Diacylglycerol and alkylacylglycerol stimulate ram sperm phospholipase A2

Pergamon

lr~r J. &o&r,~. Vol. 26, No. 7. pp. Y51~958. 1994 Copyright c 1994 Elsevier So~ence Ltd Printed in Great Britain. All rights reserved 0020.71 I X:94 $7.00 + 0.00

0020-71 lX(93)EOO60-3

DIACYLGLYCEROL AND ALKYLACYLGLYCEROL STIMULATE RAM SPERM PHOSPHOLIPASE A2 E. R. S.

ROLDAN,‘*

R. MARTINEZ-DALMAU’

‘Department of Development and Signalling, and ‘Centro

de lnvestigaciones

MOLLINEDO~

AFRC Babraham Institute, Cambridge CB2 4AT. U.K

Bioldgicas. (Received

and F.

CSIC.

ciVel6zquez

144. 28006.Madrid,

Spain

1 O~~toher 199.7)

We have investigated the susceptibility of ram sperm phospholipase AZ (PLA,) to stimulation by diacyl- and alkylacylglycerols and by monoacyl- and monoalkylglycerols. 2. PLAz activity in sonicates from ram spermatozoa was enhanced when I-stearoyl-2arachidonoyl-sn-glycerol, the diacylglycerol usually generated by polyphosphoinositide breakdown, was added to a radioactive phosphatidylcholine substrate; the effect was time- and Ca*+-dependent. 3. Both diacyl- and alkylacylglycerol considerably enhanced PLAz activity; I-0-hexadecyl-20-methyl-rat-glycerol, however. only showed slight stimulatory ability. 4. The monoradylglycerols I-monohexadecanoyl-rat-glycerol, 2-monohexadecanoylglycerol, and I-0-hexadecyl-sn-glycerol had very little effect on the enzyme’s activity. 5. Exposure of spermatozoa to I -oleoyl-2-acetyl-sn-glycerol (OAG) or I -0-hexadecyl-2acetyl-rat-glycerol ( I-0-C,,/2-CZ), when cells were stimulated with the ionophore A23187 and Ca’+, resulted in higher PLA, activity in sperm sonicates. Furthermore, parallel experiments showed that exocytosis was enhanced if spermatozoa were treated with A23187/Ca’+ and either OAG or I-0-C,,/2-Cz. Since both diacyl- and alkylacylglycerols increased PLA, activity Abstract-l.

and exocytosis, stimulation of PLA, activity by these diglycerides may take place independently from protein kinase C activation.

the mechanisms involved in its modulation. Activation of G-proteins as well as a rise in intracellular Cal+ have been reported to activate this enzyme (Burgoyne et al., 1987). In addition, diglycerides generated upon cell activation have been invoked as stimulators of PLA,; whether they act directly or via activation of protein kinase C is still a matter of active debate (Dawson et al., 1984; Kramer et ui., 1987; Schonhardt and Ferber, 1987; Burch, 1988; Bauldry et al., 1988, 1991). PLA, seems to play a fundamental role in the exocytosis of the sperm acrosome, an essential process in the series of phenomena leading to fertilization. Early studies have provided circumstantial evidence in favour of this idea (Ono et al., 1982; Llanos et al., l982), and it is only very recently that a clear demonstration of PLAz involvement in acrosomal exocytosis has been presented (Roldan and Fragio, 1993). However, the mechanisms which modulate PLAz activity in the sperm cell are virtually unknown; recent work has led to the suggestion

INTRODUCTION Phospholipase AZ (PLA2) plays a fundamental role in several cellular processes by generating an array of metabolites with various biological functions. PLA,-mediated hydrolysis of phosphoglycerides results in the release of arachidonic acid and lysophospholipids which may either have direct effects on cellular membranes or can serve as substrates for the generation of additional messengers or active metabolites (Dennis et al., 1991). Despite widespread interest in PLAz there is still controversy regarding

