J. Cell Sci. 43, 93-102 (1980)Printed in Great Britain © Company of Biologist! Limited 1080

CHARACTERIZATION AND LOCALIZATION

OF ADENYLYL CYCLASE IN MEMBRANE

VESICLES AND INTACT BOAR AND HUMAN

SPERMATOZOA

R. N. PETERSON, L. RUSSELL, L. HOOK, D. BUNDMAN AND M.FREUND

School of Medicine and Department of Physiology, Southern Illinois University, Carbon-dale, Illinois 62901, U.S.A.

SUMMARY

The enzymic properties of adenylyl cyclase in purified membrane vesicles from, human andboar spermatozoa are described. Plasma membrane vesicles, which appear to be right-side-out,show a marked increase in activity in the presence of the detergent Triton X-100, manganousion and alkaline pH. Electron-microscope cytochemical assays indicated the presence of adenylylcyclase in boar and human sperm plasma membranes and also within the axoneme of intacthuman spermatozoa. The significance and precautions in the evaluation of the cytochemicaldata are discussed.

INTRODUCTION

Adenylyl cyclase is thought to play an important role in the control of motility inmammalian spermatozoa and it has also been proposed that the enzyme may beinvolved in capacitation (Toyada & Chang, 1976; Bracket, Jeitles & Oh, 1975;Morton & Albagli, 1973). With the development of efficient, reproducible methods forthe isolation of highly purified plasma membrane vesicles from boar and humanspermatozoa (Gillis et al. 1978) it became possible to study some of the properties ofthe membrane-bound enzyme in these species. Electron-microscopic evidence for thelocalization of this enzyme is also described and evaluated.

METHODS AND MATERIALS

The procedure for preparing plasma membrane vesicles from washed human and boarspermatozoa are described in detail elsewhere (Gillis et al. 1978). Briefly, sperm suspensions aredisrupted in a Parr Bomb (650 lb/in1 (455 x io3 kN m"1) for 10 min. The ruptured cells arewashed several times in a Tris-based buffer (ph 7-4) at low centrifugal fields (2000 g, 10 min,then 8000 g, 10 min) to remove intact cells and other large debris. The supernatant, containingplasma membrane vesicles, is pelleted (100000 g, 30 min, rotor SW 501 , Model L Beckmanultracentrifuge) and then layered on a 3-8tep discontinuous sucrose gradient (i-o M, 1-30 M,1-57 M sucrose). Plasma membranes are removed from the top of the I -OM sucrose interface aftercentrifugation (100000 g, 60 min). Vesicles are stored at —79 °C in Tris-sucrose-Mg (trishy-

Address all correspondence to: Dr. R. N. Peterson, School of Medicine, Southern IllinoisUniversity, Carbondale, Illinois 62901, U.S.A.

94 R- N. Peterson and others

droxymethylaminomethane chloride, 25 mM; sucrose 0-2 M; magnesium chloride 10 mM, pH7-4) until used. Another type of membrane vesicles which are fused plasma membrane-outeracrosomal membranes (Russell, Peterson & Freund, 1979) are obtained from spermatozoa inwhich the acrosome reaction is induced by the ionophore A23187 and calcium, ions (Gillis et al.1978). These vesicles are separated from whole sperm by vigorous vortexing and are recoveredin a discontinuous sucrose gradient at the 1-30 M/ I -57 M sucrose interface. Plasma membranevesicles are also isolated from these acrosome-reacted spermatozoa after centrifugation, at thei-o M/I-3 M sucrose interface. These vesicles do not contain the plasma membranes from theprincipal piece of the head. High concentrations of the protease inhibitor trasylol BE (500/ig/ml) are added throughout both procedures.

Adenylyl cyclase activity was determined by the method of White & Zenzer (1971), as modi-fied by Counis & Mongongu (1978), which utilizes (<z-32P) ATP as substrate and chromatographyof reaction products on neutral alumina. Reactions were carried out for 30 min at 30 °C in amedium (buffered at pH 7'0) containing Tris-HCl, 25 mM; theophylline, 10 mM; ATP, 3-2 mM(1 /*Ci); and MgCl, or MnCl2 at concentrations specified in the text. Recovery of ['HjcAMPvaried between 75 and 95 %. All assays were carried out in triplicate and the Student's 't' testwas used for statistical comparisons. Protein concentration was determined by the method ofBradford (1976).

