MOLECULAR REPRODUCTION AND DEVELOPMENT 27:305-325 (1990)

Characterization and Inhibitor Sensitivity of Human Sperm Phospholipase A,: Evidence Against Pivotal Involvement of Phospholipase A, in the Acrosome Reaction ROBERT A. ANDERSON, JR.,1,2 SUSAN K. JOHNSON,' PETER BIELFELD,' KENNETH A. FEATHERGILL,' AND LOURENS J.D. ZANEVELD1v3 Departments of 'Obstetrics and Gynecology, 2Physiology, and 3Biochemistry, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois

ABSTRACT The kinetic properties and inhibitor sensitivity of human sperm phospholipase A, (PLA,; EC 3.1.1.4) were studied. Phospholipase activity was isolated from human spermatozoa by acid extraction. Hydrolysis of dipalmitoyl phosphatidylcholine was specific to the sn-2 position. Activity was sensitive to product inhibition (60% inhibition by 0.1 m M lysophosphatidylcholine). The effects of Ca2+ and sodium deoxycholate on enzyme activity were biphasic; maximal activities were observed at 0.5 m M concentration of each agent. PLA, was stimulated (135%) by 3% dimethylsulfoxide and was inhibited by elevated ionic strength (approximately 70% inhibition with either 0.2 M NaCl or 0.2 M KCI).Two molecular forms of PLA, were kinetically distinguishable, one with an apparent Michaelis constant and maximal reaction velocity of 3.0 pM and 0.64 mlU/mg protein and the other with respective constants of 630 pM and 32.0 mlU/mg protein. Both forms of the enzyme were Ca2+ dependent and heat stable; however, the low-K, activity was less resistant to 60°C preincubation at p H 7.5 (28% inactivation of low-K, activity after 45 min, as compared to no effect on high-K, activity). Quinacrine was a noncompetitive PLA, inhibitor with K,s for low- and high-K, activities of 0.42 m M and 0.49 mM, respectively. Trifluoperazine (calmodulin antagonist) inhibited the high-K, activity noncompetitively (K, = 87 pM) and the low-K, activity by a mechanism consistent with the removal of a nonessential activator. Dissociation and rate constants for inactivation of lowand high-K, activities by p-bromophenacyl bromide were 0.28 m M and 0.032 min-', and 0.73 m M and 0.066 min-', respectively. PLA, was inhibited by p-nitrophenyl-p'-guanidinobenzoate, at higher concentrations ( 10-4-10-3 M) than required to inhibit trypsinlike proteinases; paminobenzamidine, another potent trypsin/acrosin inhibitor, stimulated (approximately 40%) PLA, at concentrations from 2-5 m M but inhibited PLA,

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( 4 0 4 0 % ) at a concentration of 10 mM. MnCI, (5mM) inhibited low- and high-K, PLA, activities by 77% and 76%, respectively. Quinacrine (0.4 mM), trifluoperazine (20 pM), p-bromophenacyl bromide (20 pM), and MnCI, (5 mM) were tested as inhibitors of the ionophore A23187-induced human acrosome reaction. Inhibition was noted only with quinacrine (32%)and MnCI, (93%). The effect of MnCI, was restricted to an interaction with A231 87, rather than with PLA,; p-Bromophenacyl bromide inhibited (P < 0.05) PLA, (29%) when added to intact spermatozoa but had no effect on the acrosome reaction. PLA, inhibition was poorly correlated with the acrosome reaction. Results from this study suggest that 1 ) human sperm contain at least two kinetically distinguishable forms of PLA,, with properties similar to those of PLA, in other species and tissues; 2) PLA, inhibition is not a likely mechanism for inhibition of acrosome reactions by acrosin inhibitors; 3) sperm PLA, may be modulated by an endogenous activator; 4) the low-K, PLA, is similar to the group I (pancreatic) enzyme; and 5) PLA, is not a pivotal or rate-limiting step in the human acrosome reaction.

Key Words: Human spermatozoa, Phospholipase A2 inhibitors, Human acrosome reaction, Acrosin inhibitors, Calmodulin antagonist, Enzyme kinetics

INTRODUCTION Fertilization by mammalian spermatozoa is dependent upon their ability t o undergo the acrosome reac-

Received January 15, 1990; accepted May 14, 1990. Address reprint requests to Dr. Robert A. Anderson, OBiGyn Research, Rush Medical Center, 1653 West Congress Parkway, Chicago, IL 60612.

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tion. The release of acrosomal lytic enzymes (e.g., acrosin) from acrosome-reacted spermatozoa may facilitate sperm passage through the vestments surrounding the oocyte (for reviews, see Rogers and Bentwood, 1982; Meizel, 1984; Langlais and Roberts, 1985; Wassarman, 1987). Agents that inhibit the acrosome reaction have antifertility effects (Kaminski et al., 1985; Joyce and Zaneveld, 1985; Nuzzo et al., 1986; Joyce et al., 1987). Further, spermatozoa without acrosomes are associated with infertility (Jeyendran et al., 1985; Lalonde et al., 1988). The molecular mechanisms of the acrosome reaction are important for understanding the physiological basis of fertilization and for contraceptive research and development. Events leading to the acrosome reaction remain poorly understood. Phospholipase A, (PLA,; EC 3.1.1.4) has been implicated as a component in the cascade of reactions leading to the acrosome reaction. PLA, inhibitors exert inhibitory effects upon acrosome reactions induced in vitro (Lui and Meizel, 1979; Ono et al., 1982; Singleton and Killian, 1983). However, the sensitivity of sperm PLA, to these agents had either not been determined (Lui and Meizel, 1979; Singleton and Killian, 1983) or was found to be less than that of the acrosome reaction (Ono et al., 1982). Agents used to elucidate the involvement of PLA, in the acrosome reaction are not entirely specific for PLA,. For example, quinacrine (mepracrine) also inhibits calcium influx and energy production (Best et al., 1984; Filippov et al., 1989), processes that are likely to modulate the acrosome reaction. Similarly, the mechanism of action of p-bromophenacyl bromide (alkylation, probably of an active site histidine residue; Volwerk et al., 1974; Evenberg et al., 1977) suggests that other enzymes with a putative role in the acrosome reaction (e.g., acrosin; Lui and Meizel, 1979; Perreault et al., 1982; Nuzzo et al., 1986, 1990) may also be affected by this agent, via alkylation of an active site histidine (Lui and Meizel, 1979; Anderson et al., 1981). PLA, from other tissues is inhibited by guanidinobenzoates and amidino derivatives (Hesse and Lankisch, 1984; Nakaguchi et al., 1986; Pepinsky et al., 1986) and indomethacin (Drummond and OldsClarke, 1986; Bonney et al., 1988). These agents are generally regarded as acrosin and cyclooxygenase inhibitors. Acrosin and cyclooxygenase may also be involved in the mammalian acrosome reaction (Meizel and Lui, 1976; Perreault et al., 1982; Dravland et al., 1984; Nuzzo et al., 1986; Joyce et al., 1987). The sensitivity of sperm-derived PLA, to these agents remains to be determined. It is unknown to what extent the inhibition of the acrosome reaction by a number of inhibitors can be ascribed to their action on PLA,, or upon apparently unrelated activities. Pharmacologically relevant inhibitor concentrations (i.e., those which effectively inhibit the target enzyme from the tissue of interest, with min-

