01%Triton X-100 (P < 0.005) (N = 30) and 0.05% Triton X-100 (P < 0.05) (N = 20) models (Table 2). At pH 7.8, 7% (v/v) ethanol stimulated sperm models extracted with 0.05% Triton X-100 so that the track velocity was twofold that of the controls without ethanol (Fig. 12, Table 2). No stimulatory effect was observed with methanol.

Action of Pyrazole Pyrazole (9 mM) had no significant effect on the reactivated motility of 0.05% Triton X-100 extracted, ATP reactivated oyster sperm models with 7% (v/v) ethanol a t pH 7.8 (N = 15). Sperm still propagated asymmetrical waveforms, and moved with a similar track velocity to the ethanol-treated models (Fig. 12).

244

F.C. MOLINIA AND M.A. SWAN

Fig. 2. Longitudinal and transverse sections through the flagellar axoneme. The 9 peripheral doublet tubules (dt) are related to the central pair and sheath complex (cc) via radial spoke attachments (rs).The axoneme on the left is viewed from base to tip as the dynein arms (closed arrow) point in a clockwise direction. Scale bar: 0.2 km.

DISCUSSION Intact oyster sperm, which are not hindered by obstructions, normally rotate and swim in straight paths in filtered sea water. However like sea urchin sperm (Gray, 1955;Rikmenspoel, 19781,their resultant

pattern of movement when moving against an interface is in circular paths. Methanol (2.5%)or ethanol (5.0%) decreased velocity and increased tailwave asymmetry resulting in movement in circles of smaller radii. These results correlated with earlier findings that low concentrations of methanol, 2-propanol and ethylene gly-

EFFECT OF ETHANOL AND METHANOL ON SPERM

Figs. 3-5. Photomicrographs of intact oyster sperm obtained under darkfield and stroboscopic illumination with 1-sec exposure and strobe frequency of 16 Hz. Scale bar: 30 Km. Fig. 3. Control sample in filtered seawater. Sperm move in circular paths.

245

Fig. 4. Sperm treated with 5.0% (viv) ethanol, move in tighter circles than do control sperm in Figure 1. Fig. 5. Sperm treated with 2.5%(vh) methanol, move in circles and arcs of smaller radii as compared with the control in Figure 1.

246

F.C. MOLINIA AND M.A. SWAN TABLE 1. Effect of 5% Ethanol and 2.5 70Methanol on Motility (Mean f SE, N = 30) of Intact S. commercialis Sperm Radius of curvature (fim)

Velocity (fim/sec)

26.2 f 0.8

113.6 t 2.3

Treatment Control and 1%BSA +5% Ethanol and 1%BSA +2.5% Methanol and 1%BSA

Waveform Planar

* 0.9*

63.6 f 2.5*

Asymmetrical

20.2 f 0.5*

100.4 f 2.8*

Asymmetrical

18.2

*Significantly different from control (P< 0.001).

25 0.6

1.1

$-

20 n

0.4

0

-$

3. W

1.3

q

0.8

0.7

15

h

.c,

. I

g

10

NO ETHANOL 1% ETHANOL

s d

5

0

0.01%

0.05%

0.1%

Triton X-100 (9% concentration) Reactivation at pH 8.25 Fig. 6. Velocity profile of oyster sperm extracted with 0.01-0.1% Triton X-100 and reactivated with 1 mM ATP with or without 1%(viv)ethanol at pH 8.25. Error bars are SE (N = 30 for 0.01 and 0.1%models, and N = 20 for 0.05% models).

TABLE 2. Effect of Ethanol and Methanol on Velocity (Mean f SE) and Waveform of Triton X-100 Extracted S. commercialis Sperm Reactivated With 1 mM ATP at pH 7.8 and 8.25 No of Sperm 15 15 30 30 20 20 30 30 30

Triton X-100 pH 7.8 7.8 8.25 8.25 8.25 8.25 8.25 8.25 8.25

(%)

0.05 0.05 0.01 0.01 0.05 0.05 0.1 0.1 0.1

Organic solvent

-

7% Ethanol 1%Ethanol

-

1%Ethanol

-

1%Ethanol 1%Methanol

Velocity (pm/sec) 7.2 f 0.3 15.2 f 0.7a 16.4 f 0.4 13.5 0.7b 18.9 t 1.1 15.2 f 1.3c 19.6 t 0.6 15.6 f 0.8d 13.2 f 0.6d

+

Waveform Symmetrical Asymmetrical Symmetrical Asymmetrical Symmetrical Asymmetrical Symmetrical Asymmetrical Asymmetrical

Significantly increased by organic solvent, "P< 0.001. Significantly decreased by organic solvent, bP < 0.005, cP < 0.05, d P < 0.001.

