Animal Reproducdon Science. 27 ( 1992) 269-278 Elsw~er Science Publishers B.V.. Amsterdam

269

otility characteristics and met sperm in the presence of

ABSTRACT

Viahwanath, R.. Swan, M.A. and White. I.G.. 1992. Motility charaneristin and metabolism of ram sperm in the presence of ethanol. Aftirn. Reprod SC;., 2:: 269-278.

Ram rpcfm WCR lirsl demembranated by extracting with 0.01% T&on X-lOO,and then reactivated with adenosine 5’-triphosphaie (ATP). In the presence of 4% and 2% ethanol. the mean 1 SE reacti-

vation of demembtattatcd swrm by I mmol ATP was decrwd from 77.9%+ I.2 (control) to 56.g%? 1.2 and 5X4%+ 4.0, respectively (in five rcplicatcr). Lower concentraGons of ethanol ( 1% and 0.5%) had no effect on reactivation (76.496e4.4 and 77.6Nf4.4. respectively). Cinemicrc- graphic analysis of demcmbnnated sperm revealed no change in the mean amplitude of the wave motion at the neck and midpim junctions in the presence of 1% ethanol. There was. however. a significant increase in beat frequency which was also found in stmboscopic measurements. Methanol (I%) did na rtgttilicantly alTcct beat ftetntency whilsl n-butanol (I%) and isopmpanol (1%) com- 01e1cly inhibited teaclivalion of the sperm models.

The motilily scorn of intact sperm, washed in Krebs Ringer phosphate buffer containing &cose, was unchangedafter incubating wnh 0.01.0.1 or lbethanol for 2 h but S%cthanol decreased forward velocily. The oxygen uptake of inlacl sperm from four ejaculates incubated in the presence OF I% ethanol for I h I tS.S+O.S pl tO-a) was not rianiticantly different from the control (15.2+2.8); glycolyrio wos also ~naktcd after 2 h.

We conclude tha: the surprising increase in the beat frequency ofdemembranated, reactivated mm ,prm was owing to an ctTecl of ethanol on the moolily apparatus rather than on oxidative mnabolism.

INTRODUCTION

Ingested ethanol affects many organs including the gonads. It is capable of penetrating the germinal cell compartment in the testis and is often referred to as a male gonsdd toxin (Setchell, 19’18; Anderson et al., 1983, 1985:

Correspondence to: Dr. M.A. Swan, Department of Histology and Embryology. University of Sydney, N.S.W. 2006. Australia.

‘Present address: Livestock Improvement Curporation, New Zealand Dairy Board, Private Bag, 3016. Hamilton, New Zealand.

0 1992 Elsevierkience Publishers B.V. All rights reserved 0378-4320/92/$05.00

270

Eriksson et al., 1983). The Leydig cells are particularly vulnerable to ethanol which changes their intermediary metabolism and steroidogenesis (Ellingboe and Varanelli, 1979, Anderson et al., 1980, Van Thiel et al., 1983). The action of ethanol in vitro on spermatozoa has been studied in several species. Ethanol ( IOO-4OOmg%) inhibits fertilisation in vitro when present in mouse sperm capacitation medium (Anderson et al., !978, 1980, 1982). A similar effect was seen with hamster (Cash and Rogers, 1981) and human spermatozoa (Salonen, 1986).

The plasma membrane can be stripped from ram sperm by extracting in Triton X-lOOmedium (WbiteandVonlmavr. 1986:Visbwanathetal.. 1986) and motility reactivateh by adding a&no&e Y&phosphate (ATP). Such sperm models provide an excellent system for investigating the direct action of substances00 the motility apparatus (axooeme).

The present studies were designed to obtain information on the effect of ethanol on the motility pattern of demembranated ram sperm by cinemicro graphy and stroboscopic beat frequency and velocity measurements. The ef- fect of ethanol on the oxygen uptake of intact mm sperm has also been monitored.

MATERIALS AND METHODS

Semen was collected from Merino rams by electroejaculatioo using the bi- polar rectal electrode (Blackshaw, 1954) with a maximum stimulation of 12 V at 40 Hz (Martin and Rees, 1962 ). Ejaculates were checked for goad initial motility before 0.5 ml of the semen was diluted to 7 ml with calcium-free Krebs Ringer phosphate (pH 7.4) containing 0.1% glucose (KRPG) (Um- breit et al., 1959). The sperm were washed twice by aatrifugatioo at 8OOxg for IO min. removing the supernatant and resuspendingthe Pellet in KRPG. The fmal concentration of sperm was adjusted to 1 x 10’ sperm ml-’ with KRPG.