imal secondary effects on other enzymes and/or molecular processes) were often not used in previous studies. Several studies have appeared regarding the localization, isolation, and partial characterization of PLA, from mammalian spermatozoa (see above references). However, there has been no consensus with regard to the levels of activity found in spermatozoa or properties of the sperm-derived enzyme, in spite of reported similarities of PLA, in different tissues isolated from species throughout the animal kingdom (Slotboom et al., 1982; Kramer et al., 1989; Kuchler et al., 1989). For example, the range of PLA, activity recovered from crude extracts of guinea pig and human spermatozoa spans approximately five orders of magnitude (On0 et al., 1982; Thakkar et al., 1984; Guerette et al., 1988). While there is general agreement that sperm-derived PLA, is calcium dependent, the quantitative nature of calcium dependence is unclear. Dose-response curves have shown either hyperbolic (Thakkar et al., 1984) or biphasic (On0 et al., 1982) responses. Quinacrine and p-bromophenacyl bromide inhibit PLA, and/or the acrosome reactions of guinea pig (Ono et al., 1982) and hamster (Lui and Meizel, 1979);these findings suggest not only that PLA, may be involved in the acrosome reaction but also that the sperm-derived enzyme exhibits qualitatively similar inhibitor sensitivity to that of PLA, from other tissues. In contrast, inhibition of human sperm PLA, by p-bromophenacyl bromide appears to be independent of inhibitor concentration (Thakkar et al., 1984). Moreover, human sperm PLA, may be insensitive to inhibition by quinacrine (Thakkar et al., 1984; Langlais and Roberts, 1985). The above observations may indicate profound differences in kinetic properties between sperm-derived PLA, and that from other tissues, as well as qualitative differences among sperm phospholipases A, from several mammalian species. More likely, however, these apparent discrepancies may be explained by differences in the methods employed for measurements of PLA,. The physical form of the phospholipid substrate has profound effects on the activities of PLA, isolated from several sources. Whether the substrate is in its monomeric or aggregated (e.g., bilayer, mixed micellar) form affects not only the level of measured activity but also apparent sensitivity to inhibitors (due a t least in part to partitioning of the inhibitor in the lipid-water interface; Slotboom et al., 1982; Fawzy et al., 1988). Most of the studies of sperm-derived PLA, have utilized sonicated suspensions of phospholipid substrate, without regard to temperature of preparation or to the annealing of aggregates thus prepared. Such procedures will likely yield unstable heterogeneous particles, making interpretation of detailed kinetic data difficult (Slotboom et al., 1982). A critical evaluation of the involvement of PLA, in the acrosome reaction may be effected by comparing the effects of selected PLA, inhibitors on the acrosome

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were performed by light microscopy (400 x , phase-contrast), with a hemacytometer. All samples were processed within 4 hours of ejaculation. PLA, was isolated from spermatozoa by acid extraction, essentially as described by Thakkar et al. (1984). Spermatozoa were separated from seminal plasma by centrifugation at 1,OOOg for 15 minutes. The spermatozoa were resuspended in 50 mM Tris-HC1, pH 7.0, containing 25 mM sucrose (volume equal to th a t of the semen), and washed twice by centrifugation. The final pellet was resuspended in a volume of 0.18 N H,SO, sufficient to yield 1.5 x 10' cells/ml, and homogenized with a glass-teflon homogenizer (seven passes, up and down). The resultant suspension was extracted by constant stirring for 4.5 hours at 4°C. Particulates were sedimented by centrifugation (13,00Og, 15 min). The supernatant was dialyzed overnight (nominal molecular weight cutoff = 3,500) against 1,000 volumes of 1 mM HC1 (4°C). The dialyzed acid extract was used as the source of PLA, activity. Alternatively, PLA, activity was measured in sperm sonicates. After washing twice (see above), spermatozoa were resuspended in 50 mM Tris-HC1, pH 7.0, containing 25 mM sucrose (approximately one fourth the original semen volume), to yield 2 x lo8 cells/ml. The suspension was sonicated with a Branson (Danbury, CT) Model S-125 sonifier (three times; 15-second bursts with 30-second intervals of rest at 0°C; midrange power output with microprobe). The resultant suspension was MATERIALS AND METHODS used as a source of sperm PLA, activity. L-a-Dipalmitoyl phosphatidylcholine, palmitoyl PLA, was routinely measured by quantitating the lysophosphatidylcholine, 1,2-dipalmitoyl-sn-glycerol, release of 3H palmitate from [3H-C2-palmityl]dipalmTriton X-100, p-nitro-p'-guanidinobenzoate(NPGB), p- itoyl phosphatidylcholine, by the method of Katsumata aminobenzamidine hydrochloride, quinacrine hydro- et al. (1986). Initially, standard reaction mixtures conchloride, trifluoperazine (TFP), human serum albumin tained 50 mM Tris-HC1, 2 mM CaCl,, 2-[3H-palmityll (Fraction V), Bismark Brown Y, Rose Bengal, Ficoll dipalmitoyl-phosphatidylcholine(2.0-250 pM) and en(Type 400-DL), EDTA (tetrasodium salt), and EGTA zyme-containing protein (0.68-3.4mg for sperm soni(free acid) were obtained from Sigma Chemical Com- cate; 50-80 pg for acid extract), pH 7.5, in a total volpany (St. Louis, MO). Dipalmitoyl-[2-palmitoyl-9, ume of 2.0 ml, unless otherwise indicated. Substrate 10-3H]-L-a-phosphatidylcholine and dipalmitoyl-[1, was prepared by evaporating 27 p1 (2.7 pCi) of 3H2-dipalmitoyl-l-14C]L-a-phosphatidylcholine(specific phosphatidylcholine to dryness (ambient temperature, activities of 57Ci/mmole and 117 mCi/mmole, respec- under nitrogen), and adding 1 ml of either 0.1 mM tively) were purchased from NEN-Dupont (Boston, unlabeled phosphatidylcholine (for substrate concenMA) and Amersham Corporation (Arlington Heights, trations ranging from 2.0-20.0 pM) or 1.0 mM unlaIL), respectively. The calcium ionophores A23187 and beled phosphatidylcholine (for substrate concentraionomycin were purchased from Calbiochem (La Jolla, tions ranging from 25-250 pM), dissolved in 70% CA). Silica gel LK6D Linear-K thin layer plates (250 ethanol containing 25 mM sodium deoxycholate. Reacpm) were from Whatman Chemical Separations (Clif- tion mixtures were adjusted to yield final ethanol and ton, NJ). All other chemicals were of reagent grade deoxycholate concentrations of 7.0% and 2.5 mM, respectively, unless otherwise indicated. These condiquality. Ejaculated semen was obtained from apparently tions were later modified for evaluation of inhibitor healthy donors (ages 23-40 years) from within the sensitivity (see below). Blank reactions contained no enzyme protein. ReacMedical Center community. Donors were not receiving prescription medication a t the time of donation, and tions were incubated for 10-30 minutes a t 25°C. They they had no history of reproductive disorders. Only were terminated by the addition of 0.4 m l 5 % (v/v) Trisamples with sperm concentrations and motility in ex- ton X-100 containing 200 mM EDTA, and 2 g of anhycess of 30 x 106/ml and 40%, respectively, were used in drous sodium sulfate. Product (3H-palmitic acid) was the present studies. Cell count and motility estimates extracted into 10 ml of acidified (l%, v/v acetic acid)