Electron-microscopic cytochemical assay for adenylyl cyclase localization used the pro-cedure of Cutler, Rodan & Feinstein, (1978). This procedure uses adenylyl imidodiphosphate assubstrate and lead nitrate to precipitate the reaction product lead imidodiphosphate. Bariumchloride was used in place of lead nitrate in some experiments (Kempen, DePost, Bonting &Stadhouser, 1978). Sodium fluoride was omitted from the reaction medium since it has beenreported to increase the non-enzymic conversion of ATP to cAMP (Kempen et al. 1978).Manganous ion was substituted for magnesium in some experiments. Spermatozoa were fixedfor 5 min with glutaraldehyde (o-6 %) prior to adding substrate. Membranes were prepared forelectron microscopy (Gillis et al. 1978), and thin sections were examined with a Philips 201electron microscope.

The preparation of washed suspensions of boar spermatozoa in Tris-buffered medium wascarried out as previously described (Peterson, Bundman & Freund, 1978). Motility [percentagemotile sperm, and forward progression (Freund & Carol, 1964)], was rated at room temperatureby a trained observer who was unaware of the treatments used in particular experiments.

Radioisotopes were purchased from the Amersham Company; other chemicals were pur-chased from the Sigma Chemical Company, Aldrich Chemical Company, ICN ChemicalCompany, Boehringer-Manheim Co., and J. T. Baker Co. The ionophore A23187 was the giftof the Eli Lilly Pharmaceutical Company. The compound 9-(tetrahydro-2 furyl) adenine(Squibb 22356) was the gift of the Squibb Pharmaceutical Company.

RESULTS

Plasma membrane vesicles from boar and human spermatozoa showed very lowrates of adenylyl cyclase activity unless a small amount (o-i %) of the detergent TritonX-100 was added to the suspension. Since the active site of adenylyl cyclase is foundon the internal aspect of the plasma membrane in virtually all cells, this observationsuggested that the vesicles were right-side-out and relatively impermeable to thecharged substrate ATP. Right-side-out orientation of the vesicles was also supportedby previous studies which established that 5'-adenylic acid was rapidly hydrolysed by5'-nucleotidase without need of detergents (Gillis et al. 1978). The enzyme 5'-nucleo-tidase has been shown to have its active site on the external aspect of the plasmamembrane in other cell types (DePierre & Karnovski, 1974; Carroway et al. 1976).

Table 1 shows the results of experiments in which the adenylyl cyclase activity ofboar spermatozoa plasma membrane vesicles was determined in the presence and

Adenylyl cyclase in spermatozoa 95

Table 1. Effect of various treatments on adenylyl cyclase activity ofboar spermatozoan membrane vesicles

Adenylyl cyclaseactivity,

Addition nmol/mg/min ± s.D.

Expt. 1.(a) Untreated spermatozoa

Plasma membrane vesicles+ iomMMg1 + 0-548 ±0-012+ iomMMn 1 + 13-96 ±0-300

(b) Acrosome-reacted spermatozoaPlasma membrane vesicles

+ iomMMg1 + 0013 ±0-002+ 10 mM Mn! + 0-42810-033

•Hybrid vesicles (Mn'+, 10 mM) 0-561 ±0-052(c) Homogenate (Cavitate)

+ iomMMga+ O-OII±O-OII+ iomMMn s + 0-60910-040

Expt. 2 " .Plasma membrane vesicles 1-163 + 0099Cavitate 0-02810-029Vesicles 4-CaCl2 (01 mM) 1 079 ±0029Vesicles+ CaCla (0-5 mM) 1-306 ±0-037Vesicles + CaClE (1 -o mM) 1 -496 ± 0-040Vesicles + CaCl, (3-0 mM) 1-033 ±0-060

• Fused plasma membrane—outer acrosomal membrane vesicles.• • MgJ+(io mM) used in all treatments.

absence of various ions known to affect the activity of the enzyme. The specific activityof the plasma membrane vesicles used in these studies were always at least 10 -foldgreater than that found in the homogenate (cavitate) from which the vesicles wereobtained. This suggests that, in intact boar spermatozoa, adenylyl cyclase is concen-trated in the plasma membrane.

