MOLECULAR REPRODUCTION AND DEVELOPMENT 2855-61 (1991)

The Binding Characteristics of Cholinergic Sites in Rabbit Spermatozoa R.J. YOUNG AND J.C. LAING Toxicology Division, Chemical Research, Development, and Engineering Center, Aberdeen Proving Ground, Maryland

ABSTRACT Binding of neurotrophic ligands to rabbit spermatozoa was studied. Nicotinic cholinergic antagonists, [3H]a-bungarotoxin and [3H]dihydro-P-erythroidine (DE), bound with high affinity to different sites in the tails of rabbit spermatozoa with the former binding to 10,207 sites/cell and the latter to 562 sites/cell. a-Bungarotoxin and DE sites resemble nicotinic sites in brain in binding affinity and specificity. [3H]Quinuclidinyl benzilate (QNB), a muscarinic cholinergic antagonist, also bound with high affinity to a single class of sites located in the heads and tails of rabbit spermatozoa. The binding characteristics of the sperm muscarinic site are similar to muscarinic sites in both innervated and noninnervated cells. Rabbit spermatozoa incubated for 16-1 8 h in a medium which supported motility for an extended period possessed fewer binding sites than nonincubated spermatozoa for [3H]a-bungarotoxinand [3H]QNBand the K, for the latter ligand was also lower. Ligands specific for the kappa and delta opiate receptors showed no affinity for rabbit spermatozoa. Key Words: Binding sites, Ligands, Nicotinic, Muscorinic, Opiate

INTRODUCTION The ability of many neuroactive chemicals to alter spermatozoan behavior has raised the possibility that some spermatozoan function is under neurohormonal regulation. Several avenues of investigations support the concept that spermatozoan motility is under the control of a cholinergic system that mediates intracellular C a + levels (Nelson, 1978; Nelson et al., 1980). First, components of the acetylcholine-choline acetyltranferase-acetylcholinesterase system have been identified in mammalian and marine spermatozoa (see references in Stewart and Forrester, 1978a; Goodman and Harbison 1981), and, in mammalian spermatozoa, are present predominantly in the tail (Nelson, 1966; Chakraborty and Nelson 1976; Stewart and Forrester 1978a). Second, compounds that affect acetylcholine metabolism, or are cholinergic agonists or antagonists, change the motility characteristics of spermatozoa (Sanyal and Khanna, 1971; McGrady and Nelson, 1976; Nelson, 1978; Nelson 1976; Rama Sastry et al., 1981). +

0 1991 WILEY-LISS, INC.

Third, cholinergic agents alter Ca+ ' distribution in spermatozoa (Stewart and Forrester, 1979; Nelson et al., 1982). Recent studies that show mouse in vitro fertilization is inhibited by cholinergic agents also suggest that the cholinergic system may play a role in fertilization (Florman and Storey, 1982; Chou and Cook, 1984; Chou et al., 1989). A corollary of neurochemical participation in spermatozoan behavior is the demonstration of specific neurochemical binding sites in spermatozoa. Ligand binding studies have demonstrated the presence of a nicotinic binding site in the tails of ram and bull spermatozoa (Stewart and Forrester, 197813; Rama Sastry et al., 1979), and a muscarinic binding site in mouse spermatozoa (Florman and Storey, 1982), and that the two cholinergic sites in these spermatozoa have properties that differ from the cholinergic receptors in innervated cells. It is unknown, however, if the two classes of cholinergic binding sites are present in spermatozoa of a single species or if spermatozoa possess other types of neurotransmitter binding sites. This question has been addressed by studying the binding of neurotrophic ligands to rabbit spermatozoa. The experiments demonstrate the existence of both nicotinic and muscarinic binding sites in rabbit spermatozoa. The results of these experiments and the characterization of the binding specificity of the sites are reported in this communication.

MATERIALS AND METHODS Spermatozoa Purification. Rabbit spermatozoa were collected from New Zealand white males with a n artificial vagina. Ejaculates from 6-10 males were combined, centrifuged at 7,OOOg for 5 min at 5°C and pellet resuspended in ST buffer (50 mM Tris-HC1,150 mM NaC1, pH 7.4). Prostatic vesicular bodies, cellular debris, seminal plasma contaminants such as proteases, and less dense

Received March 19, 1990; accepted May 30, 1990. Address reprint requests to R.J. Young, Toxicology Division, Chemical Research, Development, and Engineering Center, Aberdeeen Proving Ground, MD, 21010-5423.