EFFECT OF ETHANOL AND METHANOL ON SPERM

Figs. 7-11. Photomicrographs of oyster sperm models extracted with Triton X-100and reactivated with 1mM ATP in the absence or presence of 1%(viv) ethanol or methanol at pH 8.25. Scale bars: 10 Fm. Fig. 7. Control sample extracted with 0.1% Triton X-100 and reactivated with 1mM ATP alone. The sperm moves symmetrically in a straight path. Fig. 8. 0.05%Triton X-100extracted oyster sperm reactivated with 1% (viv) ethanol. The sperm moves asymmetrically in a curved path.

247

Fig. 9. 0.1% Triton X-100extracted oyster sperm reactivated with 1% (viv) methanol. The sperm head traces the image of a sharp bend. Fig. 10. Photomicrgraph of sperm tail treated as in Figure 9. The asymmetry is apparent in the tail, which is curved and moving with high flagellar amplitude. Fig. 11. Sperm treated as in Figure 9. Sperm is immotile due to solvent damage with a sharp bend in the tail at the proximal region of the head, followed by a straight mid-region.

248

F.C. MOLINIA AND M.A. SWAN

l5 10

,

t

0.7

15.2

,

0.7

14.0

5

0 CONTROL

+7% ETHANOL

9mM PYRAZOLE +7% ETHANOL

Treatment Reactivation at pH 7.8 Fig. 12. Velocity profile of oyster sperm extracted with 0.05%Triton X-100 and reactivated with 1mM ATP in the absence and presence of 7% (v/v) ethanol and 9 mM pyrazole a t pH 7.8. Error bars are SE (N = 15).

col increase tailwave asymmetry in sea urchin sperm organic solvent (1% v/v ethanol or methanol), induced (Gibbons, 1982) and decrease beat frequency [beat a component of waveform assymetry with a concomifrequency is proportionately related to velocity in sea ta n t decrease in track velocity. These asymmetrical urchin (Gray, 1955) and oyster sperm (Denehy, 197511. waveforms were similar to those seen in sea urchin The results of this study on oyster sperm models sperm reactivated in the presence of low concentrations support earlier reports that different concentrations of of Ca2+ (Gibbons and Gibbons, 19801, and low concenTriton X-100 can either permeabilize or completely trations of methanol, 2-propanol, and ethylene glycol remove sperm membranes (Gibbons and Gibbons, 1972; (Gibbons, 1982). This implies that the solvents may be Tash and Means, 1983;Swan et al., 1980; deLamirande mimicking the action of Ca2+ in controlling flagelet al., 1983; Vishwanath et al., 1986); 0.1% Triton lar curvature and swimming pattern. Our solventX-100 was sufficient to permeabilize oyster sperm so damaged oyster sperm models also resembled the Ca2+ that no motility was recovered when extracted sperm induced quiescent sperm previously seen in sea urchin were diluted into a control medium without ATP. After sperm models (Gibbons and Gibbons, 1980). The deexposure to lower concentrations of Triton X-100 crease in flagellar beat frequency in sea urchin sperm (0.01% and 0.05%), some motility was sustained in models after treatment with solvent may be due to control media without ATP suggesting that areas of the induced alterations in the rate constants of dynein plasma membrane were still present enabling sperm to ATPase (Gibbons, 1982; Evans and Gibbons, 19861, utilize endogenous ATP (Vishwanath et al., 1986). The which may also explain the decrease in track velocity in motility of extracted sperm in media without ATP was our oyster sperm models at high pH. An interesting feature of this study was the progresthus a useful marker for determining the degree of permeabilization by the detergent in the preparation of sive increase in velocity of reactivated sperm models sperm models. with increasing concentrations of Triton X-100 at pH The optimal pH for the maintenance of the motility of 8.25. This may be due to activation of the flagellar oyster sperm models was in the same range (8.0-8.3)as dynein ATPase by the detergent (Gibbons and Gibbons, in other invertebrate sperm models (Gibbons and Gib- 1972). Vishwanath et al. (1986) found that the average bons, 1980). In this study, oyster sperm models buffered beat frequency of reactivated ram sperm progressively at pH 8.25 moved better than those buffered a t pH 7.8. decreased with increasing concentrations of Triton Recently, the motility of reactivated bull sperm were X-100; however, their model system was buffered at pH also found to increase with increasing pH (Goltz et al., 7.9. Recently, Goltz and co-workers (1988) suggested 1988). At the higher pH, treatment of sperm with that the pH may control the activity state of sperm at

EFFECT OF ETHANOL AND METHANOL ON SPERM any time, and the present study showed stimulated or inhibited motility was very dependent on the pH of the sperm model system used. In doubling the track velocity, the stimulatory effect of ethanol, which was maximal at 7% (viv), on permeabilized oyster sperm at pH 7.8 is in agreement with the doubling in beat frequency found previously in the oyster a t pH 7.2 (Guirguis and Swan, 1985) and in the ram with 1%ethanol at pH 7.9 (Vishwanath et al., 1987). Ethanol has been reported to stimulate a magnesium-activated ATPase in rat liver mitochondria (Thore and Baltscheffsky, 1965) and at concentrations of alcohol above 5% to stimulate magnesium activated dynein ATPase isolated from sea urchin sperm tails (Evans and Gibbons, 1986), so t h a t presumably ethanol affects the reactivated axoneme directly. The alcohol dehydrogenase inhibitor pyrazole (Theorell and Yonetani, 1963; Goldberg and Rydberg, 1969) prevents the inhibition of fertilization by ethanol in mice, suggesting the action of ethanol is possibly via metabolic processes mediated by alcohol dehydrogenase (Rogers et al., 1987). As pyrazole had no effect on our ethanol stimulated model we conclude t h a t ethanol affects the reactivated oyster axoneme directly.

