Stimulation of platelet-activating factor synthesis by progesterone and A23187 in human spermatozoa.

Stimulation of platelet-activating factor

synthesis by

progesterone

and

A23187

in

human

spermatozoa

Elisabetta

BALDI,t

Costanza FALSETTI,

Csilla KRAUSZ, Ginetta GERVASI, Vinicio CARLONI,* Rosaria CASANO

and

Gianni

FORTI

Dipartimento diFisiopatologia Clinica, Unita di Andrologia,and *Istitutodi Clinica Medica 11, Universitadegli Studi di Firenze, Firenze, Italy

The presence of platelet-activating factor (PAF) has been demonstrated recently in mammalian spermatozoa, together with evidence forarole of thisphospholipid in enhancing sperm motility and fertilizing ability. To investigate whether PAF synthesis and release occurs in human spermatozoa following incubation with stimuli that induce acrosome reaction, spermatozoa were incubatedwith progesterone and A23 187, two known inducers of the exocytotic event. PAF synthesis (remodellingpathway) wasassessed by[3H]acetateincorporation into PAF. Treatment of spermatozoa with progesterone and A23187 resultedin anincreaseof[3H]acetateincorporation into PAF. Mostofthe newlysynthesized[3H]PAF formedin response to acrosomereaction wasfound inthe supernatant, suggesting a release of the phospholipidfromspermatozoa. PAF-like material

INTRODUCTION

Platelet-activating factor (PAF) is a potent pro-inflammatory, lipid-derived mediator having several biological effects, including aggregation and degranulation of platelets and neutrophils and increased vascular permeability (for review see Snyder, 1990).

PAFhas been detected in rabbit and humanwashedspermatozoa

(Kumar et al., 1988; Kuzan et al., 1990; Parks et al., 1990;

Minhas et al., 1991), indicating that these cells are able to

synthesize this phospholipid. Recently, addition of PAFtosperm

incubation medium has been reported to enhance motility of humansperm(Rickeretal., 1989; Hellstrometal., 1991) andin vitrofertilization of rabbit andmouseoocytes(Roudebushetal., 1990; Kuzan et al., 1990). Conversely, treatment of rabbit

spermatozoawithPAF-receptor antagonists inhibits fertilization ability (Kumaretal., 1988;Harperetal., 1989). Atpresent,it is

not known whether PAF production by spermatozoa can be

enhancedby stimulation, in particular bystimuli thatinduce the

acrosomereaction. Thisexocytoticeventoccursin spermatozoa

in vitro following treatments that stimulate an increase in the

concentration of intracellular free calcium(Yanagimachi, 1988), such as calcium ionophores (Aitken et al., 1984) and steroids

presentinthe follicularfluid,inparticularprogesteroneand

17a-hydroxyprogesterone (Osmanetal., 1989; Thomas and Meizel, 1989; Blackmoreetal., 1990). Besidesanincrease of intracellular

calcium, ionophores and progesterone activate sperm

phospholipase C (Roldan and Harrison, 1989; Thomas and

Meizel, 1989), and ionophoresincreasespermphospholipaseA2

activity (Takkaretal., 1983;Bennetetal., 1987).Theactivation

of thesephospholipases mightaffect cellular PAFproduction.In

particular, phospholipase A2 activation might mobilize a fatty

extracted from human spermatozoa was able to induce ag-gregation of rabbit platelets and showed identical retentiontime and the same ion m/e values as authentic PAF when analysed with g.c.-m.s. Lyso-PAF: acetyl-CoA acetyltransferase (EC 2.3.1.67)activityinhumanspermatozoa wasalso studiedand showed similarkineticparameters to those described forother cell systems. Stimulation of spermatozoa with progesterone and A23187 induced an increase of [3H]arachidonic acid release, suggestinganactivation of phospholipaseA. Inconclusion,our resultsdemonstratedincreased production and release ofPAF in human sperm following stimulation with progesterone and A23187 andsuggest arole forthisphospholipid in theactivation ofspermatozoa.

acid from the sn-2 position of I-alkyl-linked phosphatidyl-choline (PC), a major phospholipid subclass in spermatozoa (Nikolopoulouetal., 1985;Aveldanoetal., 1992). This release leadstothegeneration of lyso-PAF whichmaybe acetylatedat thesn-2position by lyso-PAF: acetyl-CoA acetyltransferase(EC 2.3. 1 .67) toform PAF. Themajor aim ofthe present study was to evaluate whether incubationwith A23187 and progesterone stimulates PAF production (remodelling pathway) and release fromhumanspermatozoa. Inaddition, some kinetic parameters of lyso-PAF:acetyl-CoA acetyltransferase activity of human sperm were measured.

