357

Journal of Andrology, Vol. 9, No. 6, November/December 1988Copyright a American Society of Andrology

Ethanol Accelerates Acrosomal Loss in Human Spermatozoa

JUAN C. ALVAREZ,* MICHAEL A. LEE,* RENATO V. IOZZO4� ISABEL LOPEZ,*JOSEPH C. TOUCHSTONE,* AND BAYARD T. STOREY*4

The effects of ethanol on the loss of the human spermacrosome, as determined by the chlortetracycline fluores-cence assay and by indirect immunofluorescence assay,were assessed over 6 hours during incubation at 37 C inBWW medium containing 0 to 250 mM ethanol. Bothassays gave the same results. At the end of 6 hours, 48 ±

6% acrosomal loss was found in samples in 250 mMethanol compared with 4 ± 1% in the absence of ethanol.After 0.25 hour, the first time point chosen for sampling,the spermatozoa in 250mM ethanol showed 23±3% lossof acrosomes compared with < 1#{176}i’oin the absence ofethanol. Ultrastructural studies revealed that the eth-anol-treated spermatozoa showed complete acrosomalloss as well as loss of the equatorial segment. No exam-ples of the vesiculation characteristic of the physiologicacrosome reaction were found in the 150 cells examined.Calcium is required for the ethanol-mediated acrosomalloss: omission of Ca2*, addition of 2 mM EGTA, or 0.2

mM verapamil blocked the effect. Ethanol induced a dose-dependent effiux of cholesterol from human spermato-zoa, but the ethanol-induced acceleration of acrosomalloss occurred to the same extent in the presence of choles-terol microdispersions that prevented this efflux. Theloss of the equatorial segment, which is necessary for eggpenetration, during ethanol-induced acrosomal loss wouldexplain the known effect of ethanol in inhibiting, ratherthan enhancing, the penetration of zona-free hamstereggs by human spermatozoa.

Reprint requests: Dr. Bayard T. Storey, Department of Obstet-

rics and Gynecology, John Morgan Building 339, University ofPennsylvania, Philadelphia, PA 19104-6080.

This work was supported by NIH grants HD-15842, HL-

07027, HD-06274, and a grant from The March of Dimes BirthDefects Foundation.

Submitted for publication September 9, 1987; revised versionreceived November 16, 1987; accepted for publication February 8,

1988.

From the Departments of Obstetricsand Gynecology, *

Pathology and Laboratory Medicine,tand Physiology4

University of Pennsylvania Schoolof Medicine,

Philadelphia, Pennsylvania

Key words: human spermatozoa, acrosome reaction,ethanol-induced acrosomal loss, chlortetracycline fluo-rescence assay, cholesterol in human spermatozoa.

J Androl 1988;9:357-366.

The deleterious effects of ethanol on male repro-

ductive function have long been recognized (reviewed

by Van Thiel and Lester, 1974; Weathersbee and

Lodge, 1978; Anderson and Willis, 1981; Anderson et

al, 1983a). As pointed out in the recent critical review

by Anderson et al (1983a), these effects are expressed

at multiple locations, not only secondary to hepaticdysfunction but, more importantly, in the male

reproductive tract. The pathology is both dose- and

time-dependent (Willis et al, 1983) and is only par-

tially reversible upon withdrawal of ethanol (Ander-

son et al, 1985). The mouse has proved to be

particularly useful for studying ethanol interference

with male reproductive function (Anderson et al,

1978, 1980b, 1981, 1983a; Badr and Bartke, 1974;

Alvarez, 1985). Ethanol-induced impairment of sperm

358 Journal of Andrology November/December 1988 Vol. 9

morphology during spermatogenesis and sperm mat-

uration has been demonstrated using this animal

model (Anderson et al, 1983b).

Direct effects of ethanol on mature mammalian

spermatozoa have been less extensively examined.

Inhibition of in vitro fertilization by ethanol at concen-

trations up to 87mM (400 mg%) was reported for the

hamster by Cash and Rogers (1981) and for the

mouse by Anderson et al (1980a); it was attributed to

a capacitation deficit. At the concentration of 87 mM,

ethanol did not affect mouse sperm motility and did

not compromise the integrity and fertilizability of the

mouse egg; however, in vitro fertilization was strongly

inhibited if ethanol was present in the capacitation

medium (Joyce et a!, 1981; Anderson et al, 1982).

Salonen (1986) reported the dose-dependent inhibi-

tion by ethanol of penetration of zona-free hamster

eggs; the range of concentrations was 11 to 109 mM

(50 to 500 mg%). The inhibitory effect on both the

percent penetration and fertilization index appeared

to be more profound if ethanol was present during

capacitation and insemination. Essentially identical

results were reported for hamster spermatozoa by

Rogers et al (1987), who also documented the inhibi-

tion of penetration of zona-free hamster eggs by

human spermatozoa when ethanol was present dur-

ing sperm capacitation. Two possible actions of

ethanol on spermatozoa are consistent with the

results summarized above. One is inhibition of the

acrosome reaction, which is required for penetration

of zona-free hamster eggs by human spermatozoa

(Moore et al, 1987). The other is accelerated loss of

acrosomes accompanied by loss of other membrane

structures or components needed for gamete fusion.