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R.J. YOUNG AND J.C. LAING

nonsperm cellular material were removed by a single centrifugation (116,OOOg, 1 h, 5°C) of the sperm suspension through a discontinuous (1.8, 2.0, and 2.2 M sucrose in ST buffer) sucrose density gradient (Young, 1979; Young and Cooper, 1983). The pellet was resuspended in ST buffer and sperm heads detached from tails by sonication a t 5°C with a sonicator (Heat Systems Ultrasonics, Inc., Farmingdale, NY) equipped with a microtip and set a t 80% pulse and 20-25% maximum output. The sonicate was centrifuged at 30,OOOg for 10 min at 5°C and washed twice with the appropriate buffer for the binding experiment. Sperm concentration was determined by duplicate dilutions of the sperm suspension and counting five drops of each dilution in a Makler counter (Zygotech Systems, Inc., Springfield, MA). Head-tail separation. Detached sperm heads were separated from tails by centrifugation through a discontinuous sucrose density gradient a s previously described (Young and Cooper, 1983). Sucrose was removed from separated heads and tails by two washes (30,OOOg, 10 min, 5°C) with the buffer to be used for the binding experiment and the head and tail concentrations determined by triplicate dilutions of the suspensions and counting 5 drops of each dilution in a Makler counter. Phenylmethylsulphonyl fluoride (1mM) was present in the ST buffer when spermatozoa were used for binding experiments with a-bungarotoxin.

Ligand Binding General. Saturation binding was carried out by incubating suspensions of spermatozoa (0.5-1 ml) with either increasing amounts of ['Hllabeled ligand (hot method) or a constant amount of labeled ligand and increasing amounts of unlabeled ligand (cold method). After incubation with gentle shaking, spermatozoa were filtered through 2.5 cm GF/C or GF/B glass fiber filters (Whatman) that had been soaked in either 1% bovine serum albumin (BSA) or 1%polyethyleneimine and the filter washed three times with cold buffer. The filters were placed in scintillation vials, covered with 1 ml of 100 mM NaOH and incubated overnight at room temperature. After neutralization with glacial acetic acid, aquasol-2 (New England Nuclear, Boston, MA) was added and the filters shaken for a minimum of 4-6 h before counting in a Beckman LS 9800 spectrophotometer. Binding a t each concentration point was determined in triplicate. a-Bungarotoxin.The procedure followed was essentially that described by Colquhoun and Rang (1976) and Ben-Barak and Dudai (1979). Suspensions of spermatozoa (1-3 x lo7 cells/ml) and 1'Hla-bungarotoxin (0.04-4 nM) were incubated in 50 mM P043-, 50 mM EDTA, pH 7.4, 0.01% BSA, for 1 h a t room temperature. The reaction was terminated by filtration. When the cold method was used, unlabeled a-bungarotoxin (range 0.3-300 pmol) was added to a 0.5 ml suspension

of spermatozoa (1-3 x 107ml)and ['Hlcw-bungarotoxin (0.5 nM or 2 nM). Binding was terminated by centrifugation at 1 3 , O O O g for 10 min a t 5°C. The pellet was washed twice with buffer, dissolved in 100 mM NaOH, and the radioactivity measured a s described above. Nonspecific binding for both methods was determined in triplicate by measuring binding in the presence of nicotine (0.01-0.1 mM), or after prior incubation with unlabeled a-bungarotoxin. Dihydro-P-erythroidine. The procedure of Williams and Robinson (1984) was used to measure binding by the hot method. Suspensions of spermatozoa (16 x 106/ml) and ['HIDE (range 0.07-0.70 nM) in 50 mM Tris-HC1, pH 7.4, were incubated for 5 min a t 05°C and the reaction terminated by filtration. Nonspecific binding was assessed in the presence of 1,000-fold excess of nicotine. Quinuclidinyl benzilate. Saturation binding was measured by the hot method. Mixtures of spermatozoa (1-3 x 107/ml) and ['HIQNB (concentration range 0.6-330 nM) in 50 mM P 0 2 - , 50 mM EDTA, pH 7.4, was incubated a t 37°C for 15 min and then filtered (Yamamura and Synder, 1974a,b). Nonspecific binding was determined in the presence of 1,000-fold excess atropine. Opiates. Tubes containing increasing amounts of spermatozoa (range 2 x 106-2 x 107/ml)in 50 mM Tris-HC1, pH 7.4, were incubated with 13H]ethylketocyclazocine (1 nm) a t 37°C for 30 min, or a t room temperature for 60 min with ['Hlenkephalin (2-D-ala5-D-leu) (1 nM), and then filtered. Binding was measured in triplicate. Nonspecific binding was assessed in the presence of excess carfentanil.