*To whom correspondence should be addressed. Ahhrrriu/ions; PLAz. phospholipase A:: SAG, I-stearoyl-2. arachydonoyl-sn-glycerol: OAG, I-oleoyl-2-acetylsn-glycerol; l,2-dihexadecanoyl-sn-glycerol; diC,, , I-o-C,,!2-cz. 1-0-hexadecyl-2-acetyl-rat-glycerol; diI.2-di-0-hexadecyl-rcc-glycerol; I-0-C,,/2-Oo-c,,, C,. I-0-hexadecyl-2-0-methyl-rcrc-glycerol: I-monoc,,. I-monohexadecanoyl-rut-glycerol: 2-monoC,,. 2-monohexadecanoylglycerol; I -0-monoC,, , I-0-hexadecyl-sn glycerol: Mes. 2-(N-morpholino)ethane-sulphonic acid. 951

952

E. R. S. ROLDAN

that sperm PLAz could be directly modulated by diacylglycerol (Roldan and Harrison. I990a; Harrison and Roldan, 1990). This is an attractive possibility because protein kinase C, an important target of diacylglycerol action in other cells, does not seem to participate in events leading to acrosomal exocytosis (Roldan and Harrison, 1988, 1990b, 1992; Thomas and Meizel. 1989; Domino et ul., 1989; Roldan and Mollinedo, 1991). Evidence favouring the idea that diacylglycerol may activate sperm PLAz has been recently presented (Roldan and Mollinedo, 1991). Diacylglycerol generation in spermatozoa seems to rely on various sources and pathways. A rapid phosphoinositidase C-mediated breakdown of sperm polyphosphoinositides (Roldan and Harrison, 1989; Thomas and Meizel, 1989; Domino and Garbers, 1989) and a concomitant generation of diacylglycerol (Roldan and Harrison, 1990b, 1992), have been observed after Ca’+ entry triggered either by treatment with Cal+ and the ionophore A23187, or by putative natural agonists. Moreover, cholineand ethanolamine-containing phosphoglyceserve as additional sources rides may (Selivonchick et al., 1980; Nikolopoulou et al., 1985; Aveldano et al., 1992). because the total mass of diacylglycerol generated after cell stimulation exceeds that potentially derived from the phosphoinositides (Roldan and Harrison, 1990b, 1992). It should be noted that, in addition to diacyl-phosphoglycerides, sperm membranes contain a high proportion of alkylacyland alkenylacyl-phosphoglycerides (Selivonchick et al., 1980; Nikolopoulou et ul., 1985; Aveldaiio et al., 1992). Thus, upon hydrolysis of membrane phospholipids, molecular species of diacyl-, alkylacyland alkenylacylglycerols (i.e. diradylglycerols) could be generated (e.g. Agwu et al., 1989; Dougherty et al., 1989). These metabolites may differ from the phosphoinositidase C-derived diacylglycerol in activity, catabolism and, especially, activation of downstream processes. In particular, nothing is known about the ability of alkyl-containing diglycerides to affect molecular events leading to acrosomal exocytosis. In order to gain insight into the regulation of sperm PLAz we have compared the ability of diacyl- and alkylacylglycerols, and monoacyland monoalkylglycerols, to influence sperm PLAz activity. We have found that diradylglycerols enhanced PLAz activity and acrosomal exocytosis. We report here for the first time that

('I01.

alkylacylglycerol is able to stimulate PLAL activity directly, that it can enhance PLA, activation when added to intact sperm cells along with the ionophore A23187 and Ca’+ and, furthermore, that it can enhance exocytosis of the sperm acrosome when Ca” entry is triggered by A23 187. MATERIALS