MATERIALS

AND

METHODS

Materials

A23187, PAF (1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho-choline) and lyso-PAF (1-O-hexadecyl-2-lyso-sn-glycero-3-phosphocholine)wereobtained from Calbiochem(LaJolla, CA, U.S.A.). The sodium salts of[3H]aceticacid (1.9Ci/mmol) and

[3H]acetyl-CoA (4Ci/mmol) were purchased from New

England Nuclear (Boston, MA, U.S.A.). [3H]Arachidonic acid (207Ci/mmol) was purchased from Amersham International (Amersham, Bucks., U.K.). T.l.c. phospholipid standards [PC, lysophosphatidylcholine, phosphatidylethanolamine (PE), phosphatidylserine (PS), sphingomyelin (SM) and arachidonic acid],

fatty-acid-free

BSA, progesterone, acetyl-CoA,

N-formylmethionyl-leucylphenylalanine (fMLP), horseradish per-oxidase (Type IV) and phospholipase C (from Bacilluscereus)

were from Sigma (St. Louis, MO, U.S.A.).

tert-Butyldimethylchlorosilyl/imidazole (TBDMSI) reagent was

obtained from Alltech

Applied

Science

(Deerfield,

IL,

U.S.A.).

Abbreviations used: PAF, platelet-activating factor; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; SM,

sphingomyelin; fMLP,formylmethionyl-leucylphenylalanine; DMSO, dimethyl sulphoxide;TBDMSI, tert-butyidimethylchlorosilyl/imidazole; TNS, 2-p-toluidinylnaphthylene-6-sulphonate; m.i.d., multipleiondetection; s.i.m., selectedion monitoring; BWW,Biggers-Whitten-Whittinghammedium.

H.p.t.l.c. and t.l.c. silica gel 60 plates were from E.Merck (Darmstadt, Germany). Organic solventswerefrom Carlo Erba

(Milan,

Italy). L659,989 was a generousgiftfrom Merck Sharp and Dohme(Rahway, NJ,U.S.A.). A23187 (10 mM) and pro-gesterone (10 mg/ml) were dissolved in dimethyl sulphoxide

(DMSO).

Preparation of

spermatozoa

Human semen was collected, according to the WHO

recommended procedure (1987) by masturbation after3-4days of abstinencefrom 29normozoospermicmenundergoingsemen analysis forcoupleinfertility. Sampleswith alinearprogressive motility ofless than50

%

at60 min and withleucocytes and/or immature germ-cell concentrations greater than106/mlwere not includedinthe study. Occasionally, two to three semen samples were pooled in order to achieve a sufficient number of spermatozoa. Semen samples were processed as previously de-scribed (Baldi et al., 1991). Briefly, after complete semen liquefaction,spermatozoa were routinely separated on 40

%

and 80

%

Percoll gradients, combined and washedonce in Biggers-Whitten-Whittingham (BWW) medium (Biggers et al., 1971) containing 3mg/ml BSA, and finally resuspended in the same medium at the concentration of 5 x106 cells/ml. The presence of leucocytes was checked after Percoll separation by careful counting under an optical microscope, and by measuring for-mation ofreactiveoxygen species by luminol-loaded cells after stimulation with fMLP using a chemiluminescent method as described by Krausz et al. (1992). An increase of reactive-oxygen-species formation following fMLPstimulationwas con-sideredtobeanindication of thepresenceof activatedleucocytes in the sample (Krausz et al., 1992), and samples showing an increase ofchemiluminescence after fMLP treatment were dis-carded. Samples were always pre-incubated for 2h at37 °C in BWWmedium in anatmosphere of5

%

CO2inhumidified air before the experiments were performed. We have previously shown that such apre-incubation time wassufficienttoobtain a maximal response to progesterone (Baldi et al., 1991). Sperm motility was assessed at 60 min and after pre-incubation in

BWW medium using the HTM-2000 motility analyser

(Hamilton-Thorn Research, Danvers, MA, U.S.A.).