Ligand Displacement The specificity of binding of the ligands QNB and DE, which bound reversibly, was assessed by incubating [3H1QNB (3 nM), and ['HIDE (6 nM), with spermatozoa, 2-5 x 107/ml,and 8 x 106/ml,respectively, and increasing concentrations of displacing ligands. Filtration was used to terminate binding. For the irreversible ligand a-bungarotoxin, spermatozoa (2-5 x lo7/ ml) and increasing concentrations of displacers were preincubated for 1h before incubation with ['Hla-bungarotoxin (0.5-1 nM) for 1 h a t room temperature. Binding was terminated by filtration or centrifugation. Data Treatment The programs EBDA and LIGAND (BCTIC computer code collection, Vanderbilt Medical Center, TN) were used to analyze the saturation and displacement binding data. LIGAND (Munson and Rodbard, 19801, a nonlinear curve fitting program was used to refine the preliminary analysis of the saturation binding data by EBDA (McPherson, 1983) and compute the final best fit values for the dissociation constant, K,, and the maximum number of binding sites B,,,. The affinity constant, K,, of the competitor was calculated by EBDA

BINDING SITES IN SPERMATOZOA

57

from the displacement binding data by the equation of Cheng and Prusoff (1973).

Reagents [2,7-3H]Dihydro-P-erythroidine(1.1 TBqimmol), L[Benzilic-4,4'-3H]Quinuclidinyl benzilate (1.1 TBqi mmol), [tyro~yl-3,5-~H(N)]-Tyr-D-Ala-Gly-Phe-D-Leu (1.85 TBqimmol), and (-)-[9-3H(N)1-ethylketocyclazocine (1.11 TBqimmol) were products of New England Nuclear (Boston, MA). N-[pr~pionyl-~HlaBungarotoxin (3.44 TBqimmol) was obtained from Amersham Corp., Arlington Heights, IL. The following 0 were obtained from Sigma Chemical Co., St. Louis, MO: 0 5 10 15 20 25 30 (-)nicotine di-( + )tartrate, atropine sulfate, a-bungaBound (pM) rotoxin, d-tubocurarine chloride, hexamethonium bromide, carbamylcholine chloride, (-)scopolamine hyFig. 1. Scatchard analysis of the binding of 13Hla-bungarotoxinto drochloride, DL-propranolol hydrochloride, and ( + ,- )3- rabbit spermatozoa. Binding was carried out at room temperature for quinuclidinol. Arecoline hydrochloride and pargyline 1 h in 5 mM PO," , 10 nM EDTA, pH 7.4, 0.01%BSA with sonicated spermatozoa (1 x 107/ml) and increasing amounts of I3H1a-bungahydrochloride were purchased from Research Biochem- rotoxin. Measurement of each point was carried out in triplicate. ical, Inc., Wayland, MA. range, and again a one-site model produced the best fit. RESULTS This result was obtained regardless of whether glass or [3H]a-Bungarotoxin and [3H]DE, specific antago- plastic tubes and centrifugation or filtration were used nists of nicotinic cholinergic receptors, and [3H]QNB, a to terminate the reaction. When data from four experspecific antagonist of muscarinic cholinergic receptors iments were combined and analyzed by LIGAND for a were used to assess each cholinergic class in rabbit one site model, the binding constants shown in Table 1 spermatozoa. Each ligand bound to intact spermatozoa, were obtained. but to aid in the rapid attainment of equilibrium in This result is in agreement with the report that abinding, spermatozoa were briefly sonicated. Binding bungarotoxin binds to bull spermatozoa (Rama Sastry to sonicated spermatozoa was up to three times higher. et al., 1979), but is at odds with the observation that Since very little ligand