AND

METHODS

Materials

L-3-Phosphatidylcholine, I -stearoyl-2-[ I -‘“C]arachidonoyl (sp. act. 56 mCi/mmol), was purchased from Amersham International (Amersham, Bucks., U.K.). L-3-Phosphatidylcholine, I-stearoyl-2-arachidonoyl, poly(vinyl)alcohol, polyvinylpyrrolidone, Tris and bovine serum albumin (fatty acid free) were from Sigma Chemical Co. (Poole, Dorset, U.K.). Hepes and Mes were from BDH (Poole, Dorset, U.K.). The ionophore A231 87 was purchased from Calbiochem (Nottingham, U.K.). I-stearoyl-2arachydonoyl-sn -glycerol, I -oleoyl-2-acetyl-sn glycerol. I ,2-dihexadecanoyl-sn -glycerol, 1-Ohexadecyl-2-acetyl-rut-glycerol, I ,2-di-O-hexadecyl-rut-glycerol, I-0-hexadecyl-2-O-methylI -monohexadecanoyl-ruc-glycrat-glycerol, erol, 2-monohexadecanoylglycerol, l-o-hexadecyl-sn -glycerol. and I -0-octadecyl-rat-glycerol were purchased from Sigma and 5 mM stock solutions were made in n-hexane, kept at -20 C, and used within I week. Lipids used as standards (arachidonic acid, 1,2-dioleoyl-rucglycerol and 1,3-dioleoylglycerol) were also from Sigma. Organic solvents were of analytical grade from BDH or Merck (Darmstadt, Germany). All other general chemicals were purchased from BDH or Fluka (Buchs, Switzerland). Preparation

and

treatment

qf‘ spermatozou

Ejaculated spermatozoa from Manchego or Clun Forest rams were separated from seminal plasma by dilution in 0. I6 M-NaCI and washing through a buffer whose composition was: 10 mM-Mes, 255 mM-sucrose, 2.5 mM-KOH (pH 6.1). After centrifugation at 400~ 5 min and IOOOg,,, for IO min, most YE 2:: supernatant was removed, the cells were gently resuspended and their concentration was estimated using a hemocytometer. Spermatozoa in buffer were sonicated with three 15 set bursts (50 W). Ejaculated spermatozoa were also stimulated to undergo acrosomal exocytosis, in the absence

Diradylglycerols

stimulate

or presence of a diacyl- and an alkylacylglycerol, and then sonicated to release PLAz activity. Ram spermatozoa were separated from seminal plasma by dilution and washing through a sucrose medium consisting of 222 mM-sucrose, 2.5 mM-KOH, 10 mM-glucase and 20 mM-Hepes (pH 7.55, adjusted at 2O’C with NaOH) (Roldan and Harrison, 1988). and then incubated in a similar medium containing 142 mM-NaCl in place of the sucrose. Both sucrose-containing and saline media also contained 1 mg poly(vinyl)alcohol/ml and I mg polyvinylpyrrolidone/ml, and had an osmolality of 305 mOsm/kg. Acrosomal exocytosis was induced by incubating cells (about 5 10 x IO’ spermatozoa/ml) with Ca’+ (3 mM) and the divalent cation ionophore A23187 (1 PM) in 0.5 ml of saline medium at 37°C (Shams-Borhan and Harrison, 1981), in the absence or presence of 1-oleoyl-2-acetyl-sn -glycerol (25 p M) or 1-0-hexadecyl-2-acetyl-racglycerol (25 ,uM). After 20 min of incubation, sperm samples were rapidly frozen to -79 ‘C by placing the tubes in a mixture of 2ethoxyethanol and dry ice. Sperm samples were then thawed at 4 C, sonicated with three 15 set bmtS (50 W), and then assayed for PLAz activity as described below. Parallel sperm samples were similarly incubated and the occurrence of acrosomal exocytosis was monitored by phasecontrol microscopy of glutaraldehyde-fixed subsamples (Shams-Borhan and Harrison, 1981). A.SSUJ~,for phospholipase

A, in spermatozoa

PLA, activity was measured as described (Roldan and Mollinedo, 1991), with slight modifications. Briefly, PLAz activity was assayed in 250~1 of a buffer consisting of 100 mM-TrissHCl, 6 mM-CaCl,, 0.5 mg fattyacid-free bovine serum albumin/ml, and 50% glycerol, v/v (final pH 7.5). Radiolabelled phosphatidylcholine substrate (10 nmol; 3000c.p.m./nmol) was included in the reaction mixture as a liposomal suspension by sonication during 10 min in the buffer. Where indicated, different di- or monoradylglycerols were added to the reaction mixture and sonicated along with the substrate. The reaction was started by the addition of 5-8 ~1 of the sperm homogenate (3.5-5 x lO”cells) and, unless indicated otherwise, was carried out at 37°C for 30 min; it was stopped by the addition of 0.94ml of chloroform/methanol (1: 2, v/v). Lipids were extracted as described (Bligh and Dyer, 1959), arachidonic acid (5 p g). 1,2-dioleoylglycerol (5 pg)