The procedure for demembranation and reactivation of mm sperm, stro- boscopy and cinemicrographic analysis of partially demembranated sperm was as detailed previously, using partial demembranation with 0.01% Triton X-100 (Vishwaoath et al., 1986). The film speed for cinemicrography. was 75 frames s-‘. Darkfield Bash micrographs were obtained as described by Simpson et al. (1987).

The motility of intact sperm was scored visurdly on a scale of B-4 with a light microscope (Emmens, 1947 ) . The respiratoti activity of intact sperm was mea;_;:cd ;it 30°C by a polarographic electrode (Rank Bros., Bottisham, Cambridge) connected to a chart recorder (Leeds Northrup Speedomax, Narth Wales, USA). The oxygen uptake (fl h - * 1 O-* sperm ) was calculated from the slope of the linear trace. For estimation of glucose utilisatioo and lactic acid production, 1 ml ahquots of washed sperm suspensions (RRPG)

271

were deproteinized with 0.5 ml 5% ZnSO4 7Hz0 and 0.5 ml 0. I5 M Ba(OH)2 before and after incubation for 2 h at 30°C. After centrifugation, the super- natants were retained and glucose was estimated by the method of Bergmeyer et at. ( 1974) and lactic acid by the method of Ciutman and Wahlefeld (1974).

The forward velocity of in&t sperm was measured at 2SDC (Experiments A and B) and 37 “C (Experiment C) by darktield microscopy with Xenon arc light source (Wild, Switzerland), UV filters, 16~ Zeiss Kpl ocular, 40~ / 0.65 Zeiss objective, WL Zeiss (Germany) microscope with rotating and gliding stage. Filming was with a Wild MPS 12 camera, 35 mm Kodak TriX film, 0.5 s-exposure with stroboscope running to obtain multiple exposures for velocity measurement, as described by Molinia et al. (1988). Exposures were taked without preferentially select& motile spermatozoa.by focussing on an area but not viewing the field during exposure.

Spermatozoa were diluted with 1% bovine serum albumin in KKP (Exper- iment A) or KKPG (Experiment B and C). The velocity of all moving sper- matozoa in the Leld was measured from drawings of the projected images of film frames using a Digimatic catliper. A slide micrometer was used for calibration.

RESULTS

When ethanol was added to KBPG suspensions of washed sperm from two ejaculates to give final ethanol concentrations of 0.01, 0. I and I .O%, there was no evidence of an effect on motility scored visually after 0, 10,20,40,60 and I20 min. Likewise, I .O% ethanol had no significant effect on the oxygen up- take of washed suspensions (KRPG) of sperm from four ejaculates when measured at 10, 20, 40 and 60 min; the tinal values (meant SE) were 15.8tO.8 fl IO-* sperm and 15.2228 pt 10d8 for ethanol and control, re- spectivcly. Ethanol ( 1 .O%) also did not affect the amount of glucose utilised (ethanol 0.40f0.05. control 0.38+0.01 pmoles h-’ 1O-8 sperm) or the amount of lactic acid produced (ethanol t3.36~0.02, control 0.37 CO.01 poles h-r IO-* sperm) by washed sperm from four ejaculates.

However, in two experiments the forward velocity of sperm was r&teed after exoosure to 5% ethanol for 50 (Exueriment A) and 70 mitt (Experiment B ). Thus, the mean 2 SE. velocity (pi s- ’ ) in Experiment A was 76.6 f 3.5 (control) vs. 55823.7 (ethanol) and in Experiment B, 70.123.2 (control) vs. 51.5 F4.0 (ethanol).For Experiment A,thevarianceratio (F) was 16.20 and PcO.001; for Experiment B, F was 12.61 and PeO.01. In another exper- iment (Experiment C) the forward velocity of sperm was reduced when mea- sured immediately after adding I % ethanol. Thus, the mean 2 SE. velocity in Experiment C was 120?4.5 (control) vs. 107.8?3.6 (1% ethanol). F was 4.8 and P<O.OS.

The e&et ofincludingethanol in the ATP medium used to reactivate sperm models is shown in Fig. 1; 0.5 and 1.0% ethanol had no effect but 2.0 and

R. VISHWANATH ETAL

Fig. 1. Effect of ethanol on reactivation of partially demembnnated ram sperm. Sperm were extracted with 0.01% Triton X-100 for 2 min and transferred into a seactivation medium con- taining ethanol and I mmol ATP. Values are meanfS.E. (n=5 ejaculates).