Plasma membrane vesicles and fused plasma membrane-outer acrosomal membranevesicles (hybrid vesicles) obtained from acrosome-reacted spermatozoa were con-siderably less active than plasma membranes obtained from unreacted spermatozoa.The reason for this is not entirely clear since other studies (unpublished observationsand Gillis et al. 1978) indicated that the 3 types of membrane vesicles showed littledifference in the activity of other enzymes such as alkaline and acid phosphatase. It ispossible that adenylyl cyclase is more susceptible to proteases which may not have beencompletely inhibited by trasylol BE during the prolonged incubation times required toobtain these vesicles.

The specific activity of adenylyl cyclase in plasma membrane vesicles of humanspermatozoa (Table 2) was significantly less than that obtained with boar spermatozoa;but the membrane vesicle activity was generally 5- to 10-fold higher than that of theactivity of the enzymes in cavitates which also suggested that adenylyl cyclase ispresent in the plasma membrane of this species of spermatozoa.

96 R. N. Peterson and others

Table 2. Adenylyl cyclose activity of human spermatozoon plasmamembrane vesicles

Addition

Adenylyl cyclaseactivity,

nmol/mg/min± S.D.

Plasma membrane vesicles (10 mM Mg1+)Plasma membrane vesicles (10 mM Mn1+)Cavitate (10 mM Mga+)Cavitate (10 mM Mna +)

004610017

0-248 ±0009

0008 ±0032

002910007

10 r

0-8c

Ioi 0-6

CO

•o 0-4

0-2

6 0 6-5 8 0 8-5 9-070 7-5pH

Fig. 1. Effect of pH on the adenylyl cyclase activity of boar spermatozoan plasma mem-brane vesicles. Buffers containing Tris-HCl, 251TIM; 0 1 M NaCl and 10 mM MgCL wereused at pH levels of 7-0 and higher. Bi'j-Tris-HCl was substituted for Tris-HCl belowpH 70 .

Adenylyl cyclase activity in boar sperm plasma membrane vesicles was increasedin alkaline solution (Fig. 1) and was enhanced more than 20-fold when manganous ionwas used in place of magnesium ion (Table 1). The activity of the enzyme in hybridvesicles and plasma membrane vesicles from acrosome-reacted spermatozoa was alsomarkedly increased in the presence of manganous ion. Manganous ion also increasedthe activity of adenylyl cyclase in human spermatozoan plasma membranes, althoughnot as markedly (Table 2). Calcium ions, on the other hand, had only a small effect onthe activity of adenylyl cyclase in boar spermatozoan membranes; enzyme activityincreased slightly in the presence of 0-5-1-0 mM calcium (Table 1). In other studies

Adenylyl cyclase in spermatozoa 97

(not shown), adenylyl cyclase activity was not significantly inhibited by barium ions(4 mni) or lead ions (1 mM); lead ions at concentrations of 2 mM or higher, however,reduced enzyme activity by more than 90%. These observations have relevance to theevaluation of the cytochemical method used for the localization of adenylyl cyclasedescribed below.

Electron-microscopic observations

Cytochemical assay showed that adenylyl cyclase was localized primarily along theinner aspect of the plasma membrane of boar spermatozoa with some activity occa-sionally noted near the outer dense fibres and mitochondria of the midpiece. Theplasma membrane over the acrosomal region showed the heaviest reactivity (Figs. 2-3).Less reactivity was seen over the postacrosomal region and occasionally it was seen onthe flagellar membrane. Activity was also observed within plasma membrane vesicles(Figs. 4-5) and within hybrid vesicles (not shown). In human spermatozoa, activitywas concentrated along the microtubules within the axoneme complex (Figs. 6—7),this activity was discernible when either barium ions or lead ions were used to pre-cipitate the imidodiphosphate reaction product. Activity along the plasma membraneof human spermatozoa was seen in a minority of sperm in which reaction product wasseen along the axoneme (Fig. 7), but was less intense than that observed with boarsperm. This may be due to the much lower specific activity of the enzyme in mem-branes from human spermatozoa compared to that of the boar (see Tables 1, 2) and/orto a greater sensitivity of the membrane to the fixative glutaraldehyde.