phospholipase

A2

953

and 1,3-dioleoylglycerol (5 pg) being added as carriers, and were then separated by thin layer chromatography on silica-gel 60-coated plates (E. Merck, Darmstadt, Germany) with n-hexane/diethyl ether/acetic acid (70 : 30 : I, v/v) as the solvent system. Lipid spots were visualized by staining with iodine vapours, identified by comparison with standards run in the same plate, scraped off and the radioactivity in each determined by liquid scintillation counting. Statistics Results are means i SE. Significance of results was examined using Student’s t-test or analysis of variance (ANOVA); values of P < 0.05 were regarded as statistically significant. RESULTS

Phospholipase A, activity in sonicates of ejaculated ram spermatozoa was linear with time up to 30 min. Addition of I-stearoyl-2-arachidonoyl-sn-glycerol (SAG), the diacylglycerol usually generated by phosphoinositidase C-mediated hydrolysis of the polyphosphoinositides (Berridge, 1987), markedly increased the PLAz activity of ram sperm sonicates. As seen in Fig. l(A), addition of SAG to the radioactive phosphatidylcholine substrate resulted in a higher production of free [‘“Clarachidonic acid (2-factor ANOVA: treatment, F = 8.43, P < 0.005; time, F = 20.82, P = 0.0001). The effect of SAG was entirely dependent on the presence of Ca’+ [Fig. l(B)]. Addition of SAG by itself did not induce PLA, activity, but greatly increased the enzyme’s activity when Ca2+ was also present. The hydrolysis of sperm diacyl- and alkylacylphosphoglycerides by a phospholipase C would result in the generation of an array of diradylglycerols. We have therefore compared the effects of PLA, of diester, ester/ether and diether diradylglycerols, and compounds with substituents of varying chain length in position 2 of the glycerol backbone (all the diradylglycerols tested had fatty acids/alcohols with C,, chains in position 1). It was found that, when added to the phosphatidylcholine substrate, I ,2-dihexadecanoyl-sn-glycerol (die,,), I-0-hexadecyl-2acetyl-rat-glycerol (I-0-C,,/2-C?), I ,2-di-Ohexadecyl-rrrc-glycerol (di-O-C,,), and I-O-hexadecyl-2-0-methyl-rat-glycerol (I-0-C,,/2-OC,) enhanced the activity of PLA, (Fig. 2). In general, addition of up to 25-37.5 p M of each

E. R. S. ROLDAN et rrl.

o.4-I

0

5

10

15

20

25

0 q

-&+/-SAG +C$+ I-SAG &+/+SAG

l-

30

Time (min) Fig. 1. Effect of I-stearoyl-2-arachidonoyl-sn-glycerol (SAG) on the production of arachidonic acid by ram sperm PLA,. (A) Time~course of enzyme activity in the absence or presence of SAG. Radiolabelled phosphatidylcholine substrate was sonicated in the assay buffer (6 mM Ca” ) with or without SAG (25 )tM) and ram sperm sonicates (5 x IO” cells) were incubated for different times at 37 C, after which and methods”. (B) Effect of Ca’ ’ reacttons were stopped and lipids extracted as indicated in “Materials and/or SAG on PLA, activity. Radiolabelled phosphatidylcholine substrate was sonicated in the assay buffer (with or without 6 mM-Ca?+ ) in the absence or presence of 25 11M-SAG. and ram sperm sonicates (5 x lObcells) were then added and incubated for 15 min at 37 C. Reactions were stopped and lipids extracted as indicated in “Materials and methods”. Results are averages of duplicate determinations of two (A) and three (B) separate experiments. *P < 0.05 when compared with + Ca’+ )/- SAG.

diradylglycerol increased PLAz in a dose-dependent fashion, whereas higher concentrations resulted in lesser increases; similar biphasic effects

were found when human platelet PLA, was exposed to similar diacylglycerol concentrations (Kramer et cd., 1987). The alkyl-methyl-glycerol