bound to the 30,OOOg sonicate ram spermatozoa bound decamethonium, a nicotinic supernatant, or to the washings after sonication, all ligand, but not ['2511a-neurotoxin obtained from Naja binding experiments were carried out with sonicated naja venom (Stewart and Forrester, 1978b). Thus it spermatozoa. A series of experiments were carried out was of interest to determine if rabbit spermatozoa also with all the ligands to establish optimal conditions for showed a n affinity for the nicotinic antagonist [3H]DE, saturation binding. Binding was studied as a function which, unlike a-bungarotoxin, binds reversibly with its of time and sperm concentration to determine the time receptor. Saturation binding of [3H]DE to rabbit sperrequired to reach equilibrium, and the sperm concen- matozoa with L3H1DE by the hot method suggested that tration range over which binding was linear. These the ligand bound at a single site. Analysis by LIGAND studies showed that equilibrium was achieved in less of data from two experiments produced a one site model than 10 min at 0 4 ° C with DE, 1 h a t room tempera- as the best fit with the binding constants K, 2.6 nM ture with a-bungarotoxin, and 15 min a t 37°C with and 562 sitesicell (Table 1).Rabbit spermatozoa also QNB. Binding was linear with a-bungarotoxin, and possessed a single saturable site for L3H1QNB, a musQNB a t sperm concentrations up to 3 x lo7 cellsiml, carinic antagonist. Analysis by LIGAND of data from but was linear with DE only up to 1-2 x lo6 cellsiml. two experiments for a one site model yielded the bindSignificant amounts of the ligands bound to the glass ing constants shown in Table 1. These results show fiber filters and binding was particularly high in the that both classes of cholinergic receptors are present in case of [3Hla-bungarotoxin. This nonspecific binding rabbit spermatozoa. was reduced by 6 5 4 5 % when the filter was presoaked [3H]Ethylketocyclazocine and [3H]enkephalin-(2-Din 1%BSA or 1% polyethyleneimine (Colquhoun and ala-5-D-leu), agonist of the kappa and delta class of Rang, 1976). opiate receptors, respectively, did not bind to the 30,OOOg pellet from sonicated rabbit spermatozoa sugLigand Binding gesting that opiate binding sites are absent in these The binding of [3H]a-bungarotoxin to sonicated rab- spermatozoa. bit spermatozoa was studied by the hot method (Fig. 1). Binding to Sperm Heads and TaiIs Computer analysis of the date by LIGAND produced a The distribution of sites on rabbit spermatozoa with one site model a s the best fit. Binding was also studied by the cold method to expand the ligand concentration a n affinity for a-bungarotoxin, DE, and QNB was de-

R.J. YOUNG AND J.C. LAING

58

TABLE 1. Ligand Binding Constants for Rabbit SDermatozoa Ligand [3H1a-Bungarotoxin 13H]DE I'HIQNB

KD

nM 1.6 2 0.6 2.6 (2.3-3.0)" 5.7 (3.7-7.6)"

BMW Sitesicell 10,207 5 3,487 562 (252-871)" 4,630 (3,972-5,286)"

=Range,

termined by measuring the binding of the ligands to separated heads and tails. [3H]a-Bungarotoxin and [3H]DE bound predominantly to tails (Table 21, in agreement with Stewart and Forrester (1978b) and Rama Sastry et al. (19791, who reported that nicotinic binding sites are present in sperm tails. L3H]&NB bound to heads and tails, suggesting that muscarinic binding sites are distributed over the sperm head and tail.

