DIMETHYL SULFOXIDE A TOOL IN THE STUDY OF SPERM MOTILITY CONTROL*

Leonard Nelson

Depart men 1 of Ph ysiology Medical College of Ohio

Toledo, Ohio 43614

Spermatozoa contend with a number of environmental challenges during matu- ration and transport. The chemical composition and physical condition of the milieu vary markedly in different segments of the male and female reproductive tracts.’ In species that engage in internal insemination and also in those that practice external fertilization, substances that emanate from the oocyte may modify the sperm cells’ swimming b e h a v i ~ r . ~ , ~ In response to the varying conditions, the sperm cell may change its speed and pattern of swimming4 by changing the frequency, amplitude, wa~elength ,~ and occasionally the direction of the flagellar beat.6 Thus, a sperm cell “at rest” may begin to move in response to a mechanical stimulus, while a cell al- ready in motion may accelerate, decelerate, alter its swimming path or stop moving, according to the strength, duration and nature of the stimulus.

The property of excitability, the capacity exhibited by cells and tissues in general t o respond to environmental changes, presupposes that the stimulus received at the interface with the environment is transduced and transmitted to an effector that enables the system to react in a characteristic fashion. In unicellular or acellular organisms, the processes of reception and transmission may be amalgamated into cell membrane functions, so that environmental changes initiate responses a t this level.’ The stimulus-conduction-response phenomenon is frequently mediated by a neurotransmitter system. Hence, when Zeller and Joels found a low level of acetylcholinesterase activity in human semen, this opened the possibility that acetylcholine is involved in sperm motility. Subsequently, Sekineg reported that the highly active cholinesterase of boar semen (the level of activity approaches that of brain tissue) occurred primarily in the cellular fraction. Since that time, spermato- zoan cholinesterases with differing specific activities have been found in a wide range of species (TABLE 1).

Conjecture that the spermatozoan cholinesterase could play a significant role in the regulation of motility received preliminary confirmation when we observed that physostigmine increased the rate of flagellar wave formation in Mytilus sperm.1o No change could be detected in flagellar activity when we added acetylcholine (at con- centrations from M) to seawater suspensions of these sperm, but 10 p M physostigmine inhibited the acetylcholine hydrolysis of crude sperm homogenates by about 50%. Furthermore, eserine (physostigmine), a competitive inhibitor of cholinesterase, apparently releases the flagellum from some intrinsic control of its beat frequency, a t a concentration that reduces the enzyme activity by nearly half. Under these conditions an accumulation of acetylcholine would be ex- pected to occur within the cell, but we found that externally supplied acetylcholine had no discernible effect on the flagellar action of Mytilus sperm. The success of eserine and the failure of both acetylcholine and neostigmine to exert much influence on the performance of sea urchin spermll suggested that, unlike tertiary amines,

M to

*This work was supported by United States Public Health Service research grant HD- 03266, from the National Institute of Child Health and Human Development.

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298 Annals New York Academy of Sciences

TABLE I SPECIFIC ACTIVITY OF SPERMATOZOAN CHOLINESTERASE

Source* Manner of Preparation

Specific Activity nM/mg protein/min

Trout (a) homogenized (37°C) 0.0018 Perch (a) homogenized (37°C) 0.0126 Mussel (b) homogenized (23°C) 0.286 Mussel (b) 28% (NH,),SO, PPt (23°C) 4.56

Bull (d) naturally decapitated (20°C) 15.4 (motile flagella) Bull (c) homogenized, epididymal(2O"C) 5.53

ejaculated 29.7 (non-motile flagella)

*The sources for these figures were (a) Tibbs," (b) Applegate and Nelson," (c) Nelson,'* (d) Nelson."

quaternary ammonium compounds did not have ready access to sensitive sites within or below the plasma membrane. Dimethyl sulfoxide (DMSO) reportedly in- creases the rate of absorption of such compounds,'2 and Steinbacht3 noted that DMSO "activates Arbacia sperm."

EXPERIMENTAL PROCEDURES

The following experiments were designed to determine the sensitivity of sperm cells to substances that act on their acetylcholine metabolism, and to differentiate between the effects of those that have and those that do not have the quaternary am- monium configuration. At the same time, the nature of the cell's response to drug interactions might provide insights into the process of motility control.

Motility, as a characteristic feature of sperm activity, serves as a convenient index by which to quantitate the sperm cell's responses. The highly condensed DNA in the head of the mature spermatozoon confers a positively geotropic orientation on the free-swimming cell. In a mild gravitational field in the horizontal rotor of a clinical centrifuge, spermatozoa become aligned and the motile cells swim to the centrifugal pole, Sea urchin sperm cells that have been killed with 0.2% formal- dehyde sediment minimally, if at all, provided that the centrifugal force does not exceed 120 x g. The displacement of experimentally treated or untreated control cells was monitored in a simple colorimeter, and correction was made for any changes in the optical density of the suspension of killed cells.