reaction with their inhibition of the isolated enzyme. This objective was addressed in the present study. The assay conditions chosen include the use of substrate in a form amenable to kinetic studies. The data suggest that phosphatidylcholine hydrolysis by acid extracts of human spermatozoa is due entirely to PLA,. The enzyme preparation appears to be free from endogenous inhibitors and contaminating substrates and is suitable for analysis with exogenous inhibitors. In contrast to previous reports, the present data indicate that human sperm PLA, is similar to that of other species and somatic tissue. Kinetic evidence is presented for the presence of a t least two forms of PLA, in human spermatozoa; each form can be differentiated on the basis of its Michaelis constant for phosphatidylcholine and its sensitivity to heat inactivation. The data further suggest a similar sensitivity of both forms of the enzyme to several PLA, inhibitors. Kinetic data suggest that one form may interact with calmodulin or some as yet undefined modulator. The complex interaction of PLA, with high concentrations of acrosin inhibitors suggested that inhibition of the acrosome reaction by these agents is due to their effects on acrosin, rather than on PLA,. Finally, a poor correlation was obtained between sperm PLA, inhibition and effects on the acrosome reaction. If involved, PLA, is not a rate-limiting step in the cascade of events leading to the human acrosome reaction.

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volume of 2.0 ml, unless otherwise indicated. Substrate was prepared by evaporating 2.7 pCi of 3H-phosphatidylcholine to dryness (ambient temperature, under nitrogen), and adding l ml of either 0.1 mM unlabeled phosphatidylcholine (for substrate concentrations ranging from 2.0-5.0 pM) or 2.5 mM phosphatidylcholine (for substrate concentrations ranging from 50-250 pM), dissolved in 70% ethanol containing 5 mM sodium deoxycholate. All reaction mixtures were adjusted to yield final ethanol and deoxycholate concentrations of 7.0% and 0.5 mM, respectively. Identification of Products of Reversibility of inhibition was determined for all inPhosphatidylcholine Hydrolysis hibitors by either dialysis or by dilution. For studies by Acid Extracts with reversible inhibitors (quinacrine, trifluoperazine, The specificity of hydrolytic cleavage of phosphati- MnCl,), enzyme was preincubated in the presence of dylcholine by acid extracts of human spermatozoa was inhibitor for 10 minutes before initiating reactions determined by incubating 0.1 mM dipalmitoyl-[ 1,2-di- with substrate. For the irreversible inhibitor, p-bropalmitoyl-l-'4C]-L-c-phosphatidylcholinewith acid ex- mophenacyl bromide, inhibition was evaluated after tract (80 pg protein) at pH 7.5 (50 mM Tris-HC1) in a preincubation of enzyme with different concentrations total reaction volume of 0.25 ml for 40 minutes at 25". of inhibitor for varying periods, ranging from 15-200 Reactions were terminated by the addition of 50 ~ 1 5 % minutes. Stock solution of p-bromophenacyl bromide, Triton X-100, containing 200 mM EDTA. Portions (10 at concentrations ranging from 5-50 mM, were prep1) from each reaction were either applied to silica gel pared in dimethylsulfoxide (DMSO). The final DMSO G plates or counted by liquid scintillation spectrome- concentration in reactions containing p-bromophenacyl try. The plates were developed with CHC1,:MeOH:ace- bromide was 1% (v/v) in all instances except reactions tic acid:H,O (25:15:4:1). Authentic dipalmitoyl phos- containing 1 mM inhibitor, which contained 2% phatidylcholine, palmitoyl lysophosphatidylcholine, DMSO. Control reactions contained equimolar concendipalmitoyl glycerol, and palmitic acid were chromato- trations of DMSO to those of reactions containing ingraphed on the same plate. Lanes containing only stan- hibitor. After the desired preincubation period, enzyme dards (10 pl each of 10 mM solutions in ethanol) were activity was measured by adding 9 vol of buffered subsprayed with a 10% solution (in acetone) of saturated strate solution, such that final concentrations of phospotassium dichromate in 70% H,SO,. Plates were phatidylcholine were either 5 pM (measurement of developed at 100°C for 40 minutes, resulting in visual- low-K, activity) or 100 pM (high-K, activity). ization of the standards (dark brown). Relative migraCapacitation and Induction of Acrosome tions (R,) for lysophosphatidylcholine, phosphatiReaction of Human Spermatozoa dylcholine, palmitic acid and dipalmitoylglycerol were 0.07 t 0.01 (SD; n = 4), 0.24 2 0.03, 0.91 -+ 0.05, and Acrosome reactions were evaluated on pools of four 1.00 t 0.01, respectively. separate ejaculates per determination, essentially as To achieve maximum separation and recovery of described by De Jonge et al. (1989a,b). Spermatozoa each of the four compounds, segments, ranging from were separated from seminal plasma by layering whole 0.35-1.0 cm, were scraped from the plates and placed semen in borosilicate tubes (approximately 5 x lo6 in scintillation vials. The radiolabel present in each s p e r d t u b e ) over 11% Ficoll, containing 120 mM NaCl segment was expressed as the percentage of the total and 25 mM HEPES (N-2-hydroxyethylpiperazine-N-2radiolabel recovered from each lane. Corresponding ethanesulfonic acid), pH 7.4, and centrifuging at 500g segments from blank reactions (containing no acid ex- for 30 minutes (ambient temperature). Spermatozoa tract) were similarly expressed and subtrated from from each tube were resuspended in capacitation methose of incubation that contained enzyme. dium, consisting of 35 mg/ml human serum albumin, 94.6 mM NaC1, 4.8 mM KC1, 1.2 mM sodium phosEvaluation of Human Sperm PLA, Sensitivity phate, 1.7 mM CaCl,, 2.4 mM MgSO,, and 25 mM to Inhibition by Various Agents NaHCO,, pH 7.4. The sperm were washed once by cenBased on studies in which optimal conditions for trifugation (500g, 2 minutes) and resuspended in 1ml PLA, activity were determined (Results), reaction mix- capacitation medium (37°C). tures for kinetic evaluation of inhibitors contained 50 Motility of each sample was assessed by light microsmM Tris-HC1, 0.5 mM CaCl,, 2-[3H-palmityl] dipalmi- copy after 15 minutes and after 3 hours incubation at toyl-phosphatidylcholine (2.0-5.0 pM for measure- 37°C (under 95% air, 5% CO,; 100% relative humidity). ment of low-K, activity; 50-250 pM for measurement After 15 minutes at 37"C, 0.5 ml of each sample was of high-K, activity; see Results), and enzyme- fixed and examined for acrosomal integrity (see below). containing protein (50-80 pg) at pH 7.5, in a total The remainder of each sample was incubated at 37°C