Cell

labefling

with

[3H]acetate

and

[3H]PAF

production

[3-H]PAF formationwasmeasuredby quantitative incorporation of

[3H]acetate

into PAF as described previously (Baldi et al., 1990; Villani etal., 1991).Briefly,spermatozoa werecentrifuged

at600g for 1-0 min, resuspendedin BWWmediumsupplemented with 3mg/ml fatty-acid-free BSA at a concentration of 5 x106cells/ml andincubatedfor 30 min at 37 °C in thepresence of

[3H]acetate

(20,tCi/ml). Stimuli or vehicle (0.1% DMSO) werethen added atthe indicated concentrations for theindicated times. The reaction was stopped by adding 3 ml of ice-cold chloroform/methanol/acetic acid

(1:2:

0.04, by vol.), andlipids wereextractedtwice inchloroform/methanol (1: 1, v/v) accord-ing to the Bligh and Dyer (1959) extraction procedure. The combined chloroform extracts were washed once withmethanol/

water (10:9, v/v), dried under nitrogen and reconstituted in chloroform/methanol (9:1,

v/v).

Samples werethenappliedon

heat-activated h.p.t.l.c. silica gel 60 plates and developed in

chloroform/methanol/water

(65:35:6, by vol.). The labelled phospholipid products were identified by co-chromatography with known standards. The RF value of PAFwas0.2.Thelipid

fractions were visualized under a u.v. lamp after 2-p-toluidinylnaphthylene-6-sulphonate (TNS) exposure, and areas corresponding to PAF carefully scraped and the radioactivity countedby liquid scintillation. For [3H]PAF-release experiments, spermatozoa, after incubation with stimuli, were centrifuged for 10 min (600 g, 4°C) and lipids were separatelyextracted from supernatant and cell pellet.

Assay of lyso-PAF:

acetyl-CoA acetyltransferase

activity

PAF acetyltransferase activity was determined in lysates of spermatozoa by the incorporation of [3H]acetyl from radiolabelled acetyl-CoA into [3H]PAF, using lyso-PAF as substrate, as described previously (Villani et al., 1991). Spermatozoa, after incubation with stimuli for the indicated times, were centrifuged and washed in a buffer containing 250 mM sucrose, 0.5 mM EGTA, 1 mM dithiothreitol and 10 mMHepes (pH7),andfinallyresuspendedin the same buffer containing 1 mM CaCl2. Cells were then sonicated (3x15s

bursts)andefficiencyof sonication was checked each time under anopticalmicroscope. The homogenate was centrifuged at 600 g for5min at 4°C andaliquots ofsupernatant wereincubatedfor 5min at 37 °C,in ashakingwaterbath,inthe presenceof30

,uM

lyso-PAF or otherwise indicated. [3H]Acetyl-CoA (5 ,uCi, 100 ,uM, orotherwiseindicated)wasthenaddedandincubation proceeded for theindicated times. The reaction wasstopped by addition ofchloroform/methanol/aceticacid(1: 2:0.04, byvol.); lipids were extracted and PAF separated by t.l.c. as described above.

Identification of

[3H]PAF-like material

as authentic PAF

Wetestedsperm-derivedPAF-likematerial, isolatedasdescribed above, for itsability to aggregate washed rabbit platelets. The assay wasperformedasdescribedpreviously (Wangetal., 1988; Baldi etal., 1990). Briefly,5x 107platelets were stirred at 37 °C in an aggregometer in Tris-buffered Tyrode's solution supplemented with

0.250%

gelatin, in the presence of 10,M indomethacin. A calibration curve with PAF standard was performedandPAF-like material present inthe samplecalculated accordingly. In someexperiments, aliquots of the sample were pretreated with 30 mMsodiumhydroxideinmethanol at22°C for5min toinactivate PAFbydeacylation. Also, theability of L659,989 (10 ,uM), athird-generation PAF-receptor antagonist (Ponpipometal., 1988),toblockplateletaggregation induced by the samplewas tested.