TABLE I

CinemiorDgraphic analysis afthc effect of Slhanol on motility channeristicsof partially demembran- atcd sperm

Region Mean amplitude Beat frequency um ) (Hz)

0

I%

Neck Midpieee

Neck Midpiea

3.720.4 6.3kO.5 5.3co.7 6.2tO.8

3.9f0.3 13.2+0.7’

6.7f0.6 13.2kO.9’

Each value is the moan + S.E. of five sg~m~ analyscd for three cyclesof movement. In addition. for I I sperm fmm the same experimcnl, cineminaOraphic an&&s gave a mid&e beoc frawmcy of5.9 f 0.4 HZ for conlrol (noethanol) and 12.1 +O.d Hz for I% ethanol (Fz69.26. PcO.CCIOI 1.

4.0% ethanol decreased the percentage of sperm that were reactivated. Ci- nemicrographic analysis ofdemembraaated sperm reactivated by 1 mmol ATP in the presence of I% ethanol. revealed no statistically significant change in the mean amplitude of the wave motion at the neck and midpiece junctions (Table 1). Wave motion and wave envelope traces showed an increased asymmetry during forward progression which gave a peculiar circular motion to the sperm (Fig. 2, 3, 4 and 5). Surprisingly, ethanol (1%) induced an increased beat frequency in the sperm models which was recorded cinemicro- grrtphically (Table I ) . As well as an increased beat frequency and asymmetry of the spew-tail, many sperm also had looping of the tail-end and shortening

273

!

Fig 2. Effect of ethanol on wave motion of reactivated ram sperm models. The traces were obtained fmm the lilm used for cinemicmgraphic analysis. Arrows show the pronounced asym- merry under the influence of ethanol.

ETHANOL

(1%)

Fig. 3. Wavcenvelope:nresofpanially demembranated sperm in the presenceof ethaoal. Tbia was obnincd by aligning the neck regions of progressively motile sperm and tracing a:~cna:c frames. Arrows show Ihe pronounced asymmetry as seen in Fig. 2.

of the wavelength towards the distal part of the tail in the presence of 1% and 2% ethanol (Fig. 5). Stroboscopic measurements on five ejaculates con- firmed that the beat frequency (Hz) of sperm models was significantly greater (P-&05) inthepresenceof l%ethanol (l2.8!10.4) thancontrols (9.3S.6). Methanol ( 1%) did not significantly affect the beat frequency (8.620.4)

Fip 5. Electronic flesh. darkfield micrographs ofdcmembraoated, reactivntedram spermatozoa in the pVS%PX Of 2% ethanol to demonstrate the effect of ethanol on bendingot” the sperm tail.

In these _iVe micrqmphs, the two sperm to Ihe right oftbe field have an abnormally high number of waves pmpagnting distally (i.e. wavelength is decreasing) and the sperm on the fa: right in (a)-(d) h~s~loopedtail-end.

while n-butanol ( 1%) and isopropanol ( 1%) completely inhibited reactiva- tion of the sperm models by 1 mmol ATB.

There are many descriptions in the literature of inhibitory, toxic or delete- rious effects of ethanol on the male reproductive system and on spermatozoa (for review, see Mann and L&v&-Mann, 198 1). In vitro inhibition of fertil- isation by ethanol appears to be mediated by inhibiting sp”“n capacitation, while having no effect on the motility of the intact spermatozoa at the cotrcen-

Fig_ 4. Electronic flash. d&field micrographs of demembranated, reactivated ram spermato- zoa. (s,b) Soccz&ve lmmesof control ram sperm models (noethanol). (c,d) Successive frames of ram sperm models treated as in (a,b), but with the addition of I# ethanol. Note that one sperm in c (ielI sick) and another in d (upper right side) arc demonstratingthe excessive pmx- imal bending ofthe til which is typically Seen in the preeseotx of ethanol.

276 R. “lSHvL4F ;H ETA%_

trations used (Anderson et al., 1982; Saionen, 1986; Rogers et al., 1987). The manner in which ethanol inhibits capacitation is not known, but as pyrazole prevents the inhibition of fertilisation by ethanol (Anderson et al., 1982) it is presumed to act via metabolic prccesses mediated by alcohol dehydrogen- ase. We found no effect of 1% ethanol on oxygen uptake of ram sperm at 1 h, or on glycolysis during 2 h; however, over longer periods ethanol may depress oxygen uptake (Scott et al., 1963).