n-hexane, a s described by Katsumata et al. (1986). A portion (1ml) of the organic phase was taken for scintillation counting. Results were quantitated on the basis of cpm contained in 10 pl of stock substrate (0.1 or 1.0 mM), and were expressed as nmoles palmitate formeamin (mIU) per mg protein or pmoles palmitate formed/min/106 spermatozoa. Protein content was estimated by the Biuret method (Gornall et al., 1949) with bovine serum albumin as a standard, or by the absorbance of the acid extracts at 280 nm.

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I I I I I I I I I 1 for 3 hours. Various additions were made at the indilysophosphatidylpalmitate cated times during capacitation. Sperm motility was 10 choline unaffected under these conditions. Longer incubation periods in the presence of the respective inhibitors or higher concentrations of inhibitors produced significant loss of sperm motility (data not shown) and were not included in data analysis. After incubation, acrosome reactions were induced by addition of calcium ionophore (either A23187 or ionomycin, final concentration, 10 pM); after 15 minutes, sperm were fixed and stained for acrosomal integrity (see below). Samples containing ionophore were compared to samples that received a n equal volume of vehicle only (DMSO; final concentration, 1%). Results were expressed as ionophore-dependent acrosome reaction. I Spermatozoa were fixed by addition of an equal vol-20' Oli 012 013 014 015 016 017 018 019 t 0 ume of 3.0% glutaraldehyde, containing 100 mM soRf dium cacodylate, pH 7.4. After 30 minutes, sperm were pelleted and washed once in water by centrifugation Fig. 1. Distribution of products of phosphatidylcholine hydrolysis (500g, 2 minutes). The washed pellet was resuspended by acid extracts of human spermatozoa. PLA, activity in acid extracts in 50 pl H,O; the suspension was smeared onto micro- (n = 3) was measured with 0.1 mM dipalmitoyl-[1,2-dipalmitoyl-l''Cl-La-phosphatidylcholine as substrate, as described in Methods. scope slides and allowed to air dry. Portions of each reaction (3.3%of total incubation) were applied to Spermatozoa were stained for acrosomal integrity by thin layer (silica gel G ) plates and developed in CHC1,:MeOH:acetic a modification of the method of Talbot and Chacon acidH,O (25:15:4:1). Segments of each lane to which samples were (1981). Slides were placed in 0.8%Bismark Brown-HC1 applied were scraped from the plates and placed into scintillation (pH 1.8) for 10 minutes at 37"C, washed twice in dis- vials, and the radiolabel was quantitated. Blank reactions (containing acid extract) were similarly treated. The radiolabel contained in tilled H,O, and counterstained with 0.8% Rose Bengal no each segment was expressed as a percentage of the total radiolabel in 100 mM Tris-malate (pH 5.3) for 20 minutes at am- recovered from the plate. Results were expressed as the difference in bient temperature. After washing twice with H,O, the percentage of total radiolabel recovered between incubations with and spermatozoa were dehydrated with increasing concen- without acid extract (x ? SEM). R,values for authentic lysophosphatiphosphatidylcholine, palmitic acid, and diacylglycerol trations of ethanol and cleared in xylene. Stained sper- dylcholine, were 0.07 t 0.01,0.24 ? 0.03,0.91 ? 0.05,and 1.00 ? 0.01,respecmatozoa were examined by light microscopy (1,000 x , tively. The ratio of lysophosphatidylcholine formed to palmitate oil). Intact spermatozoa were identified as those with formed was 0.94 ? 0.04;radiolabel comigrating with authentic palmred-stained anterior portions and brown-stained poste- itic acid accounted for 99.7 ? 6.6% of the hydrolysis of phosphatidylrior portions of the sperm head. Spermatozoa with un- choline (assuming that the specific activity of recovered palmitate stained anterior portions of the sperm head were con- was one half that of the substrate). sidered acrosome reacted. The percentage of acrosome reacted sperm was based upon 200 sperm counted per transformation, with 90% confidence limits indicated slide; duplicate slides were prepared for each assay. in parentheses (Sokal and Rohlf, 1981a). The associaStatistical Analyses tion of the efficacy of various agents as PLA, inhibitors PLA, activity was reported as average values ? with their ability to inhibit the acrosome reaction was determined with Kendall's coefficient of rank correlastandard errors of the mean. The effectiveness of Ca", ionic strength, p-aminobenzamidine, and NPGB as tion (Sokal and Rohlf, 1981b). Differences were considmodulators of PLA, and of different concentrations of ered significant a t the 0.05 level of confidence. Values p-bromophenacyl bromide on PLA, in situ were evalu- of P greater than 0.10 were used a s a n argument ated by analysis of variance. Differences among indi- against significant differences. vidual groups were identified with the Newman-Keuls RESULTS multiple range test (Woolf, 1968a). Data obtained with Quantitation and Identification of Phospholipase inhibitors were presented as double-reciprocal plots of Activity in Human Spermatozoa average values, with the reciprocal of the upper 90% Whole human sperm sonicates hydrolyzed 0.1 mM confidence limit for reaction velocity indicated. Data were fit to linear curves, and differences between dipalmitoyl phosphatidylcholine a t a rate of 5.2 0.17 curves were identified, where indicated, by regression (SEM; n = 3 6 ) pmoles/min/106 sperm (0.070 * 0.002 analysis (Woolf, 196813). Data regarding acrosome re- mIU/mg protein). This was contrasted with a n average actions were first subjected to arcsine transformation activity of 29.2 0.65 pmoles/min/106 sperm equivabefore further parametric analysis. Values are re- lents, or 5.4 0.12 (n = 36) mIU/mg protein in the acid ported as the back transform of the average arcsine extract, representing a 75-fold increase in specific ac-

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different amounts of sodium deoxycholate, as indicated. Results are expressed as averages 2 SEM of three determinations at each concentration of deoxycholate.