Wealsoperformedg.c.-m.s.analysis ofanA23 187-stimulated

sperm sample by using a Perkin Elmer 8420 gas

chromatography-ion-trap mass spectrometer. The sample, extractedasdescribedabove,wasre-extracted from t.l.c.powder andexposedtophospholipaseC

(1

unit/mlin 0.5 MTris,pH7.6) hydrolysis. The obtained diglyceride was thenderivatized with TBDMSIaccordingtoTriolo et al.(1991)andappliedtothegas chromatograph-ion-trap mass spectrometer. Chromatographic separation was obtained using a DB-1

(1000%

methylsilicone) fused-silica capillarycolumn. Injection was performed using a

splitless programmed temperature vaporizer (250°C). Acquisitions were made both inmultiple ion detection (m.i.d.) andselected ionmonitoring (s.i.m.)by comparisonwithaPAF

C16:0

standard. Whenanalysed bym.i.d., the ions selectedwith PAFC16

:Owere

m/e1 7,131, which are formedbyrearrangement of theacetylgroup anddimethylsilanol (CH3CO+74), and 415 (M-57),which isproduced

by

cleavage

of the

tert-butyl

radical from the parent ion. Whenanalysiswasperformed withs.i.m., the ion m/e 415 (M-57) was selected. These three ions are

representative of 1-O-alkyl-sn-2-acetylglycerophosphocholine (PAFC16:0).

2.79+0.39 (n= 11) fold increase over basal respectively] was

observed (Figure 2). A similar effect ofprogesterone(10

,ug/ml)

was also observed after 60min incubation (1.7+0.17-fold

in-[3H]Arachidonic acid release from prelabelled spermatozoa

In thisset of experiments, spermatozoa (5x 106 cells/ml), after

separation on a Percoll gradient, were pre-incubated in BWW

medium supplemented with fatty-acid-free BSA. [3H]Arachidonic acid (1 ,Ci/ml)wasaddedtothe incubation medium during the last 90minand incubation proceeded inashakingwaterbath. As

shown by Bennetetal. (1987), arachidonate incorporation into

sperm lipids was dependent on the total concentration of

arachidonicacid, with maximal incorporation into phospholipids occurring at a concentration of 8-10 ,uM. Accordingly, we

incubated spermatozoa with a final [3H]arachidonic acid

con-centrationat8,uM. At the end of incubation, spermatozoawere

centrifuged, washedoncein fresh BWW medium, and incubated

in the presence of stimuli for 120 min. Total radioactivity

incorporated into spermatozoa was determined by counting

aliquots of the final cell suspension. The reactionwasstopped by

placing tubesonice and centrifugingat600gfor 10minat4 'C. Supernatants were collected and lipids extracted as described

above. The chloroform phase was dried under nitrogen,

reconstituted in chloroform/methanol (9: 1,v/v),and appliedto

heat-activated silica gel 60 t.l.c. plates. The solvent system

used was hexan/ethyl ether/acetic acid (70:30: 1, by vol.).

[3H]Arachidonic acidwasidentified by co-chromatography with aknownstandard and visualizedundera u.v.lamp afterexposure

to fluorescein. Areas corresponding to arachidonic acid were

scraped andcounted by liquid scintillation.Insomeexperiments,

cell pellets (corresponding to 1.5x 107 spermatozoa) were

reconstituted in buffer, an aliquot was counted to determine residualradioactivity, and the remaining sample was extracted

according to the procedure of Bligh and Dyer (1959). The chloroform phase was dried under nitrogen, reconstituted in

chloroform/methanol (9:1, v/v) and applied to heat-activated silicagel 60 t.l.c. plates. The solventsystemusedwaschloroform/

ethanol/triethylamine/water (30: 34: 35: 8, by vol.), which allows separation of phospholipids, free fatty acids and neutral lipids (Touchstone et al., 1980). Different spots were identified by

parallel chromatography with known standards visualized under

a u.v. lamp, after exposure to TNS. Areas corresponding to

differentphospholipids (PC, PE and PS) and free fatty acidswere

carefully scraped and counted.

Statistical analysis

Resultsareexpressedas means+S.E.M.Statisticalcomparisons

were performed using paired Student's t-test. For time-course experiments, results were analysed using ANOVA. The

pro-gesteronedose-response curve was analysed with the ALLFIT

program(De Leanet al., 1978).