0.5 m 5 rg % is! ‘E

0.4

0.3

NT

Y sai

0.2

B f z*

0.1

E a 0.0

diC16

l-0-qfj2-C*

di-O-C16

l-O-C~~2-O-C~

Fig. 2. Effect of diradylglycerols on the production of ardchidonic acid by ram sperm PLA,. Diradylglycerols were added to the reaction mixture and sonicated with the radiolabehed phosphatidylcholine substrate. Assays were carried out by incubating the sperm sonicate (5 x IOh cells) for 30 min at 37 C as described in Materials and methods section. Results are averages of duplicate assays carried out on tive different occasions. DiC,,: 1.2-dihexadecanoyl-sn-glycerol; I-0-C,,/2-C,: I-0-hexadecyl-2-acetyl-ru(,glycerol: di-O-C,,: l.2-di-0-hexadecyl-rrrc-glycerol; l-0-C,,:‘2-O-C, : I-O-hexadecyl-2-O-methyl-rcrc-glycerol. ‘P <0.05 when compared with OicM.

Dimdyiglycerols

1-monoCl6

stimulate

2-monoC 16

phospholipase

955

A,

1-o-mOnOC 16

diCr6

Fig. 3. Effect of monoradylglycerols on the production of arachidonic acid by ram sperm PLAz. Monoradylglycerols were added to the reaction mixture and sonicated with the radiolabelled phosphatidylcholine substrate. Assays were carried out by incubating the sperm sonicate (5 x IOhcells) for 30 min at 37°C as described in the Materials and methods section. Results are averages of duplicate assays carried out on at least three different occasions. I-monoC,,: I-monohexadecanoyl-rag-giycerol~ 2-monoC,h: 2-monohe~decanoylgiycerol~ I-O-monoC,6: I-0-hexadecyl-stt-glycerol; diC,,: 1,2-dihexadecanoyl-snglycerol. *P < 0.05 when compared with 0 ,uM.

1-0-&,/2-O-C, departed from the general stimulatory effect seen with the diradylgIy~erols because its inclusion only showed a slight stimulation of PLA, activity at the concentrations used in this study. To further examine the specificity of the diradylgiycerol effect, we compared the ability of three monoradylglycerols to influence PLAz activity with that of diC,,, a diacylglycerol shown to enhance the enzyme’s activity (see above). Figure 3 shows that the presence of only one acyl group was not sufficient to grant stimuiatory ability because neither 1mono-hexadecanoyi-~uc-glycerol (I -monoC,,) nor 2-mono-hexadecanoylglycerol (ZmonoC,,) enhanced PLA, activity. On the other hand, the presence of a fatty alcohol in position 1 seemed to confer a slight stimulatory ability to I-O-hexadecyl-sn-glycerol ( I-0-monoC,,); this slight stimulatory effect was seen with ail the concentrations tested (12.5-50 p M). In a different set of experiments, ram spermatozoa were stimulated with the ionophore A23187 and Ca’+, in the absence or the presence of the permeable diacy~glycerol 1-oleoyl-2acetyl-glycerol (OAG) or alkylacyigtycerol I-0-C,,/2-CZ, before they were sonicated to release PLA, activity. Sonicates from spermatozoa which had been stimulated with A231 87 and Ca”+ and higher PLAz activity than those that

received no treatment (Table 1). Sonicates from cells that were exposed to A23187/Ca” and either OAG or l-0-C,,/2-C, exhibited even higher PLAz activity (Table 1). To examine the effects of diacyl- and alkylacylglycerol on acrosomal exocytosis, parallel sperm samples were incubated in a saline medium and stimulated with A23187iCa’” in the absence or presence of OAG or I-0-C,,j2Cz. Spermatozoa examined I5 min after the beginning of ionophore treatment revealed that both diacyl- and aIkylacy~glycero1 enhanced consjderably the percentage of cells undergoing exocytosis (Table 1).