Fig. 3. Biphasic effect of Caz+ on PLA, activity. PLA, activity of acid extracts was determined (see Methods) in the presence of 0.1 mM phosphatidylcholine and different concentrations of CaCl,, as indicated. Values represent the average SEM of four determinations at each concentration of CaC1,.

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tivity and a 462% increase in total activity. No detectable activity was present in the particulate fraction after acid extraction. The increased total enzyme activity suggests either the presence of a n endogenous phospholipase inhibitor, or latent enzyme activity, in the sperm sonicates. Either factor could confound the interpretation of data regarding the effect of various agents on enzyme activity. For this reason, all subsequent studies were carried out with acid extracts a s the enzyme source. It was assumed that activity measured in acid extracts represented the total sperm phospholipase content. Product recovery was quantitative, as indicated by the recovery of added 14C-palmitate to incubation mixtures (107 9.5%; SEM, n = 4). Product formation was linear over time from 10-60 minutes (r = 0.991; n = 5),

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and over enzyme protein from 31-155 pg (r=0.998; n = 5). Assays were routinely conducted for 10-15 minutes with 50-80 pg enzyme protein. The rate of hydrolysis of 0.1 mM phosphatidylcholine was insensitive to inhibition by 0.1 mM palmitate (P > 0.05, NewmanKeuls multiple range test); rates were 6.5 0.20 for control incubations, and 8.1 ? 1.17 mIU/mg protein in the presence of 0.1 mM palmitate (n = 9 and 4, respectively). Conversely, inhibition was significant (60%; PO.l). However, when the pH of the enzyme preparation was raised to 7.5, a small but significant decrease (28.6%) in the low-K, activity was observed following a 45 minute preincubation at 60°C (Table 1). No effect of 60°C preincubation at pH 7.5 was observed on high-K, activity.

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Reversibility of Inhibition of Human Sperm PLA, by Phospholipase Inhibitors Inhibition of PLA, by 1.0 mM quinacrine (59%; 90% confidence limits = 56-62%, n = 8) and by 50 pM TFP (43%; 36-50%, n = 8) was completely reversed by dialysis against 1,000 vol of 50 mM Tris-HC1, pH 7.5, containing 2 mM CaC1,. Enzyme assays were performed with 0.1 mM phosphatidylcholine. Similarly, inhibition of PLA, by 10 mM quinacrine (98%; 96loo%, n = 5) and 50 pM TFP (38%; 36-41%, n = 5) was reduced by 52% and 87%, respectively, by a 1:lO dilution of the preincubation medium prior to enzyme assay. However, inhibition of PLA, by 0.2 mM p-bromophenacyl bromide was irreversible. Inhibition (43%; 32-55%, n = 3) of enzyme activity after a 60 minute preincubation and subsequent assay in the presence of 0.2 mM p-bromophenacyl bromide was essentially the same as th a t obtained following a 1 : l O dilution of the enzyme after preincubation with 0.2 mM inhibitor prior to assay (47%; 37-57%, n = 3). Inhibition of PLA, by Quinacrine Inhibition of both low-K, and high-K, PLA, activities by quinacrine appeared to be noncompetitive (Figs. 6 and 7). Replots of 1/V, vs. inhibitor concentration were fit to linear curves for the low- (r = 0.982) and high-K, (r = 0.992) activities (data not shown, but derived from those presented in Figs. 6 and 7); the x intercepts of these curves yielded Ki values of 0.42 mM and 0.49 mM, respectively.

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Fig. 4. Stimulation of PLA, activity by dimethylsulfoxide. PLA, activity of acid extracts was determined in the presence of 0.1 mM phosphatidylcholine (see Methods) and different concentrations of dimethylsulfoxide (DMSO), as indicated. Values represent the average i SEM of three determinations at each concentration of DMSO.

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1/ [ phosphatidykholine](rnM)

Fig. 5. Kinetic evidence for the presence of two forms of human sperm PLA,. Data represent the reciprocal of average reaction rate as a function of reciprocal substrate concentration (n = 5 per concentration). Error bars represent the reciprocal of the upper 90% confidence limit for each rate. The solid curve represents the theoretical response

of two Michaelis-Menten enzymes, one with a n apparent K, and V, of 3.0 pM and 0.64 mIU/mg protein, and the other with a K, and V,, of 0.63 mM and 32.0 mIU/mg protein, respectively (see Results for procedure used to derive kinetic constants).

Inhibition of PLA, by Trifluoperazine TFP behaved as a noncompetitive inhibitor of the high-K, activity (Fig. 8). A replot (not shown) yielded a Ki of 87 pM (r = 0.988).In contrast, a different pro-

file of inhibition by trifluoperazine was observed for the low-K, activity (Fig. 9). The curves fit to the data by linear regression had a common point of intersection (l/v = 0.50 2 0.006 nmoles-'-min-mg protein; SEM, n

SPERM PHOSPHOLIPASE A, AND THE ACROSOME REACTION I

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TABLE 1. Inhibitory Effect of 60” Preincubation at pH 7.5 on Low-K,,, Phospholipase A, Activity, but not on High-K, Activity* Preincubation time at 60°C (minutes) 0 15 30 45

11 [ phosphatidylcholie] (pM)

Fig. 6. Inhibition of “low-K,” PLA, activity by quinacrine. PLA, activity in acid extracts was measured in the presence of different concentrations of phosphatidylcholine (2.0-5.0 pM) and quinacrineHCl (0-2.0 mM). Double reciprocal plots of reaction velocity as a function of substrate concentration were constructed at each concentration of quinacrine. Values represent the reciprocal of average reaction rate (n = 3 for each point); error bars represent the reciprocal of the upper 90% confidence limit for the average rate. Pearson’s product-moment correlation coefficients for curves generated with 0, 0.5 mM, 1.0 mM, and 2.0 mM quinacrine are 0.994, 0.993, 0.996, and 0.985, respectively.

I

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[Quinacrine]= 1.OmM

[Qumacrine]=O.BmM

[~uinacrine]=~

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-8

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[ phosphatidylcholine1 (mM)

Fig. 7. Inhibition of “high-K,” PLA, activity by quinacrine. PLA, activity was measured in the presence of different concentrations of phosphatidylcholine (0.05-0.25 mM) and quinacrine-HC1 (0-2.0 mM). Additional information is provided in the legend to Figure 6. Pearson’s product-moment correlation coefficients for curves generated with 0,0.5 mM, 1.0 mM, and 2.0 mM quinacrine are 0.998,0.988, 0.995, and 0.980, respectively.