Since the method using lead ion as a precipitant has been criticized by Kempen et al.(1978), controls were critical to the interpretation of these experiments. The followingpoints are noted: (1) no activity was discerned in plasma membrane vesicles or in intactspermatozoa in the absence of substrate; and (2) where a pattern of activity was dis-cernible using barium ions as precipitant, it was essentially the same as when lead wasused as precipitant barium only slightly inhibited enzyme activity in membranepreparations. Kempen et al. (1978) noted that lead ions markedly inhibited adenylylcyclase activity in the cells employed in their study; a similar observation was made inthis study. However, the concentration of lead ions appeared to be critical, as reportedabove. At 1 mM Pb2+, enzyme inhibition was slight, but at 2 mM Pb2+ activity wasreduced more than 90%. This probably accounts for our early observations that inthe majority of experiments carried out at the higher concentration of lead ions, noactivity in either vesicles or intact cells were found. It is also noted that nucleotidepyrophosphohydrolase activity has been detected in plasma membranes, but theenzyme appears to be located on the external surface of cells (Riordan & Foastner,1978) and not on the cytoplasmic surface where activity was found in these experi-ments. Adenylyl cyclase also appears to be located on the inner surface of thesevesicles as indicated by direct measurements of enzyme activity which require thepresence of a detergent such as Triton X-100.

We note also, however, that it was difficult to obtain complete inhibition of enzymeactivity as measured by the cytochemical assay in the presence of large concentrationsof the Squibb reagent 22356. Cutler et al. (1978) reported that this compound was an

R. N. Peterson and others

2 3

\

t

4t

X r

f

K', \• • • - * * > ,

4f

• >•• • * , • < ••

V*4

Adenylyl cyclase in spermatozoa 99

effective inhibitor of adenylyl cyclase activity in platelets at a concentration of 1 mM.Our enzymological studies indicated that significant inhibition of adenylyl cyclaseactivity in membrane vesicles required very high concentrations of Squibb 22356(> 10 mM). These larger concentrations of Squibb 22356, however, also decreasedcytochemical reactivity in vesicle preparations (compare Figs. 8 and 9). Cytochemicalreactivity in membrane vesicles was also decreased when magnesium ions weresubstituted for manganous ions (compare Figs. 8 and 10). This was also observed inenzymological assays and provides additional support for the validity of the localizationof enzyme activity by the cytochemical procedures used.

DISCUSSION

Plasma membrane vesicles from spermatozoa prepared from washed sperm sus-pensions by nitrogen cavitation and sucrose density gradient centrifugation provide arich source of the enzyme adenylyl cyclase. The enzyme in both human and boarmembranes is not unlike that which has been reported for other species in that itsactivity is markedly enhanced by manganous ion and alkaline pH (Morton & Albagli,1973; Casillas & Hoskins, 1971; Herman, Zahler, Doak & Campbell, 1976). Theseexperiments provide the first cytochemical evidence for the location of adenylyl cyclasein mammalian spermatozoa. The cytochemical experiments support the enzymologicalexperiments that adenylyl cyclase is present in high concentrations in the plasmamembranes of boar spermatozoa and that it is present in the plasma membranesof human spermatozoa. However, the cytochemical experiments suggest that adenylylcyclase is also present within the axonemal complex of human spermatozoa. This is ofinterest especially in view of the work of Herman et al. (1976) which indicated thatmore than one form of adenylyl cyclase exists in bull spermatozoa and that the enzymeis not entirely confined to membranes. Adenylyl cyclase has also been located at sitesin addition to the plasma membrane in other types of contractile tissue (Raible,Cutler & Roadan, 1978; Dhalla, Sulahke & McNamarra, 1973). We note too thatTamblyn & First (1977) have reported reactivation of motility in detergent-treatedbovine sperm models with caffeine, and Lindemann (1978) has shown that cAMP mayinteract directly with the motile apparatus in such demembranated sperm. However,we emphasize that caution is required in interpreting these experiments as well as