DISCUSSION

The results of this study show for the first time that both diacyl- and alkyla~yIglycerol stimulate directly the activity of a Ca”-dependent PLA, from ram spermatozoa, and that both enhance the exocytosis of the sperm acrosome to the same extent. On the other hand, various monoradylglycero~s showed no or very little effect on the enzyme’s activity. Our previous work has shown that there was a positive relationship between the chain length of the fatty acid in position I of the glycerol backbone and the ability of diacylglycerol to enhance PLA, activity (Roldan and Mollinedo,

E. R. S. ROLDAN PI al.

956 Table

I. Stimulation

of ram spermatozoa

Cells treated Control A23187,;Ca’+ +OAG (25 FM) + l-0-C,,&Cz (25 I’M) OAG (25 PM) alone I-0-C,,/2-Cz (25 PM) alone

with A23187/Ca’+ cxocytosis

PLAz activity* (pmol arachidonic acid/mini 0.091 0.276 0.458 0.630 0.104 0.95

k + 7 : ) *

0.01”1 0.05”,‘,” 0.07”’ 0.12” 0.05 0.04

and diradylglyccrols

IOh cells)

enhance

PLA,

activity

and

% of cells undergoing acrosomal cxocytosis after 15 mint 4.3 43.5 62.1 14.2 5.2 6.5

+ 0.9’4’ + 0.6@.‘.” ; 1.3”’ & I .41h’ + 1.2 * 0.8

*Ram spermatozoa in saline medium were incubated with or without A23187 (1 PM) and CaZ+ (3 mM). in the absence or presence of I-oleoyl-2-acctyl-sn-glycerol (OAG) or I -O-hexadecyl-2-acctyl-rLlr-glycerol (I 0 C,,/2-C), for 20 min at 37 C. At the end of the incubation sperm samples were rapidly frozen to -79 C. thawed at 4 ‘C, and sonicatcd as described in the Methods section. Assays for PLAZ activity were carried out by incubating the sperm sonicate (2 x 10” cells) for 30 min at 37-C as described in the Materials and Methods section. Results arc means (&SE) of duplicate assays carried out on three occasions. Wpcrmatozoa treated as indicated above were incubated for 15 min at 37’ C. At the end of the incubation, sperm samples were fixed in a glutaraldehyde fixative and examined by phase contrast microscopy. Results arc means (_+ SE) of three different experiments. Similar superscripts indicate significant differences: “‘P < 0.01; “‘P < 0.05; “‘P < 0.03; 14’P < 0.000 I: “‘P < 0.00I : ‘h’f < b.OOl.

1991). In this study we have found that diradylglycerols containing either ester or ether groups in position 1 have equal stimulatory ability on sperm PLA, (see Fig. 2 and Table 1) and, similarly, that both 1,2-diester- and I ,2-dietherdiradylglycerols exhibited comparable stimulatory ability on PLAz activity (Fig. 2). The only diradylglycerol which differed considerably was I-0-C,,/2-O-C,; it was found that the presence of a methyl group in position 2 resulted in a reduction in the stimulatory ability of this diglyceride. Furthermore, the lack of a second acyl or alkyl group (either in position 1 or 2) as seen in the three monoradylglycerols tested, greatly diminished the stimulatory ability of these compounds. Work by others has shown that various diacylglycerols can enhance PLA, activity when added directly to the substrate (Dawson ct d., 1984; Kramer et al., 1987; Burch, 1988) or when used to “prime” human neutrophils prior to a second stimulus (Bauldry et al., 1988, 1991) but, to the best of our knowledge, so far only one alkylacylglycerol has been found to enhance PLAz activity (measured as arachidonic acid release in labelled cells) (Bauldry rt al., 1988, 1991). Many agonists cause their effects by triggering the hydrolysis of membrane phosphoinositides, which results in the generation of a predominant type of diacylglycerol (1 -stearoyl2-arachidonoyl-glycerol; SAG) (Berridge, 1987). On the other hand, some agonists induce hydrolysis of membrane phospholipids to form both diacyl- and alkylacylglycerol molecular species (Agwu rt ul.. 1989; Dougherty ct al., 1989). The results presented here suggest that