313

PLA, activity (mIU/mg protein) 5.0 pM PC 100 pM PC 0.70 0.012” 6.8 2 0.84d 0.70 k 0.007” 7.5 2 0.36d 0.55 f 0.006b 7.1 2 0.54d 0.50 k 0.008‘ 6.9 2 0.96d

*Acid extracts were preincubated in the presence of 50 mM Tris-HC1 for various times, as indicated, after which, portions (55 pg protein) were removed and assayed for PLA, activity. Assays were conducted with either 5.0 pM phosphatidylcholine (PC) or 100 pM PC, to estimate the effect of elevated temperature on low-K, and high-Ki, activities, respectively. Enzyme reactions were carried out at 25°C for 15 minutes; other details of the assay are described in Methods. Values represent the average 2 SEM of triplicate determinations. ,-Values with different superscripts differ (PO.l).

= 4;1/[S] = -0.056 ? 0.003 p,M-’). This is consistent with either 1) the removal by TFP of a nonessential activator (Segel, 1975a) or 2) a mixed type of inhibition by TFP (Segel, 1975b). For the former mechanism, K, for the fully activated enzyme would be decreased by a factor a,and V,, increased by a factor p. For the latter mechanism, K, would be increased by a factor (Y (l 0.1).

*

*

Inhibition of PLA, by p-Bromophenacyl Bromide Inhibition of both low-K, and high-K, PLA, activities by p-bromophenacyl bromide increased as a function of time and of inhibitor concentration (Figs. 11and 12). The decrease in activity over time followed first order kinetics. As a n alkylating agent, it is assumed that p-bromophenacyl bromide inactivates PLA, according to the following equation:

SPERM PHOSPHOLIPASE A, AND THE ACROSOME REACTION

315

and 0.68, respectively; df = 5; P > 0.1). The ratio of k,/KD, which is a measure of the efficacy of the inhibitor for the enzyme, was 0.11 mM-'.min-' and 0.09 mM-'.min-' for the low- and high-K, activities, respectively.

Fig. 10. Secondary replot of inhibition of "low-K," PLA, activity by trifluoperazine. l / A slope and l / A intercept were plotted as a function of reciprocal estimated calmodulin (or other activator) concentration in the enzyme preparation, expressed in arbitrary units; A slope (K,/Vmax) represents the difference between this parameter at a given concentration of calmodulin (estimated, on the basis of the concentration of trifluoperazine and the dissociation constant for the calmodulin-trifluoperazine complex) and that of the completely unactivated enzyme. Slopes were derived from the data in Figure 9. A intercept represents the difference between the y intercept in the presence of a given concentration of calmodulin and that of the completely unactivated enzyme. Intercepts were calculated from the data in Figure 9. The common point of intersection of these plots on the x axis is equal to -P/uK,, where PV,,, and uK, are the maximal velocity and apparent Michaelis constant, respectively, for the fully activated enzyme, and K, is the dissociation constant for the activator-enzyme complex (Segel, 1975b). In this instance, (Y = 0.089 and P = 4.27. These values were calculated based upon estimated values of K, and V,, of the unactivated enzyme of 13.9 pM and 0.46 mIU/mg protein, respectively (see Results), and y intercept values for the plots of l / A intercept [0.60,equal to !3Vmax/(!.-1)] and l / A slope (0.034, equal to PV,,,/K,(P-a). Theoretical considerations, and the assumptions upon which calmodulin (or other activator) concentration and kinetic constants for the completely unactivated enzyme were estimated, are presented in the Results. Pearson's product-moment correlation coefficients for the l / A slope plot ( 0 )and the l / A intercept plot ( 0 ) are 0.982 and 0.978, respectively.

Effect of Other Agents on Human Sperm PLA, PLA, was inhibited by the trypsin/acrosin inhibitor, NPGB, with approximately 40% inhibition of high-K, activity observed at 1 mM NPGB (Table 2). However, inhibition did not increase as a function of time, arguing against PLA, inhibition by NPGB as a pseudoirreversible inhibitor (Chase and Shaw, 1970). The low-K, activity was more sensitive to inhibition by NPGB; 1 mM NPGB inhibited the hydrolysis of 5 FM phosphatidylcholine by over 80%.Another trypsidacrosin inhibitor, p-aminobenzamidine (Walsh, 1970), had no apparent effect on PLA, activity at a concentration of 5 mM (Table 2). A more detailed evaluation of the effect of p-aminobenzamidine on low- and high-K, PLA, activities showed biphasic effects on enzyme activity, with maximal activities occurring at approximately 5 mM and 2 mM p-aminobenzamidine (45 2 4% and 47 2 13% increase over control rate; Fig. 131,respectively. In contrast, 10 mM p-aminobenzamidine reduced the rate of phosphatidylcholine hydrolysis by both low- and highK, activities (Fig. 13). Manganese chloride inhibited both low- and high-K, activities to approximately the same extent. Reaction rates in the absence and presence of 5 mM MnC1, were 1.1 2 0.06 and 0.25 2 0.010 mIU/mg protein, respectively, when PLA, was assayed with 5 FM phosphatidylcholine and 4.2 2 0.12 and 1.0 2 0.08 mIU/mg protein when assayed with 100 pM phosphatidylcholine (n = 3 for each group; P < 0.001, 2-tailed t test). No further analysis of this effect was conducted.

Effect of PLA, Inhibitors on Ionophore-Induced Acrosome Reaction Based on the above data, the effects of quinacrine, trif luoperazine, p-bromophenacyl bromide, and MnC1, where [I] is the concentration of inhibitor, KD is the on the human acrosome reaction were examined (Table reversible dissociation constant of enzyme-inhibitor 3). Of the four inhibitors, only 0.4 mM quinacrine and complex, E-I, and k, is the first-order rate constant for 5 mM MnC1, significantly inhibited (32% and 93%, reformation of the irreversibly inhibited enzyme, E-1'. spectively) the A23187-induced acrosome reaction. In contrast, 5 mM MnCl, had no effect on the acrosome [El, represents the concentration of free enzyme. Replots of the reciprocals of the slopes of the linear reaction induced by ionomycin. Due to the irreversible nature of PLA, inhibition by curves in Figures 11and 12 as a function of reciprocal inhibitor concentration yielded similar linear curves p-bromophenacyl bromide (see above, Reversibility of for both low- and high-K, activities (r = 0.985 and Inhibition of Human Sperm PLA, by Phospholipase In0.981, respectively; data not shown), from which KD hibitors), it was possible to test its effectiveness when and k, were derived (y intercept = Uk,; slope = KD/k,; added to whole-cell suspensions. In a separate set of Ganu and Shaw, 1981). Values of KD and k, were 0.28 experiments, spermatozoa were incubated in capacitamM and 0.032 min-' for the low-K, activity, and 0.73 tion medium for 90 minutes, and either DMSO (solvent mM and 0.066 min-' for the high-K, activity. Neither for p-bromophenacyl bromide; 1% final concentration), slopes nor intercepts for the replots differed (t = 1.36 0.02 mM or 0.2 mM p-bromophenacyl bromide was