Figs. 2, 3. In Fig. 2, showing an unstained section, the reaction product (lead imidodi-phosphate) is primarily localized over the acrosomal region of this boar sperm withminor reactivity over the postacrosomal region, x 34000. In Fig. 3, a moderately high-magnification micrograph of an unstained section ( x 54000), reactivity is seen associa-ted with the plasma membrane (arrows) and in some cases it appears to have diffusedinward.Fig. 4. Isolated plasma membrane vesicles of boar spermatozoa (stained with uranylion and lead) show dense lead imidodiphosphate reaction product associated with theinterior aspect of each vesicle (arrows), x 60000.Fig. 5. Unstained preparation showing imidophosphate reaction product (arrows) onthe internal aspect of boar sperm plasma membrane vesicles, x 92000.

100 R. N. Peterson and others

8\

X\

10

Adenylyl cyclose in spermatozoa 101

some of the cytochemical experiments reported here. Definitive proof of the exis-tence of adenylyl cyclase within the axoneme must await development of methodswhich can assay the enzyme directly in isolated axoneme preparations.

Finally we note the ability to prepare pure right-side-out membrane vesicles in highquantity, tightly sealed and enzymically active should provide an approach to thestudy of ion and sugar transport in spermatozoa which has heretofore eluded detailedinvestigation because of the rapid metabolism and small intracellular volumes pre-sent in intact spermatozoa.

This work was supported by grants HD 10947 and HD 11823 from the National Institutes ofHealth.

REFERENCES

BRACKETT, B. G., JEITLES, G. G., JR. & O H , Y. K. (1975). Fertilization by sperm treated withhigh ionic strength and N'-O'-dibutyryl-cyclic-monophosphoric acid. Fedn Proc. Fedn Am.Socs exp. Biol. 34, 256.

BRADFORD, M. (1976). A rapid and sensitive method for the quantitation of microgram quanti-ties of protein utilizing the principle of protein/dye binding. Analyt. Biochem. 72, 248-254.

CARROWAY, K. L., FOGLE, D. D., CHESTNUT, A. W., HUGGINS, J. W. & CARROWAY, C. A. C.(1976). Ectoenzymes of mammary gland and its tumors: Lectin inhibition of 5' nucleotidaseof the 13762 rat mammary ascites carcinoma. J. biol. Chem. 251, 6173-6178.

CASILLAS, E. R. & HOSKINS, D. D. (1971). Adenyl cyclase activity and cyclic 3, 5'AMP contentof ejaculated monkey spermatozoa. Archs Biochem. Biophys. 147, 148-155.

COUNIS, R. & MONGONGU, S. (1978). Adenylate cyclase assay with [a"P] ATP as substrate.Analyt. Biochem. 84, 179-185.

CUTLER, L., RODAN, G. & FEINSTEIN, M. B. (1978). Cytochemical localization of adenylatecyclase and of calcium, ion-activated magnesium ATPase in the dense tubular system ofhuman blood platelets. Biochim. biophys. Acta 542, 357-371.

DEPIERRE, J. W. &KARNOVSKI,M. L. (1974). Ectoenzymes of the guinea pig polymorphonuclearleukocyte. I. Evidence for an extoadenosine mono-phosphatase-adenosine triphosphatase andP-nitrophenyl phosphatase. J. biol. Chem. 249, 7111-7120.

Figs. 6, 7. Human spermatozoa showing a portion of the flagellum in longitudinalsection.

Fig. 6. Lead imidodiphosphate reaction product is seen primarily along theaxonemal complex of the flagellum of this stained preparation. Less reactivity isobserved along the plasma membrane (arrowhead), fibrous ring, dense outer fibres andmitochondrial sheath, x 36000.