TABLE 2. Ligand Binding to Rabbit Sperm Heads and Tails* Ligand I3Hla-Bungarotoxin 1 'HIDE 13HlQNB

Head (DPMIIO') 114.2 34.4 77.3

Tails (DPMilO') 235.7 284.5 63.2

"'Binding was carried out by incubation of separated sperm heads or tails with radioligand. Measurements were carried out in triplicate. Nonspecific binding was assessed in the presence of excess nicotine (r3Hla-bungarotoxin and L3HlDE) or atropine VHIQNB).

tion. The acrosome, visible as a dark crescent under phase-contrast microscopy (Young, 19791, was absent from nonmotile spermatozoa. Incubated spermatozoa showed no change in binding affinity for C3Ha-bungarotoxin, a one site fit of combined data from two experiments gave the K, as 1.5 0.3 nM, but a decrease in Binding Specificity the number of binding sites to 4,731 3,288 sitesicell The specificity of ligand binding to cholinergic sites was found. There was also little change in the ability of was studied by competitive displacement with ligands tubocurarine to displace [3Hla-bungarotoxin as the KI of known specificity. Nicotine, the nicotinic antago- after incubation was 0.41 pM. However, carbamyl nists tubocurarine and hexamethonium, a cholinergic choline (K, 0.43 p M ) appeared to be more effective in agonist, carbamyl choline, a s well as scopolamine, a displacing [3Hla-bungarotoxin from incubated spermamuscarinic antagonist, all displaced [3H]DE (Table 3). tozoa. The K,, 1.03 nM, for L3H]QNB binding after inIt is interesting to note that scopolamine was appar- cubation was lower and there were also fewer binding ently more effective in displacing L3H1De than car- sites. 663 2 580 sitesicell. bamyl choline. Nicotine, tubocurarine, and carbamyl DISCUSSION choline also displaced [3Hla-bungarotoxin, but with The present experiments demonstrate that the nicomuch higher K, values. As expected, unlabeled a-bungarotoxin with a K, of 0.00038 pM was more potent tinic antagonists a-bungarotoxin and DE bind to the than the other three compounds in displacing [3Hla- tails of rabbit spermatozoa. The binding affinity and bungarotoxin from the sperm nicotinic site (Table 3). rank order of displacement of a-bungarotoxin binding Hexamethonium and scopolamine were ineffective in by competing ligands are in good agreement with those displacing [3H]a-bungarotoxin. Atropine, another mus- found for binding to the electric tissues of E . electricus carinic antagonist, inhibited binding of the two nico- and T. marmorata iWeber and Changeux, 1974a,b; tinic ligands somewhat a t a concentration of 1-10 mM. Franklin and Potter, 1972), mammalian skeletal musScopolamine and atropine were much more potent cle (Brockes and Hall, 1975; Colquhoun and Rang, 1976; than the cholinergic agonists arecoline and carbamyl Patrick et al., 1977; Vogel et al., 19721,and mammalian choline in displacing ['HIQNB. Indeed 3-quinuclidinol, brain (Green et al., 1973; Eterovic and Bennett, 1974; a QNB analogue, propranolol, a P-adrenergic ligand, Schmidt, 1977; Marks and Collins, 1982; Nukina et al., and hexamethonium were more effective than the ag- 1985; Marks et al., 1986). Binding of a-bungarotoxin onists in displacing [3H1QNB. Nicotine inhibited occurred with high affinity to a single class of sites and [3H]QNB binding a t 1 mM. Physostigmine, a n inhibi- was inhibited by the nicotinic ligands nicotine, tubotor of acetylcholine esterase, had no effect on the bind- curarine, and carbamyl choline, but not by muscarinic ligands. Hexamethonium, a n antagonist of nicotinic reing of the three ligands to rabbit spermatozoa. ceptors a t ganglia and muscle, had little effect even at Ligand Binding After Incubation a concentration of 1 mM on a-bungarotoxin binding to The possibility that the binding properties of the nic- rabbit spermatozoa. This concentration of the ligand otinic and muscarinic binding sites change during the was required to inhibit a-bungarotoxin binding to brain lifetime of the sperm was investigated by measuring (Eterovic and Bennett, 1974; Schmidt, 1977; Marks and binding after incubation in a medium which main- Collins, 1982) while much lower concentrations were tained motility of rabbit spermatozoa for a n extended sufficient to inhibit binding to muscle or electric tissue period (Brackett and Oliphant, 1975). The percentage (Franklin and Potter, 1972; Weber and Changeux, of motile spermatozoa decreased from 80 to 90% a t the 1974b; Colquhoun and Rang, 1976; Patrick et al., 1977). start of incubation to 10-25% after 16-18 h incuba- In this respect a t least, the sperm toxin binding site