RESULTS

Effect ofprogesterone and A23187 on

[3H]PAF

production by spermatozoa

PAFproduction ([3H]acetate incorporationintoPAF) byA23 187

(10

#tM)-stimulated

human spermatozoa was time-dependent

(Figure 1), with a maximal response at 60min followed by a

plateau. Whenspermatozoafromdifferentmenweretreated for 2 h with progesterone (10,ug/ml) and A23187 (10 ,uM), an

increase of [3H]PAF formation [1.76+0.26 (n=9) and

0L 0 CCo 00 0. c)0 , a <o.X D < Qi 1 C) x 6 60 120 Time(min)

Figure 1 Timecourse of[3H]acetateincorporation into PAF in control and A23187-stimulated human sperm

Spermatozoawereincubated at 37°C forthe indicatedtimes with(A)orwithout(0)A23187 (10 uM) in the presence of[3H]acetate. After lipid extraction and t.l.c. separation, areas

corresponding to PAF were scraped and radioactivity counted. n=four experiments.

*P< 0.005versuscontrol spermatozoa;rawc.p.m.werefive timeshigher.

0:2 CrCJ C ,- 2 o~ 0 0 Coo 0 E

,

a) -n0 T i X C3 q 6 0

TL

T

Control 1) P A23187 (9) (11)

Figure2 Effectofprogesteroneand A23187on [Hjacetateincorporation Into PAF in human spermatozoa

Spermatozoawereincubated at 37°C for2 h withprogesterone (P) (10,ag/ml)and A231 87

(10 1M)in the presenceof[31H]aceticacid. After lipidextractionandt.i.c.separation,areas

corresponding to PAFwere scraped and counted. The number ofexperiments isgiven in

parentheses. Control=non-stimulatedspermatozoa;rawc.p.m. datawerefivetimeshigher.

Statistical signiticance:*P<0.005versuscontrol.

U-0L 0 4.--C c U0= ,PX.0M 7Dmm I E -0.32 3.2 Log[progesterone(,uM)]

Figure 3 Dose-response curve of progesterone-stmulated

[3H]acetate

incorporation into PAF in spermatozoa

Spermatozoawereincubatedat370Cfor2 hwithincreasing concentrations ofprogesterone in the presenceof[3H]acetate.Afterlipid extraction andt.i.c.separation,areascorresponding to PAF were scraped and radioactivity counted. C.p.m. in the control samples were 2306+333/5x106spermatozoa.The number of experiments is given in parentheses.

2

Oa- 4--CN g 0 1.5 , CL - O E2 8E 1 u MD 0O c) (DCD

0.E

05

O'2

0 Control P A23187 14) (4) (41

Figure4 [3H]PAFrelease inthemedium by humanspermatozoa Spermatozoa wereincubated at 37 0C for 2 h with progesterone (10 1ug/ml) and A23187 (10IM)inthepresenceof[3H]acetic acid. At the end of incubation, sampleswerecentrifuged andlipids extracted from supernatants and cell pellets. Aftert.l.c.,areascorresponding to PAF

were scraped and radioactivity was counted. The number of experiments is given in

parentheses. Control=non-stimulatedspermatozoa; rawc.p.m.datawerefivetimes higher.

Statistical significance: *P<0.05versuscontrol.

crease over basal, n=3). The effect ofprogesterone was

dose-dependent (Figure 3), reachingaplateauattheconcentration of

5 tg/ml (16,M). The EC50 of the curve (defined as the concentration with an expected half-maximal response) was 1.12,tg/ml (3.56,M).

When cellpellets andsupernatants were analysed separately,

mostofnewly synthesized[3H]PAFwasfound inthesupernatants

of both unstimulated and stimulated spermatozoa (Figure 4).