Fig. 7. High-magnification micrograph of an unstained preparation showingbarium imidodiphosphate reactivity primarily along the axonemal micro tubules of theprincipal piece. Some activity is also seen near the plasma membrane (arrows) althoughthis is a somewhat atypical example since the plasma membrane overlying most flagelladid not usually stain this heavily, x 135000.

Figs. 8-10. Barium imidodiphosphate precipitate in unstained preparations of plasmamembrane vesicles of boar spermatozoa after various treatments, x 55000.

Fig. 8. Reactivity is indicated by arrows in this preparation which includedmanganous ion.

Fig. 9. Less reactivity is seen after incubation with Squibb inhibitor (Mn'+ plus20 mM Squibb 22356).

Fig. 10. Reactivity in the presence of Mg s + ion is light (compare with Fig. 8).

102 R. N. Peterson and others

DHALLA, N. S., SULAHKE, P. V. & MCNAMABRA, D. B. (1973). Studies on the relationshipbetween adenylate cyclase activity and calcium transport by cardiac sarcotubular membranes.Biochim. biophys. Acta 323, 276-284.

FREUND, M. & CAROL, B. (1964). Factors affecting haemocytometer counts of sperm con-centration in human semen. J. Reprod. Fert. 8, 149-155.

GILLIS, G., PETERSON, R., RUSSELL, L., HOOK, L. & FREUND, M. (1978). Isolation and charac-terization of membrane vesicles from human and boar spermatozoa: Methods using nitrogencavitation and ionophore induced vesiculation. Prep. Biochem. 8, 363-378.

HERMAN, C. A., ZAHLER, W. L., DOAK, G. A. & CAMPBELL, B. J. (1976). Bull sperm adenylatecyclase: Localization and partial characterization. Archs Biochem. Biophys. 177, 622-629.

KEMFEN, H. J. M., DEPOST, H., BONTING, S. L. & STADHOUSER, A. M. (1978). The cytochemicallocalization of adenylate cyclase, fact or artifact. J. Histochem. Cytochem. 26, 298-312.

LINDEMANN, C. B. (1978). A cAMP induced increase in the motility of demembranated bullsperm models. Cell 13, 9-18.

MORTON, B. & ALBAGLI, L. (1973). Modification of hamster sperm adenyl cyclase by capacita-tion in vitro. Biochem. biophys. Res. Commun. 50, 697-703.

PETERSON, R. N., BUNDMAN, D. & FRFUND, M. (1978). Use of a fluorescent dye to measure drug-induced changes in the membrane potential of boar spermatozoa. Life Sci. 22, 659—666.

RAIBLE, D. G., CUTLER, L. S. & ROADAN, G. A. (1978). Localization of adenylate cyclase inskeletal muscle sarcoplasmic reticulum and its relation to calcium accumulation. FEBSLetters, Amsterdam 85, 149-152.

RIORDAN, J. R. & FOASTNER, G. G. (1978). In Current Topics in Membranes and Transport (ed.F. Bronner & A. Kleinzeller), pp. 145-231. New York: Academic Press.

RUSSELL, L., PETERSON, R. & FREUND, M. (1979). Direct evidence for formation of hybridvesicles by fusion of plasma and outer acrosomal membranes during the acrosome reaction inboar spermatozoa. J. exp. Zool. 208, 41-56.

TAMBLYN, T. M. & FIRST, N. L. (1977). Caffeine stimulated ATP reactivated motility in adetergent treated bovine sperm model. Archs Biochem. Biophys. 18, 208—215.

TOYADA, Y. & CHANG, C. (1976). Capacitation of epididymal spermatozoa in a medium withhigh K/Na ratio and cyclic AMP for the fertilization of rat eggs in vitro. J. Reprod. Fert. 36,125-134.

WHITE, A. A. & ZENZER, R. V. (1971). Separation of cyclic 3', s'-nucleoside monophosphatefrom other nucleotides on aluminum oxide columns: Application to the assay of adenylcyclase and guanyl cyclase. Analyt. Biochem. 41, 372-396.

{Received 23 November 1979)