*

*

BINDING SITES IN SPERMATOZOA

59

TABLE 3. Effect of Competitors on Ligand Binding to Rabbit Spermatozoa* Receptor class Nicotinic

Ligand [3H]a-Bungarotoxinb

13H1DEc

Mu s carin ic

[3HIQNBd

Competitor Unlabeled Bungarotoxin Nicotine Tubocurarine Carbamyl choline Hexamethonium Nicotine Tubocurarine Scopolamine Carbamyl choline Scopolamine Atropine Propranolol Quinuclidinol Hexamethonium Arecoline Carbamyl choline

K, (FMY 0.00038 0.089 ? 0.052 0.58 ? 0.39 1.44 ? 0.29 0.015 0.068 0.081 3.53 ? 2.1 31.2 0.11 ? 0.03 0.58 ? 0.29 50.9 ? 3.5 54.55 26.7 221.3 k 84 1,450 t 669 4,696 ? 1,561

*

"Inhibition of [3H]QNB and 13H1DE was carried out by incubation of sonicated spermatozoa and ligand with increasing amounts of displacers. In the case of [3Hla-bungarotoxin, sonicated spermatozoa, and displacers at different concentrations were preincubated for 1 h, and then incubated for a further 1 h after addition of [3H]a-bungarotoxin.Experimental points were measured in triplicate, and experiments were carried out up to three times. "Average 2 S.E.M. bAtropine inhibited a t > 10 mM; scopolamine, hexamethonium, and physostigmine had no effect. 'Atropine had no effect a t 1 mM. dPhysostigmine had no effect; nicotine inhibited a t 1 mM.

resembles the brain binding site more closely than the muscle or electric tissue toxin binding sites. DE also bound with high affinity to a single class of sites in rabbit spermatozoa. Binding was sensitive to scopolamine and the site thus appears to possess some muscarinic-like character, a property shared by the DE site in rat brain where binding was also inhibited by muscarinic ligands (Williams and Robinson, 1984). The observation t h a t the number of [3H]DE binding sites in rabbit spermatozoa is only 5% of the number of ['Hlabungarotoxin binding sites is surprising as the two ligands are antagonists a t nicotinic sites. The distribution of DE and a-bungarotoxin binding sites are different in rat brain (Williams and Robinson, 1984; Clarke, 1987; Wonnacott, 1987a; see also Loring and Zigmond, 1988). This situation may exist in rabbit spermatozoa and would account for the difference in the number of ['HIDE and [3H]a-bungarotoxin binding sites. The dissociation constants for the binding of the muscarinic antagonist L3H]QNBto noninnervated cells such as rabbit and mouse spermatozoa (Florman and Storey, 19821, erythrocytes (Tang, 1986) and lymphocytes (Gordon et al., 1978) are similar but are higher than the dissociation constants for binding to a variety of innervated tissues and glands (brain: Yamamura and Snyder, 1974a; Gilbert et al., 1979; Marks and Collins, 1982; lung: Joad and Casale, 1988; ileal smooth muscle: Yamamura and Snyder, 197413; pineal: Taylor et al., 1980; nasal: Klaassen et al., 1985; pituitary: Mukherjee et al.,

1980). However, the rank orders of inhibition of ['HIQNB binding to rabbit spermatozoa and innervated tissues and glands by the muscarinic antagonists, scopolamine and atropine, and the agonists, arecoline and carbamyl choline, were similar. The nicotinic antagonist hexamethonium inhibited binding of ['HIQNB to rabbit spermatozoa. Hexamethonium also inhibited 13H]QNBbinding in heart and brain (Gies et al., 1987) and may also have the same effect in erythrocytes (Tang, 1986).Thus, the properties of the muscarinic site in rabbit spermatozoa are similar to the properties of the muscarinic receptor in both innervated and noninnervated cells. The inhibition of [3H]QNB binding to rabbit spermatozoa by the P-adrenergic ligand, propanolol, was a n unexpected result, but is not inconsistent with the recent demonstration of significant amino acid and structural homology between the P-adrenergic and muscarinic receptors (Hall, 1987; Wang et al., 1988; Shorr e t al., 1986). Centrifugation of spermatozoa through a sucrose gradient dislodges the acrosome but leaves the plasma membrane essentially undamaged (Young and Cooper, 1983; Cooper and Young, unpublished results), and i t is likely that the cholinergic ligands bound to sites on the plasma membrane of intact spermatozoa. Detachment of the sperm head from the tail by brief sonication exposes sperm intracellular structures. Binding would occur to detached plasma membrane, to plasma membrane that remain attached to heads and tail pieces