Thetreatmentwithprogesterone(10 ,tg/ml)andA23187(10 1M)

stimulated anincrease of[3H]PAFrelease in thesupernatant of

1.78 + 0.59- and 2.2+0.54-fold respectively. We suggest that [3H]PAF release occurs from theacrosome as aconsequence of the acrosome reaction. Indeed, although no PAF activity has been found in seminalplasmafrom both rabbit(Kumaret al., 1988)and human spermatozoa(Minhasetal., 1991),release of PAF occurs in mouse spermatozoa after incubation for 3 h (Kuzan et al., 1990). As mouse spermatozoa undergo spon-taneous acrosome reaction during in vitro incubation, PAF release might be duetothisexocytoticevent.Moreover,Daviset

al.(1980)reportedanincrease ofphospholipids in theincubation medium of rat spermatozoa when incubated for prolonged periods of time, that they suggest is due to spontaneous exocytosis. Spontaneous acrosomereaction,to acertain extent, may occur in the absence of stimulation in human sperm (Takahashi et al., 1992;Falsettietal., 1993). However,whether PAF is released into the mediumas aconsequence ofacrosome reaction remains to be established.

Lyso-PAF: acetyl-CoA

acetyltransferase

activity in human spermatozoa

The results of the experiments designed to characterize and optimizethe enzyme assayarereported inFigure5.Figure 5(a) shows the time course of the reaction, indicating that, in our

conditions, PAFsynthesis waslinearup to90 min,followed by

aplateau. Increasing the number ofspermatozoainthe sonicate up to 1.5x107 spermatozoa/tube, PAF synthesis increased linearly up to a level of5x106 spermatozoa/tube (Figure Sb). Accordingly, experiments were performed using Sx106 spermatozoa.The reactionwaslinearly dependenton lyso-PAF concentration upto50,tM(Figure Sc); however,asshown in the figure, significant incorporation of[3H]acetate from [3H]acetyl-CoAwasalso present in the absenceof exogenously added lyso-PAF,suggestingthathighconcentrationsoflyso-PAFarepresent inunstimulated cells.Thedependenceofacetyltransferase activity

ontheconcentrationofacetyl-CoAis shown inFigure 5(d).The observed Km value for acetyl-CoA, obtained from a double-reciprocal plot (Figure Sd, inset) was 46.6,M. These results indicate that kinetic parameters of lyso-PAF: acetyl-CoA acetyltransferase of human spermatozoa are similar to those described for different cell preparations (Wykle et al., 1980; Snyderetal., 1987). Thetreatmentwithprogesterone(10 ,ug/ml) and A23187

(10

M) enhanced lyso-PAF: CoA acetyl-transferaseactivityatearlytimes(Table 1).

Identification

of

sperm-derived

PAF-like material as authentic PAF PAF-like material co-migrating with authentic PAF isolated from A23187-stimulated human spermatozoawasabletoinduce aggregationof rabbitplatelets,whichwasinhibited bythe PAF-receptorantagonist L659,989(10lM)orby alkaline methanolic hydrolysis (resultsnotshown). Basedonthe PAFdose-response curve,weestimated that 1 x 108A23187-stimulated spermatozoa produce 0.37pmol of bioactivePAF/2h.

G.c.-m.s. analysis ofphospholipase C-treated and

TBDMSI-derivatized PAF-like material isolated from human spermatozoa, showed the presenceofa chromatographic peakwith identical retention time of standard PAF

C16:

o Ionsm/e117,131and415, with identical ratiostothose of PAF standardions,werepresent in the sample when analysed by m.i.d. [Figures 6(a) and 6(b)]. When monitoredbys.i.m.,the ion

mle

415(M-57)showed the

same retention time as the PAF standard (results not shown). These results confirm the presence of 1 -O-alkyl-sn-2-acetyl-glycerophosphocholine, the biologically active PAF, because the acyl-PAF analogue

(1-O-acyl-sn-2-acetylglycerophospho-30 60 90 120 Time (min) 0.15 c

.E

0 E a. 0.1 as

cm(

0.05 -U. C<L I2- I I I I 0 5 10 15 Spermatozoa/tube (x 106) 20 40 60 Lyso-PAF concn.(pM) I E (c) E CU 0. 0 0 _ Q X E CU ax _ 400 E

t0

CU 2 m &- 200 CU C U-I0 l 2-I 80 ^- 0 80 100 150

Acetyl-CoAconcn.(guM)

Figure 5 Lyso-PAF:acetyl-CoA acetyltransferase activityInhuman spermatozoa

Acetyltransferaseactivitywasassessed insonicated spermatozoa by measuring[3H]acetyl-CoA incorporation into PAFas afunctionof incubation time (a), cell number (b), lyso-PAF concentration (c)andacetyl-CoA concentration (d). The insetin(d)showsthedouble-reciprocal plotof substrate dependence ofacetyltransferaseactivity towards acetyl-CoA. The resultsarerepresentative of at leasttwo differentexperiments done in duplicate.