The motility index (the percentage of the control swimming speed) may be ex- pressed as M = (AODJAOD,) x 100, where AOD, is the change in optical density of the treated, uniformly dispersed specimen less that of the treated specimen, after 4 min of centrifugation at 120 x g (corrected for any displacement of the formal- dehyde-killed cells), and AOD, is the corrected change in optical density of the con- trol (untreated) sperm cells before and after 4 min of centrifugation at 120 x g. All experiments were performed at 22.5-23.5"C.I4

Sea urchins (Arbacia punctulata) were induced to spawn by the injection of 0.53 M KCl (which is isotonic with seawater). The sperm were diluted with filtered seawater just before the tests. Five ml of the diluted sperm suspensions were added to 10 ml round cuvettes that contained 0.5 ml of the reagents to be tested, and the contents were mixed by inversion of the cuvettes. The initial optical densities were recorded, and after the cuvettes had been centrifuged for 4 min, the optical densities

Nelson: T h e Study of Sperm Motility Contro l 299

were again recorded. In the 4 min of centrifugation the swimming speed of actively motile control cells averaged 280 pm/sec.

RESULTS AND DISCUSSION

DMSO, in concentrations of 50 mmol/l seawater and less, did not affect the swimming speed of the sea urchin sperm, but 100 mM DMSO reduced their speed by 40% and 1 M DMSO caused virtually complete cessation of progressive move- ment. Eserine, on the other hand, elicited a biphasic, dose-dependent response. At the low concentration of 1 pM, eserine increased the swimming rate by 40%, while the high concentration of 1 mM depressed the swimming rate by an equivalent amount. Neostigmine and acetylcholine also acted in a biphasic manner, but the extent of the sperm cell response was much diminished (to between 5 and 20% of the control rate). In the presence of 10-20 mM DMSO, however, neostigmine at 1 mM/I filtered seawater depressed the swimming speed by 6095, while a t 100 pM/I, neostigmine increased the speed t o 20% above control. Acetylcholine, a t a concen- tration of 10 mM/l in filtered seawater that contained DMSO, increased sperm motility to 8% above the control rate, and at 10 pM/1 it raised the speed to 70% above the control rate.

Thus far, it seems that the stimulatory component involved in the biphasic response to eserine may indeed depend on a transitory accumulation of acetylcholine. One of the criteria used in classifying an esterase as an acetylcholinesterase is the substrate optimum effect. When acetylcholine exceeds the optimum concentration, the enzyme activity is depressed below its maximum. This is graphically illustrated in FIGURE 1. It is also evident from FIGURE 1 that “excessive” inhibition of the acetylcholinesterase (for example, by eserine or neo- stigmine) may further depress motility. DMSO, in facilitating the penetration of the quaternary ammonium compounds neostigmine and acetylcholine, accentuates their actions at the level of acetylcholine hydrolysis.

Sperm motility and its regulation appears to depend on a critical concentration (as yet undetermined) of acetylcholine, or else on the acetylcholine: cholinesterase ratio. A substance that blocks acetylcholine synthesis and thereby shifts the equilib- rium in the direction of substrate deprivation produces a similarly drastic inhibition of sperm motility. Hemicholinium interferes with the transport of choline, and so ul- timately limits the synthesis of acetylcholine. Hemicholinium at a concentration of 10 mM/l filtered seawater, causes a reduction of more than 50% in sperm motility, but the same concentration in filtered seawater that contains DMSO almost com- pletely blocks swimming. As with neostigmine, lower concentrations of hemi- cholinium increase the swimming rate to about 20% above that of untreated con- trol sperm.

A central element of the physiological action of acetylcholine is its depolariza- tion of the cholinergic receptor. The spermatozoan receptor molecule or site remains to be identified. However, indirect evidence that a receptor does exist in the sperm cell (which we obtained by using agents known to act a t or on the receptor) is encouraging. Of these agents (FIGURE 2), the competitive receptor blockers d-tu- bocurarine and atropine were tested, as were the receptor depolarizers suc- cinylcholine and decamethonium. The effects of curare (d-tubocurarine) may be overridden in neuroeffector systems by an increase in the concentration of acetylcholine relative to the amount of curare. In the spermatozoon, under the assay conditions, the sequence of addition of the acetylcholine and the curare appeared to determine the outcome of the drug interaction. First curare, added without DMSO

300

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2 0 0

100

NEO!