60

R.J. YOUNG AND J.C. LAING

(Cooper and Young, unpublished results), to membrane sites that may have been hidden or inaccessible prior to detachment of the tail by sonication, and to the exposed intracellular sites. The additional binding sites would account for the increase in ligand binding observe with sonicated spermatozoa. Although [3H]QNB bound to separated heads and tails i t is possible that the ligand bound only to the head or tail of intact spermatozoa. The significance of the alteration in binding properties after incubation of spermatozoa for 18 h in unclear. The composition of the sperm surface macromolecules change throughout the life of the spermatozoa and this may in t u r n alter the binding properties of the binding sites. Ligand binding studies have now demonstrated the presence of cholinergic sites in spermatozoa of the ram, bull, mouse, and rabbit. There is, however, little understanding of the possible roles these sites play in sperm function. The ability of nicotinic cholinergic ligands, a t concentrations approaching the KDfor binding, to alter sperm motion characteristics (see Nelson e t al., 1980) show that neurotrophic ligands are able to modify spermatozoan function, presumably via cholinergic receptors. As enzymes of acetylcholine metabolism are present in spermatozoa, the opportunity for neurotrophic agents to activate sperm cholinergic receptors is present from the time of ejaculation to fertilization. Nicotinic cholinergic receptors in innervated tissues act as a ligand-gated channel regulating passage of monovalent ions, while muscarinic cholinergic receptors mediate several responses in innervated cells by activation of membrane-bound transducing proteins, the G-proteins. Recent studies have demonstrated that G-proteins are present in the membrane of spermatozoa from several species, and in bovine spermatozoa, a t least, several G-protein types are distributed into defined regions of the head and tail (Bentley et al., 1986; Kopf et al., 1986; Garty e t al., 1988). The G-proteins regulate several biochemical processes that result, depending on the receptor, in changes in inositol phospholipid metabolism, and in the intracellular levels of cyclic nucleotides, Ca’ +,and monovalent ions (Nathanson, 1987; J a r v and Bartfai, 1988). Cyclic nucleotides, Ca’ +,N a + , and K ’ cations are important for sperm motility, sperm capacitation, and the acrosome reaction (see Bavister, 1986; Fraser and Ahuja, 1988). If sperm G-proteins regulate effector systems in the same way as their counterparts in somatic cells, then the putative sperm receptors to which they are coupled and the activators of these receptors may control sperm motility and fertilization. However, it should be noted that both muscarinic agonists and antagonists inhibit mouse in vitro fertilization (Florman and Storey, 1982), a result inconsistent with the supposition that the mouse muscarinic binding site functions in the same manner a s muscarinic receptors in innervated tissue. Although a-bungarotoxin binds with high affinity to certain sites in brain, and to au-

tonomic ganglia, the antagonist does not block nicotinic transmission in the central nervous system, and the function of the binding site is unknown (Schmidt et al., 1980; Wonnacott, 1987b; Clarke, 1987). Thus equating the rabbit sperm nicotinic and muscarinic binding sites with the pharmacological nicotinic and musarinic receptors would be premature. Until further biochemical, pharmacological, and physiological characterization, the rabbit sperm binding sites should be regarded a s neurotrophic binding sites rather than neurotrophic receptors. Nevertheless the existence of the sites in a number of mammalian spermatozoa is supportive of the possibility of a role for neurotransmitters in the regulation of sperm function.

ACKNOWLEDGMENTS Comments on the manuscript by Dr. G.W. Cooper is gratefully acknowledged.

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