Table 1 Time-courseofIyso-PAF:acetyl-CoA acetyltransferaseactivation

(pmol/5x106spermatozoa permin)Inhuman spermatozoaafterincubation

withprogesterone (10

pg/mI)

andA23187 (10 uM)

Spermatozoa were incubated with progesterone and A23187 for the indicated times and acetyltransferase activitymeasured in sperm sonicates as described in the text. Values are the mean+S.E.M.of twoexperiments.

Acetyltransferase activity(pmol/5x 106spermatozoa permin)

Time(min)... 2 5 10 20 60 120 Control 0.05 0.07 0.07 0.09 0.10 0.10 +0.004 +0.007 +0.014 +0.009 +0.000 +0.014 Progesterone 0.08 0.11 0.14 0.11 0.12 0.11 +0.014 +0.015 +0.000 +0.004 +0.009 +0.021 A23187 0.09 0.12 0.13 0.12 0.11 0.11 +0.019 +0.007 +0.007 +0.000 +0.014 +0.009

choline)hasdifferent electronimpactmassspectraandadifferent

gas-chromatographicretention time in oursystem.

Effect of progesterone on arachidonic acid metabolism of human spermatozoa

Under steady-state conditions, 310+41.6c.p.m./106 spermato-zoa were recoveredin the materialco-migratingwith authentic

arachidonic acid of the supernatantofunstimulated, prelabelled

spermatozoa after 2 h. Free [3H]arachidonic acid represented

21.2+1.1%of totalradioactivity. Exposureof [3H]arachidonic-acid-prelabelled spermatozoa to progesterone (10ltg/ml) and

A23187

(10,uM)

for2h,stimulated asignificant increaseinthe

release of [3H]arachidonic acid (Figure 7) and an increase of

radioactivity in the free-fatty-acid fraction of the cell pellet (Table 2).Infourdifferentexperiments,the effect ofprogesterone

wastestedatconcentrations of1, 5,and 10,ug/ml,whichelicited cU 0 N 0 E Q CU 0 to0 x 0 E 0. V C-(L 02-25 20 15 10 5 E a 0.2 m 0 0 01 m a, 0.15 c 0 x E 0. "0.05 C U-12. 0 200

100 IS, .1 am .i ._a r-50 100 100 Co o-0 ._' c a) (U 6) .(1 50 0 200 150 300 250 150 co (u cn -0h (. a) V) -o C., (U) u I 400 350 450 mle

Figure 6 G.c.-m.s.analysis of sperm-derived PAF-likematerial PAF-like material extracted from human spermatozoa, after exposure to phospholipase C

hydrolysis and derivatization with TBDMSI, was injected into a Perkin Elmer 8420 gas

chromatograph-ion-trap mass spectrometer, and acquisition performed with m.i.d. mode.

Identificationwasmadeby comparisonwith PAF

C16:0

standard.Full-scanacquisitionof PAF

C16:0standard isrepresentedin(a). Masses117,131and415,fragmentions characteristic

of PAFC160,werepresentinthesampleat thesameretention timeasthe standard (b).

a stimulation of

1.06+0.23-,

1.09+0.3- and 1.19+0.35-fold

increase overbasalrespectively.

Among the cellular phospholipids examined, a decrease of

radioactivitywasobserved in PC after stimulationwith A23187

(Table 2), whereas the changes in PE and PSwerenotsignificant. Progesterone determined a slight decrease in PC

radio-activity (4.9%), which was approaching statistical significance

(P=0.07) (Table 2).