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111111111111

1

2.2

100

200

Time (mid

Fig. 11. Inhibition of “low-K,” PLA, activity by p-bromophenacyl bromide. PLA, activity was measured in the presence of 5 KM phosphatidylcholine. Other details are presented in the legend to Figure 12. Pearson’s product-moment correlation coefficients for linear curves generated with 0.05 mM (A),0.065 mM (01, 0.10 mM (o), and 0.20 mM (A) p-bromophenacyl bromide are 0.988, 0.999, 0.991, and 0.999, respectively.

added. After a n additional 90 minutes under capacitating conditions, spermatozoa were sedimented and washed twice by centrifugation, and extracted for PLA, (Methods). Both concentrations of p-bromophenacyl bromide inhibited PLA, when measured either with 5 pM or 100 pM phosphatidylcholine (Table 4). Inhibition of PLA, in situ by 0.02 mM (28%; 90% confidence limits = 21.6-36.0%) and 0.2 mM (88%;84.391.1%) p-bromophenacyl bromide agreed reasonably well with inhibitions (21% and 78%, respectively) predicted from kinetic constants derived from isolated PLA, (see above, Inhibition of PLA, by p-bromophenacyl Bromide). Percent inhibitions of the acrosome reaction and of PLA, activity were calculated from the data in Table 3 and from the kinetics of inhibition of PLA, activity in acid extracts. Percent inhibition of PLA, by 0.4 mM quinacrine hydrochloride (47%) was calculated based upon the assumption that quinacrine acts as a noncompetitive inhibitor, with a n average Ki for both low- and high-K, activities of 0.45 mM. Percent inhibition of PLA, by TFP (24%) was approximated, based upon two types of inhibition: 1)noncompetitive, for the high-K, activity (Ki= 87pM) and 2) “mixed type” (or removal of

a nonessential activator), where inhibition varies as a function of substrate concentration. The calculated inhibition for the high-K, (24%) was used, since the endogenous substrate and its concentration at the enzyme active site in spermatozoa is unknown. The estimate is reasonable for the low-K, activity as well, since the predicted inhibition by 20 pM TFP ranged from 17-35% as the substrate concentration decreased from 10 to 0.25 K, (Fig. 9). PLA, inhibition by 5 mM MnC1, (77%) was directly determined for PLA, in acid extracts (see above, Effect of Other Agents on Human Sperm PLA,). Inhibition of PLA, by 20 FM pbromophenacyl bromide (29%) was calculated from the data in Table 4. The effects of all four agents on the acrosome reaction (Table 3) were poorly correlated with their inhibitory activities toward human sperm PLA, (Kendall’s tau = 0.316; P > 0.1). The effect of MnC1, on the ionomycin-induced acrosome reaction, rather than on that induced by A23187, was used in the correlational analysis. The latter effect most likely represents a n artifact specific to the properties of A23187 (see Discussion). DISCUSSION Intracellular phospholipase A, has been found in every mammalian tissue examined (Van den Bosch, 1980). PLA, has been implicated in the generation of second messengers and in physiologically important processes, such a s the inflammatory response (Van den Bosch, 1980; Alonso e t al., 1986; Fawzy et al., 1987) and the release of secretory granules (Best et al., 1984). PLA, has been detected in spermatozoa from several species, including man (Thakkar et al., 1984; Vainio et al., 1985; Weinman et al., 1986; Bennet et al., 1987; Hinkovska et al., 1987; Antaki et al., 1988; Guerette et al., 1988). Further, the in-vitro acrosome reaction of nonhuman spermatozoa is decreased by PLA, inhibitors (Lui and Meizel, 1979; Ono et al., 1982; Singleton and Killian, 19831, while being stimulated by products of PLA, action (Kyono e t al., 1984; Meizel and Turner, 1984; Yanagimachi and Suzuki, 1985). These findings make a role of PLA, in the acrosome reaction a n attractive hypothesis. However, the PLA, inhibitors used in the above studies are not specific (Introduction), and potential products of PLA, action that stimulated the acrosome reaction are not necessarily, either qualitatively or quantitatively, those produced by spermatozoa. Further, previous studies (On0 et al., 1982; Thakkar et al., 1984; Langlais and Roberts, 1985) that addressed the inhibitor sensitivity of sperm PLA, were not amenable to kinetic interpretation, due to the use of aggregated substrate for PLA, assay (Introduction). It was therefore necessary to reexamine the catalytic properties of PLA, from human spermatozoa, such th a t subsequent kinetic evaluation of known PLA, inhibitors was possible. These properties are described in the present report.

SPERM PHOSPHOLIPASE A, AND THE ACROSOME REACTION

O

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40

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ao

100 Time (mid 120 140

Fig. 12. Inhibition of "high-K," PLA, activity by p-bromophenacyl bromide. The decrease in reaction velocity (measured in the presence of 0.1 mM phosphatidylcholine) followed first-order kinetics, with a linear time-dependent decrease in In v. Values represent the natural logarithm of the average of triplicate determinations. Standard error

180

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bars represent 90% confidence limits, where indicated. Pearson's product-moment correlation coefficients for linear curves generated with 0.05 mM (A), 0.065 mM (01, 0.08 mM (A), 0.10 mM (01, and 0.20 mM ( 0 ) p-bromophenacyl bromide are 0.973,0.998,0.999,0.992,and 0.998, respectively.

TABLE 2. Inhibition of Human Sperm PLA, by NPGB, but not by P-Aminobenzamidine" ~~~

~

PLA, activity (mIU/mg protein)

Additions

* *

5.3 0.37 (8)b None NPGB (10-4 MI 4.2 f 0.08 (3)" NPGB (10-3 MI 3.1 0.16 (7)d p-Aminobenzamidine (5mM) 5.2 t 0.33 (4)b Evidence against irreversible inhibition of human sperm PLA, by NPGB" Preincubation time PLA, activity (min) (mIU/mg protein) [Phosphatidylcholine] 5 CLM 100 pM No additions 1 mM NPGB No additions 1 mM NPGB 5 0.78 t 0.02 0.13 t 0.02 7.4 k 0.55 4.2 0.64 10 0.74 0.01 0.08 0.02 5.7 t 0.26 4.0 0.55 20 0.79 f 0.01 0.14 f 0.02 6.6 t 0.26 4.1 f 0.10 40 0.73 0.04 0.10 2 0.01 6.7 f 0.66 4.3 0.64

* *

317

*

* * *

"PLA, activity was measured as described in Methods. Enzyme was preincubated for 5 minutes at pH 7.5 in the presence of the indicated additions prior to initiating reactions with 100 pM phosphatidylcholine. Data are expressed as averages f SEM, with the number of determinations indicated in parentheses. MValues with different superscripts differ (P< 0.05, Newman-Keuls test). eEnzyme was preincubated in the presence or absence of 1 mM NPGB at pH 7.5 for the indicated period, prior to initiating the reaction with either 5 pM or 100 pM phosphatidylcholine. Data are presented as averages f SEM of three separate determinations. Analysis of variance yielded significant effects of NPGB on PLA, activity when assayed in the presence of 5 pM or 100 pM phosphatidylcholine (F:,=2,133 and F:,=47.6, respectively; P < 0.001). No time/inhibitor interactive effect was apparent at either concentration.