As much as 5% ( 1.09 mol) ethanol had no effect on motility of dog sperm (Ivanov, 1931, cited by Mann, 1964) and 87 mmol bad no effect on motility of mouse sperm (Anderson et al., 1982). Similarly, in this study, up to 1% ethanol had no effect on the motility of intact washed ram sperm, assessed visually at intervals up to 2 h. However, when velocity of individual intact ram spermatozoa was measured we found that both I% and 5% ethanol de- creased forward velocity. Similarly, Molinia and Swan ( I99 1) found that 5% ethanol decreased the forward velocity of intact oyster spermatozoa.

It was surprising therefore to find that 1% ethanol bad a profound stimu- latory effect OP the beat frequency of partially demembranated ram sperm models. A similar increase in beat frequency was found in oyster sperm models, with the maximum stimulatory effect occurring at a concentration of 7% ethanol (Guirguis and Swan, 1985; Molinia and Swan, 1991). Molinir and Swan ( 19? 1) found that 7% ethanol doubled the velocity of oyster sperm models at pH 7.8 but not at pH 8.25 where the velocity was initially higher than at pH 7-S and decreased after adding 7% ethanol.

It should be noted that the reactivation frequency of ram sperm models was lower than we obtained previously (Vishwanatb et al., 1986). This could be owing to differences in initial motility of sperm samples or slight differences in demembranation and reactivation technique.

Stimulation of the motility of demembranated ram sperm is presumably owing to a direct effect of ethanol on the motility apparatus; with the mem- brane made permeable by the partial demembranation procedure, ethanol has direct access to the axonemal components. The ATP produced by the middle- pieces in this sperm model leaks out of the permeable cells, but is replaced in the reactivation procedure.

A stimulatory effect of ethanol on magnesium-activated ATPase activity in rat liver mitocbondria was reported (Tbore and Bahscheffsby, 1965). This may be relevant, as dplein which comprises the arms on doublet A microtu- bules, is also a magnesium-stimulated ATPase. Evans and Gibbons ( 1986) found that prior incubation of dynein isolated from sea urchin sperm-tails with alcohol at concentrations above 5% led to stimulation of dynein ATPase activity when the alcohol was diluted to 0.25% of its preincubation concentration.

A solvent effect can be excluded as the mechanism of action of ethanol, as e-butanol, which is four to live times more effective than ethanol as a lipid

THE EFFECT OF ErnAN0L0N MOTllJTY OF RAM SPERM 277

solvent (McComb and Goldstein, 1979), did not stimulate the beat fre- quency of our ram sperm models. Similarly, methanol and isopropranol bad no stimulatory effect on their beat frequency.

ACKNOWLEDGEMENTS

Thanks are due to the IJniversity of Sydney for a Postgraduate Studentship (R. Yishwanath) and to Dr. I.C.A. Martin (Laboratory Animal Services) and Dr. K. O’Toole (Histology and Embryology) for help with statistical analysis. We thank Roland Smith for printing the flash micrographs.

REFERENCES

Anderson, R.A., Beyler, S.A. and Zaneveld, L.J.D., 1978. Alterations of male reproduction in- duced by chronic ingestion of ethanol. Development of an animal model. F&l. Steril.. 30: 103-105.

Anderson. R.A.. Reddy, J.M.. Joyce, C.. Willis. B.R. and Zaneveld, L.J.D., 1980. Decreased male fertility induced by chronic alcohol ingestion. Fed. Pmt., 39: 542.

Andenon. R.A.. Reddy, J.M., Joyce, C., Willis, B.R., Vandefvcn, H. andZanweld, L_J.D., 1982. Inhibition ofmouse sperm capacilation by ethanol. Biol. Repmd., 27: 833-840.

Anderson. R.A.. Willis. B-R., Oswald, C. and Zaneveld, L.J.D., 1983. Male reproductive tract sensilivily to ethanol: A critical overview. Pharmacol. Bieehem. B&v., ( 18 Suppl.). I: 30% 310.

Anderson, R.A., Quig& J.M., Oswald, C. and Zmeveld. L.J.D., 1985. Demonstration ofa fiw~ tional blood-testis barrier 10 acetaldehyde. B&hem. Phamtacd., 34: 685-695.

Bcrgmeyer, H.U., Bcmt, E., Schmidt, F. and Stork, H., 1974. D-glocox determination with hexokinase and glucose-&phosphatedehydrosenasc. In: H.U. Bergmcycr (Editor), Methods of Enzymic Analysis. Vol. 3, 2nd edn. Chemie Weinheim. New York, pp. I 196-1201.

Blacksha=, A.W., 1954. A bipolar electrode for the electrical production of ejaculation in sheep. Aust. Vet. J., 30: 249-250.

Cash, K.M. and Rogers, B.J.. 1981, The effect ofalcohol on fertilization by human and!umster spermatozoa. Biol. Repmd., 24: 233 (Abstract).