DISCUSSION

The resultspresented in thispaperdemonstrate thatincubation

of humanspermatozoa,either withphysiological (progesterone) and non-physiological (A23187) stimuli ofacrosome reaction,

producesasignificant increase of PAF production,asassessedby

[3H]acetate incorporation into PAF. Moreover, we report the

presence oflyso-PAF: acetyl-CoA acetyltransferase activity in

human spermatozoa. Suchactivity wasdependent ontime and substrate(lyso-PAFandacetyl-CoA) concentrations, with simi-lar kinetic parameters to those found in other cells. The oc-currenceofPAF inmammalian spermatozoa hasbeenreported

inseveral studies(Kumaretal., 1988; Kuzanetal., 1990; Parks

etal., 1990; Minhasetal., 1991); however,thesestudies donot

140 |-130 1-120 e-110 1-100 Progesterone (9) A23187 110)

Figure7 Effect of progesterone and A23187 on [HJarachidonic acid release from prelabelled humanspermatozoa

Spermatozoawereincubated with[3H]arachidonicacid for 90min,washed and stimulatedor

not with A23187 (10 ,uM) and progesterone (10 ,ug/ml) for 2 h. After centrifugation, supernatantwascollected andlipidsextracted andsubjectedtotilc. Areas correspondingto standardarachidonic acid werescraped and counted. C.p.m. in the control samples were

1550 +205/5x106spermatozoaper2 h.Thenumberofexperimentsis giveninparentheses. Statisticalsignificance: *P<0.05;**P<0.005.

provide information concerning the enzymicalroutesinvolvedin PAFsynthesisin thesecells. Indeed,PAF canbe synthesized by atleasttwodifferentpathways, namely the remodelling andthe de novo anabolic pathways (Snyder, 1990). The evaluation of

rH]acetate

incorporation

into PAF is considered a reliable method to study the anabolic, remodelling pathway of PAF synthesis. Our results indicate that PAF synthesis in human spermatozoa may occur at leastthrough the remodelling path-way, and that this route canbe increased by stimuli that induce

Ca2+

entryand theacrosomereaction.

Perturbations of membrane phospholipid metabolism after induction of the acrosomereaction ofspermatozoa have been reported.Inparticular,progesteroneandfollicular fluidareable to stimulate phospholipase C, with increased production of inositolphosphates and, presumably, of diacylglycerol (Thomas

Table 2 Changes In

radloacfivty

of

[3H]arachidonle

acid-labelled lipids In progesterone- (1.0

pcg/ml)

and A23187- (10

pM)

stimulated human spermatozoa

[3H]Arachidonicacid-labelled spermatozoawerestimulated with progesterone and A23187at 37°C for 2 h. Results are expressed as a percentage of total cellular radioactivity

(5837+500.3c.p.m./106spermatozoa) andaremeans+S.E.M.of fiveexperiments donein triplicate. Statistical significance:**P <0.005,*P <0.05,versuscontrol.

Changeinradioactivity(%)

Lipid Control Progesterone A23187

Freefattyacids 6.6+1.1 8.15+1.4* 10.2+1.3**

PC 40.3+3.3 35.4 + 3.2 21.3+4.5* PE 17.5+1.8 16.7+2.2 139+1.4 PS 7.04+2.1 5.1 +1.5 5.4+1.7 I I I I I I I 131 (b)_ 117 415

I

L

I

I

T

and Meizel, 1989).Similarly, stimulation of acrosome reaction of In summary, progesterone and A23187 elicitan increase of mammalian spermatozoa with A23187 induces an increase of PAF production and arachidonic acid release in human phospholipase C (Roldan and Harrison, 1989), phospholipase A spermatozoa, which may play a physiological role in the process (Takkar et al., 1983; Bennet et al., 1987) and lipoxygenase (Lax of fertilization.

et al., 1990) activities. Futhermore, the induction of acrosome

reaction of sea-urchin sperm with fucose sulphate activates both This paper was

supported

by a grant from Consiglio Nazionale delle Richerche a phospholipase C (Domino and Garbers, 1988) and a (Progetto FATMA, contratto no. 92.00097.PF41). We thank Professor Mario Sefio phospholipase D which stimulates phosphatidic acid and and Dr. MarioMaggi (Endocrinology Unit, University ofFlorence, Florence, Italy),and diacylglycerol accumulations (Domino et al., 1989). All these

Dr.

Fabio Marra

and Dr. Massimo Pinzani(ClinicaMedica II,

University

ofFlorence, activities are entirelydependent upon calcium influx in the cells,

indicating that an increase of intracellular calcium is the primary;.

necessary event for stimulation of phospholipases. Our.results -- i.ERENCES

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