ACKNOWLEDGMENTS The authors thank Roland Smith for assistance in the preparation of the figures and the printing of photomicrographs.

NOTE ADDED IN PROOF Ethanol also had a stimulatory effect on ram sperm models. Vishwarath R, Swan MA, White IG. Motility characteristics and metabolism of ram sperm in the presence of ethanol Anim Reproduction Science (in press).

REFERENCES Anderson RA, Reddy JM, Joyce C, Willis BR, VanderVen N, Zanaveld LJD (1982):Inhibition of mouse sperm capacitation by ethanol. Biol Reprod 273334340. deLamirande E, Bardin CW, Gagnon C (1983): Aprotinin and a seminal plasma factor inhibit the motility of demembranated reactivated rabbit spermatozoa. Biol Reprod 28:788-796. Denehv MA (1975): The propulsion of nonrotating - ram and oyster spermatozoa. Biol Reprod i3:17-29.

249

Evans JA, Gibbons IR (1986): Activation of dynein 1 adenosine triphosphatase by organic solvents and by Triton X-100. J Biol Chem 261:14044-14048. Franzen A (1956): On spermiogenesis, morphology of the spermatozoon and biology of fertilization among invertebrates. Zoo1 Bidrag Uppsala 31:355-482. Gibbons BH, Gibbons IR (1972):Flagellar movement and ATP activity in sea urchin sperm extracted with Triton X-100. J Cell Biol 54:75-97. Gibbons BH, Gibbons IR (1980): Calcium induced quiescence in reactivated sea urchin sperm. J Cell Biol 84:13-27. Gibbons BH, Gibbons IR (1981): Organic solvents modify the control of flagellar movement in sea urchin sperm. Nature 29295-86. Gibbons BH (1982):Effects of organic solvents on flagellar asymmetry and quiescence in sea urchin sperm. J Cell Sci 54:115-135. Goltz JS, Gardner TK, Kanous KS, Lindemann CB (1988): The interaction of pH and cyclic adenosine 3’,5’-monophosphate on activation of motility in Triton X-100 extracted bull sperm. Biol Reprod 39:1129-1136. Goldberg L, Rydberg U (1969): Inhibition of ethanol metabolism in vivo by administration of pyrazole. Biochem Pharm 18:1749-1762. Gray J (1955): The movement of the spermatozoa of the bull. J Exp Biol 35:96108. Guirguis A, Swan MA (1985): A study of the effects of gossypol and ethanol on oyster sperm models. J Anat 143:232. Healy JM, Lester RJG (1991):Sperm ultrastructure in the Australian oyster Saccostrea commercialis (Iredale & Roughley) (Bivalvia: Ostreoidea). J Mol Stud 57:219-224. Ivanov J (1913): Action de I’alcohol sur les spermatozoides des mammiferes. CR SOC Biol Paris 74:480. Rikmenspoel R (1978):Movement of sea urchin sperm flagella. J Cell Biol 76:310-322. Rogers BJ, Cash MKM, Vaughn WK (1987): Ethanol inhibits human and hamster sperm penetration of eggs. Gamete Res 16:97-107. Salonen I (1986):Exposure to ethanol during capacitation impairs the fertilizing ability of human spermatozoa in vitro. Int J Androl 9:259-270. Swan MA, Linck RW, Ito S, Fawcett DW (1980): Structure and function of the undulating membrane in spermatozoan propulsion in the toad Bufo marinus. J Cell Biol 85:866880. Tash JS, Means AR (1983): CAMP,calcium and protein phosphorylation in flagellar motility. Biol Reprod 28:75-104. Theorell H, Yonetani T (1963): Liver alcohol dehydrogenase-DPNpyrazole complex: A model of a ternary intermediate in the enzyme reaction. Biochem Z 338537-553. Thore A, Baltscheffsky H (1965): Inhibitory effects of lower aliphatic alcohols on electron transport phosphorylation systems. Acta Chem Scand 19:1591-1599. Vishwanath R, Swan MA, White IG (1986): Effect of Triton X-100 on ultrastructure, reactivation, and motility characteristics of ram spermatozoa. Gamete Res 15:361-371. Vishwanath R, Swan MA, White IG (1987): Effect of ethanol on motility characteristics and metabolism of ram spermatozoa. In Proceedings of the Nineteenth Annual Conference of the Australian Society of Reproductive Biology, August 24-26, Sydney, pp 96.