both phosphoinositide-derived SAG as well as diradylglycerols derived from choline- or ethanolamine-containing diacyl- or alkylacyl-phosphoglycerides could have a role in modulating the activity of sperm PLAz under physiological conditions: the direct addition of SAG and other diradylglycerols to the substrate increased PLAz activity, and treatment of cells with A231 87/Ca’+ and a diacyl- or alkylacylglycerol resulted in higher PLAz activities in the sperm sonicate. This effect on PLA, may be physiologically relevant because exocytosis was enhanced by treatment of spermatozoa with permeable diacyl- or alkylacylglycerol. The mechanisms by which diradylglycerols could stimulate PLAz are not clear. Evidence has been gathered in other cell systems in favour of a diacylglycerol-mediated activation of protein kinase C that would, in turn, lead to activation of PLA, (Halenda et al., 1985). However, many studies have supplied good evidence against the mediation of protein kinase C and in favour of a more direct action. Thus, kinase C inhibitors did not affect the diacylglycerolmediated stimulation of PLAz from Swiss 3T3 fibroblasts, HL60 granulocytes or human neutrophils (Burch, 1988; Billah and Siegel, 1987; Bass er ul., 1987) and, moreover, both I ,2- and 1,3-isomers enhanced the activity of PLA, from rat intestinal mucosa and human platelets (Dawson rt al., 1984; Kramer et ul., 1987); it is well documented that only 1.2-diacylglycerol activates protein kinase C (Rando and Young, 1984). Similarly, both diacyl- and alkylacylglycerol were found to enhance the activity of human neutrophil PLAz (Bauldry et rd., 1988,

Diradylglycerols

stimulate

1991) although alkylacylglycerol does not have any effect on, or actually inhibits, protein kinase C (Molleyres and Rando, 1988). Diradylglycerol action may also be related to the translocation of PLA, from cytosol to membranes (Schonhardt and Ferber, 1987), in a manner similar to the effect of diacylglycerol on protein kinase C (Maraganore, 1987). Lastly, another mechanism by which diradylglycerols may affect PLAz activity could be by disruption of cell membranes (Dawson ef al., 1984) therefore allowing increased access of the enzyme to the phospholipid substrate. The results we found in our studies on sperm PLAz suggest that diradylglycerol effects are unlikely to be mediated by protein kinase C for two reasons. Firstly, I ,3-diacylglycerol, which does not stimulate protein kinase C (Rando and Young, 1984), enhanced enzyme activity (Roldan and Mollinedo, 1991) and stimulated acrosomal exocytosis (Roldan and Harrison, 1990b). Secondly, the alkylacylglycerol I-OC,,,/2-Cz, which does not activate PKc (Molleyres and Rando, 1988) enhanced both PLAz activity in vitro and exocytosis (this study). However, whether the diradylglycerol effect is directly on PLAz or by perturbing sperm membranes to make the substrate more available to the enzyme is at present unknown and will have to be resolved in future studies. In summary, we have found that (a) when added to a phosphatidylcholine substrate, both diacyl- and alkylacylglycerols stimulate Ca’+dependent PLA, activity of ram sperm sonicates; (b) treatment of spermatozoa with A23 187/Ca’+ results in an enhancement of PLAz activity in sperm sonicates; (c) treatment of cells with A23 187/Ca’+ and diacyl- or alkylacylglycerol enhanced PLA, activity even further; and (d) spermatozoa treated with A23187/Ca’+ and either diacyl- or alkylacylglycerol revealed higher rates of acrosomal exocytosis than cells treated with A23187/Ca”+ alone. Our findings therefore suggest that the activation/stimulation of sperm PLAz is probably mediated by both diacyl- and alkylacylglycerol via mechanisms not involving protein kinase C. They also imply that both polyphosphoinositides and cholineor ethanolamine-containing phosphoglycerides may contribute to the diradylglycerol pool that modulates PLAz activity. We believe that the enzyme plays a key role in the generation of metabolites that will be instrumental in bringing about membrane fusion (Roldan and Harrison, 1990a; Harrison and Roldan, 1990).

phospholipase

Az

957

Acknonbdgmenrs-We thank Robin Harrison for advice and discussions, Roy Jones for critically reading the manuscript, Isabel Vazquez and Julian Garde for the supply of semen from Manchego rams (Madrid), and Tony Tilley for collecting samples of semen from Clun Forest rams (Cambridge). This work was supported by Ministerio de Education y Ciencia (Spain), a British Council/Spanish Ministerio de Education y Ciencia “Accibn Integrdda”, Grant PM89-0003 from DGICYT (Spain), and by the Agricultural and Food Research Council (U.K.).

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