U I-

. --

Annals New York Academy of Sciences

R / / ' '. / \

+ O M S 0 / A C E T Y L C H O L I N E /

\

/ \ ' \

/ i T l G M I N E

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CNE \ \ \

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\ \

\ I

-7 -5 -3

L O G c FIGURE I . Acetylcholinesterase inhibitors. The abscissa shows the log molar concentration

of agents in seawater suspensions of Arbacia sperm; the ordinate shows the sperm swimming speed in the first 4 min centrifugation interval; the control rate (the horizontal dashed line) was 280 pmlsec. +---+ = Eserine (physostigmine); x - - - x = neostigmine plus DMSO; u- - ~ D = acetylcholine + DMSO. The inset shows rates with no DMSO: 0- ~ - 0 = neo- stigmine; 0----0 = acetylcholine.

or acetylcholine, elicited a slight biphasic dose-dependent response, which ranged from 20% below to about 20% above the control swimming rate. But curare in the presence of DMSO, over the limited range tested, depressed the motility a little fur- ther; acetylcholine then added to the curare-DMSO mixture reduced motility dras- tically, t o only 25% of the control rate. On the other hand, when added to sperm suspensions previously exposed to acetylcholine and DMSO, curare increased the acceleratory effect noted above still further (FIGURE 3). It may be worthy of note here that d-tubocurarine, once regarded as a bisquaternary compound, is now thought to contain both a monoquaternary amine and a tertiary nitrogen. One may speculate that while the tertiary nitrogen may tend to permit the molecule a readier passage through the cell membrane than would be predicted because of the qua- ternary amine, the addition of DMSO probably facilitates the penetrability of the

Nelson: The Study of Sperm Motility Control 30 1

latter. What could not be anticipated was that acetylcholine and curare would interact synergistically rather than in an antagonistic fashion, or that the direction of the effect on motility would depend on the sequence of pretreatment of the sperm with DMSO or acetylcholine. That is, acetylcholine accentuates the depressant effect of curare, while curare potentiates the stimulatory effect of acetylcholine. Ad- ditional investigation is required to sort out the details of these atypical interac- t i o n ~ . ' ~

Atropine, the antimuscarinic cholinoceptive blocker, evidently penetrates the sperm cell membrane readily and exerts a considerably biphasic, dose-dependent effect. The stimulatory phase persists down to the nanomolar level, where it causes a 50% increase in motility, but millimolar atropine causes a 75% decrease. The extent of the sperm cell's sensitivity to atropine suggests that a muscarinic type of receptor is involved in the control of Arbacia sperm motility, but the evidence to substantiate this conclusion must be considered preliminary (FIGURE 2).

Among the agents that act a t the receptor level, only succinylcholine had no con- sistent effect (except in the presence of DMSO). Succinylcholine possesses a diqua- ternary structure, which would account for its lack of access to intracellular or in-

5 0 0

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SUCC I~YLCHOLINE

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FIGURE 2. Receptor blockers and depolarizers. The abscissa shows the log molar concen- tration of agents in seawater suspensions of Arbacia sperm; the ordinate shows the sperm swimming speed. v- - - v = d-Tubocurarine in filtered seawater (FSW); A---A = atropine in FSW; o---o = decamethonium in FSW; A--J- = succinylcholine in FSW and DMSO.

302 Annals New York Academy of Sciences

Ac t i v ~ C U O L I N I

\ b \

\

\ + DMSO

a

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l o o L A c nl

C U R A R E

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FIGURE 3. Drug interactions: acetylcholine and curare. The abscissa shows the log molar concentration acetylcholine (left) and of curare (right); the ordinate shows the sperm swim- ming speed. A---A = Acetylcholine in FSW; 0----0 = sperm pretreated with 20 mM DMSO + 100 p M curare, then acetylcholine added; o---n = acetylcholine plus 20 m M DMSO; A----A = curare in FSW; t - - 0 = curare plus 20 mM DMSO in FSW; m-m = sperm pretreated with 20 mM DMSO + 100 fiM acetylcholine, then curare added.

traplasmalemmar sites, but DMSO reveals succinylcholine’s remarkable potency. Even in nanomolar concentrations, succinylcholine plus DMSO raises the motility by about 25% over the control level, while micromolar concentrations increase the swimming rate to more than 75% over the control level. At the opposite extreme, 5 mM succinylcholine depresses motility by nearly 20% below the control level.

However, millimolar acetylcholine introduced into sperm suspensions pretreated with DMSO and 5 mM succinylcholine lowers motility to about half the control rate (FIGURE 4); while in 100 nM acetylcholine, the succinylcholine-treated sperm swim just over 50% faster than do the control sperm cells. These interactional effects are striking, in view of the fact that the 80% increase in motility caused by millimolar acetylcholine (with DMSO) was converted to a 50% decrease by the inclusion of 5 mM succinylcholine. Under these conditions, the presence of 5 mM suc-

Nelson: The Study of Sperm Motility Control 303

cinylcholine/l shifts the optimum from millimolar acetylcholine (which otherwise causes an increase of 80%) to lo-' M acetylcholine (which here causes an increase of 50%). Conversely, the optimum for succinylcholine with DMSO occurred at 0.5 pM, while in DMSO and 1 .O pM succinylcholine, 0.5 pM acetylcholine lowered the optimum response from 75% to 40% above the control swimming rate.