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Fig. 13. Biphasic effect of p-aminobenzamidine on PLA, activity. a) Effect on high-K, activity. Values represent the average 2 SEM of triplicate determinations of reaction rates a t each concentration of p-aminobenzamidine, measured in the presence of 0.05 mM (A),0.067 mM (o),0.10 mM (A),or 0.25 mM ( 0 )phosphatidylcholine. b) Effect on low-K, activity. Values represent the average f SEM of triplicate determinations of reaction rates at each concentration of p-aminobenzamidine, measured in the presence of 2.0 pM (A),2.5 pM (O), 3.33 pM (A),or 5.0 pM ( 0 ) phosphatidylcholine.

Use of PLA, Assay Suitable for Kinetic Studies PLA, was measured in the presence of a modified monomeric form of phosphatidylcholine, essentially as described by Katsumata et al., (1986). Sodium deoxycholate was added at levels below its critical micelle concentration (CMC; 4-6 mM, Dawson et al., 1986a). Sodium deoxycholate forms coordination complexes with the hydrophobic tails of phospholipids, preventing the formation of substrate aggregates (Smith et al., 1983; Dawson et al., 1986b). Inclusion of ethanol a t a final concentration of 7% likely contributed to the maintenance of substrate dispersion in the assay mixture, resulting in a n optically transparent homogenous solution of substrate at all concentrations examined. While the physiological significance of monomeric dispersions of phospholipid substrate may be argued, this state allows for the measurement and kinetic evaluation of PLA, under defined conditions. These conditions are not achievable with aggregated substrate, es-

pecially when prepared in a n uncontrolled manner. Good agreement between kinetics of inhibition of isolated human sperm PLA, by p-bromophenacyl bromide with inhibition of the enzyme in situ (Figs. 11 and 12, Table 4) supports the validity of the enzyme assay used herein. Unstable, heterogeneous particulate forms of the substrate (as contained in preparations sonicated at room temperature) are not likely to be more physiologically similar to the structured lipid domain of cellular membranes a s compared to the form of substrate used in the present study. Acid extracts used in the present study, as described by Thakkar et al. (19841, are suitable for the characterization of PLA, with regard to inhibitor sensitivity. Linear increases in product (palmitate) formation as a function of time and amount of enzyme (Results) indicate that the acid extract is free from confounding factors that may hinder accurate data interpretation. Total and specific enzyme activities were higher in the acid extracts than in sonicated sperm preparations. This apparent increase agrees with previous studies of human sperm PLA, (Thakkar e t al., 1984). Similar findings have also been made for PLA, from rat spleen (Nakaguchi et al., 1986) and lung (Lindahl et al., 1989). These data result in part from partial purification of the enzyme. Extraneous protein exerts nonspecific inhibitory effects on phospholipases A (Di Rosa et al., 1988; Kunze et al., 1988). Inhibitory substances may also have been removed, a s reported for other tissues (Hostetler et al., 1986; DiRosa et al., 1988). An apparent increase in activity may also be caused by the removal of endogenous phospholipids from the enzyme preparation that can act as substrates and effectively compete with the radiolabeled substrate used for PLA, assay (Kramer et al., 1986; Pierik et al., 1988).

Identification of PLA, Activity in Sperm Extracts The phospholipase activity of sperm acid extracts is of the A, type (Fig. 1). Hydrolysis of the acyl group at the sn-2 position of phosphatidylcholine can result from either the action of PLA, or the sequential actions of PLA, and lysophospholipase, or of phospholipase C (PLC) and diacylglycerol lipase (Lapetina and Crouch, 1989). If the PLA, pathway were involved in hydrolysis a t the sn-2 position, the appearance of radiolabeled palmitate from phosphatidylcholine hydrolysis when both 1- and 2-acyl groups were labeled would exceed the radiolabel associated with lysophosphatidylcholine. This was not observed (Fig. 1).The ratio of radiolabel comigrating with palmitate obtained with L3H2-palmitoyl] phosphatidylcholine to that obtained with [14C-1,2-dipalmitoyl] phosphatidylcholine of the same specific activity would be less than 2:l if PLA, activity were contributing to the release of radiolabeled palmitate. However, this ratio was 2.0:l (data not shown). Finally, the absence of radiolabel a t the solvent front (corresponding to the migration of diacylglycerol) pre-

SPERM PHOSPHOLIPASE A, AND THE ACROSOME REACTION

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TABLE 3. Effects of Phospholipase A, Inhibitors on the Human Acrosome Reaction* Additions during capacitation

Preincubation time (minutes)

None

180

0.4 mM Quinacrine (n = 5) 0.02 mM Trifluoperazine (n = 5 ) 0.02 mM p-bromophenacyl bromide (n = 3) 5 mM MnC1,

5 90 90 180

Ionophore-induced acrosome reaction A23187 Ionomvcin (10 pM) (10 i M ) 18.9 (17.4-20.4)" 23.6 (21.8-25.4Id n = 2 n = 3 12.9 (10.8-15.2Ib 18.0 (15.4-20.8)" 15.7 (11.3-20.7)"~~ 1.3 (0.5-2.6)' 23.9 (17.5-30.5)d n = 7 n = 3

*Washed spermatozoa from fresh ejaculates were capacitated for 3 hours, as described in Methods. Various additions were made during capacitation and were incubated for the indicated periods immediately preceeding induction of the acrosome reaction by the addition of ionophore. The actual frequency of acrosome-reacted spermatozoa observed in each experiment was higher (by approximately 14%) than reported in the Table, since only the ionophore-induced acrosome reaction was considered. Ionophore-induced acrosome reaction was taken as the difference in acrosome-reacted spermatozoa between samples with and without ionophore. Data were subjected to arcsine transformation prior to further analysis. Values reported are the backtransform (measured in percent) of the average arcsine transform, with 90% confidence limits indicated in parentheses. After 3 hours, 14.4 (13.3-15.51% of spermatozoa had acrosome-reacted in the absence of ionophore. None of the agents, at the concentrations indicated, affected this response. Two-way analysis of variance of data regarding the effects of ionophore type and MnCl, on the acrosome reaction showed significant ionophore (F:6 = 49.08; P