Ellingboe, 1. and Varanclli. CC., 1979. Ethanol inhibits testosterone biosynthesis by direct ac- tion on Leydig cells. Res. Commun. Chem. Pathol. Pharmacol., 24: 87-102.

Emmens. C.W.. 1947. The motility and viability of rabbit spermatozoa at different hydrogen ion concentrations. 1. Physiol. Land., 106: 471~-t81.

Eriksson, C.J.P.. Wideoius. T.V.. Vlikahri, R.H., Harkonen, M. and Leinonea. P., 1983. Inhi- bition of testostemne synthesis by ethanol. Biochem. J.. 210: 29-36.

Evans. J.A. and Gibbons. I.R. 1986. Activation of dynein I adenosine triphosphate by organic solvents and by Triton X-100. J. BioL Chem., 261: 14044-14048.

Guirguis, A. and Swan, M.A.. 1985. A study of the effects ofgossypol and ethanol on oyster sperm models. I. Anat., 143: 232.

Gutman, I. and Wablefeld, A.W.. 1974. L( + )-lactate determination wrth lactic dehydrogenase and NAD. In: H.V. Bergmeyer (Editor), Methods of Enzymic Analysis. Vol. 3, 2nd edn. Chemie Weinheim, New York, pp. 1464-1468.

Mann, T., 1964. The Biochemistry of Semen and the Male Reproductive Tract. Methuen, London.

Mann, T. and Lutwk-Mann, C.. 1981. Male Reproductive Fuoction and Semen. Springer, Berlin.

278 R. VlSHWANATH ET AL.

Martin, I.C.A. and Rees, D., 1962. The use of direct current pulses for the electro ejaculation of the bull. Awl. Vet. J., 38: 92-98.

McComb, J.A. and Goldstein, D.B., 1979. Quantitative comparison of physical dependence on leniary butanol and ethanol in mice. Correlation with lipid solubility. J. Phannacol. Exp. Ther., 208: 113-l 17.

Molinia, F.C. and Swan, M.A., 1991. Effect of ethanol and methanol on the motilily of Saccm- lwa commercialis sperm and sperm models. Mol. Reprod. Dev., 30: 24 l-249.

Molinia, F.C., Howe, P.A. and Swan, M.A., 1988. A techr.iqu c of simul:asrously esrimating forward velocity, beat frequency, % live and the nature of flagellar waveforms in spermat- zoa. Pmt. Aust. Sot. Reprod. Biol., 20: 41.

Rogers, 8.1.. Cash, M.K. and Vaughn, W.K., 1987. Ethanol inhibits human and hamster sperm penetration ofeggs. Gamete Res., 16: 97-l 07.

Salonen. I.. 1986. Exposure to ethanol during capacitation impairs the fenilizingability of bu- man spermatozoa in vitro. Int. J. Andml., 9: 259470.

Scolt. T.W., Baggett, B. and White, LG., 1963. Mctaboliim of lesmstemne by xmen and the effect oftestosterone on oxidative metabolism ofspermatozoa. Ausl. J. Exp. Biol. Med. Sci.. 4 I: 363-367.

Sewhell, B.P., 1978. The Mammalian Testis. Elek Books, London. Simpson, A.M., Swan. M.A. and White, LG., 1987. Succeptibilily of epididymal boar sperm to

cold shock and proteclive action of phoaphaIidylcholine. Gamete Res., 17: 355-373. There, A. and Baltscheffaky, H., 1965. Inhibitory &ecis of lower aliphaiic alcohols on electron

trensporl phoaphorylation systems. Acla Cbem. &and., 19: 1591-1599. Umbreit, W.W., Buris, R.H. and Slauffer, J.F., 1959. Manometric Techniques Bmgcss, Min-

neapolis, MN. Van Thiel, D.H., Gavaler, J.S., Cobb, D.F.. Santucci. L. and Graham, T-0.. 1983. Ethanol, a

Leydigcell toxin: evidence obtained in-viva and iwvitro. Pharmacol. Biochem. Behsv.. ( 18 suppl.), 1:317-323.

Vishwanath, R., Swan, M.A., White, LG., 1986. E&I of T&on X- 100 on ultraawucture. reac- tivatioo and motility characlerirlics ofmm sperm. Gamnc Ra.. 15: 361-371.

White, LG. and Voglmayr. J.K., 1986. ATP.inducod reactivation of ram testicular. cauda epi- didymal and ejaculated spermatozoa extracted with Ttilon X-100. Biol. Reprod.. 34: 183- 193.