Decamethonium seems to depolarize the receptor irreversibly in neuroeffector systems. Although decamethonium is a bisquaternary ammonium compound (as is succinylcholine), its pronounced inhibitory effect on the motility of sea urchin sperm did not require any assistance from DMSO. Slight enhancement of motility is caused by 10 r M decamethonium, while 1 mM decamethonium brings about 75% depression; this pattern bears a formal resemblance to the transitory increase in ex- citability that precedes the marked inhibition induced by decamethonium at neuro- muscular junctions (FIGURE 2).

One may conclude from these observations that environmentally induced altera- tions in acetylcholine metabolism are causally involved in the regulation of the motility of sea urchin spermatozoa. In another phase of the work, we have found that bull sperm membrane potentials range from -10 m V in very active cells down to -2 mV in quiescent cells.16 These potentials, which depend on the partition of K, Ca, and CI ions across the cell membrane, may be modified by the addition of

5 O(

V

u* 3 0 1

10

SUCCINYLCHOLINE

\/*\ /

x \ X \

'X

FIGURE 4. Drug interactions: acetylcholine and succinylcholine. The abscissa shows the log molar concentration of acetylcholine or succinylcholine; the ordinate shows the sperm swim- ming speed. 0- - -0 = Acetylcholine + DMSO; +---. = succinylcholine t DMSO; x---x = acetylcholine in sperm suspensions pretreated with DMSO and 5 mM Suc- cinylcholine.

304 Annals New Y o r k Academy of Sciences

A 7 R 0 PINE I I

DEWMETHONIUM

I I

FIGURE 5. A motility index (in summary) of the effects of agents assayed on Arbuciu sperm. The abscissa shows the percentage of control motility. The range from left to right goes from the highest effective concentration to the lowest optimum concentration.

eserine and acetylcholine to the suspending medium. Eserine also causes marked de- viation from the normal swimming pattern of mature bull sperm cells. Factors that affect the membrane potentials also cause changes in the frequency, amplitude, and length of the flagellar wave. It appears that the acetylcholine-mediated control of sperm motility is not limited to marine organisms. Thus the sperm cell warrants the following description: an excitable system that mediates its behavioral adjustments to environmental alterations through acetylcholine control processes (FIGURE 5).

A cautionary note must be appended here. While neurobiological terminology and methodology can provide useful insights into sperm motility control mechanisms, it must be borne in mind that a flagellum is neither a nerve nor a muscle, and so a sperm cell must serve as its own model.

REFERENCES

I . NELSON, L. 1974. Spermatozoan Motility. Handbook of Physiology, Vol. 7. American

2. YANAGIMACHI, R. 1966. J. Reprod. Fertility 11: 359. 3 . MILLER, R. L. 1966. J. Exp. Zool. 162: 23. 4. GADDUM, P. 1968. Anat. Record 161:471. 5. MCGRADY, A. V. & L. NELSON 1973. Exp. Cell. Res. 76: 349.

Physiological Society.

Nelson: The Study of Sperm Motility Control 305

6. BROKAW, C. J., S. F. GOLDSTEIN & R. L. MILLER 1970. In Comparative Spermatology.

7. DRYL, S. 1970. Acta Protozool. 7: 325. 8. ZELLER, E. A. & C. A. JOEL. 1941. Helv. Chem. Acta 24: 968. 9. SEKINE,T. 1951. J . Biochem. (Tokyo)38: 171.

10. APPLEGATE, A. & L. NELSON 1962. Biol. Bull. 123:475. 11. NELSON, L. 1972. Biol. Reprod. 6: 319. 12. STOUGHTON, R. B. & W. FRITSCH 1964. Arch. Dermatol. 90: 512. 13. STEINBACH, H. B. 1966. Biol. Bull. 131: 166. 14. NELSON, L. 1972. Exp. Cell. Res. 74: 269. 15. NELSON, L. 1973. Nature242: 401. 16. MCGRADY, A. V. & L. NELSON 1972. Exp. Cell. Res. 73: 192. 17. TIBBS, J. 1960. Biochim. Biophys. Acta 41: 115. 18. NELSON, L. 1964. J. Reprod. Fertility 7: 65. 19. NELSON, L. 1966. J. Cell. Physiol. 68: 113.

B. Baccetti, Ed.: 475. Accademia Nazionale de Lincei. Rome, Italy.