The recent development of a rapid fluorescence assay

for assessing the acrosomal status of human sperma-

tozoa (Lee et al, 1987) has provided a means for

distinguishing between these two possibilities. Re-

sults supporting the second possibility are reported

in this article.

Reagents

Materials and Methods

Clutaraldehyde was obtained as a 25% aqueous solutionfrom Polysciences, Inc (Warminster, PA). The divalentcation ionophore A23187 was purchased from Calbio-chem (La Jolla, CA). Cholesterol, acetaldehyde, chlortet-racycline, human serum albumin (HSA, Fraction V), andbovine serum albumin (BSA, Fraction V Powder) werefrom Sigma Chemical Co (St. Louis, MO). The monoclo-nal antibody HS-19 (Florman et al, 1984; Wolf et al, 1985)

was generously provided by Dr. Kathleen Bechtol (Genen-tech Inc, South San Francisco, CA). Verapamil (lsoptin-

HC1) was kindly provided by Dr. Fernando Sende (Fitz-gerald Mercy Hospital, Yeadon, PA). Solvents were EMScience chromatographic grade. High Performance ThinLayer Chromatography (HPTLC) 10 X 10-cm precoated

silica plates (250-tim thick) were obtained from WhatmanInc (Clifton, NJ).

Spermatozoa

The culture medium used for incubation of washedspermatozoa was the modified Krebs Ringer bicarbonatemedium (BWW) of Biggers et al (1971). The medium con-tained human serum albumin (HSA) at 3 mg/mI or 30mg/ml as indicated below. Human sperm ejaculates wereobtained from healthy adult donors by masturbation after36 to 48 hours of abstinence, and were allowed to liquify at37 C for 15 to 30 minutes. Ten donors of proven fertilityfrom the AID program provided 60 samples for this studyover the course of 2 years. All ejaculates used in these

experiments had at least 65% motile cells, 60% normalmorphology, and cell counts ranging from 60 to 280 X 106

cells/ml. After the ejaculate had liquified, it was mixedwith 2 vol BWW containing 3 mg/ml HSA. The Spermato-

zoa were washed by centrifugation in 12-mi conical tubes

at 600 X g for 10 minutes. The sperm pellet was gently

overlayed with 2 ml BWW containing 30 mg/mI or 3mg/mi HSA, depending on the experiment. The centri-

fuge tube was inclined at 45 degrees, and the motile sper-matozoa were allowed to “swim up” out of the pellet for 30

minutes. This “swim-up” technique is a slightly modifiedversion of the one described by Overstreet et al (1980).

Spermatozoa were scored for the percentage of motilityand forward progression (scale 0 to 3; Amelar, 1966) byphase contrast microscopy at 400 X. Sperm viability wasdetermined by incubating 20 �tl of the sperm suspensionand 20 M’ of eosin B at 5% (w/v) in physiologic saline at 25

C for 5 minutes. Viable spermatozoa excluded the dye;dead spermatozoa stained orange. Spermatozoa werecounted in a hemocytometer after a 20-fold dilution andabnormal morphologies were scored. At least 100 cells

were examined in each sample. The sperm samplesobtained by the swim-up method consistently showedmotility percentages over 90%, forward progression of 2+

or more, eosin B viability scores over 95%, and less than10% abnormal cell morphologies; cell counts ranged from16 to 100 X 106 cells/mI.

Sperm Incubations

The sperm suspensions obtained by the swim-up meth-od were added in 0.1-mI aliquots to 0.3 ml BWW contain-ing 3% HSA and ethanol concentrations from 0 to 250mMin 1.0-mi screw-cap vials. The tubes were tightly sealedwith Teflon-lined screw caps to prevent loss of ethanoland CO2. and the samples were incubated at 37 C for thedesired times in a shaking water bath. At the end of theincubation time, 20-Al aliquots were taken to score motil-

ity and viability, and 5-Al aliquots were taken for thechiortetracycline fluorescence assay (Lee et al, 1987).

Ethanol concentration was checked at this time by gasliquid chromatography (GLC), using the same procedure

for acetaldehyde described below. In those experimentswhere lipid peroxidation was determined, 0.3-mi aliquots

No.6 ETHANOL AND ACROSOMAL LOSS . Alvarez et al 359

were taken and analyzed for maionaldehyde by the thio-

barbituric acid assay (Alvarez and Storey, 1982; Alvai�ez #{233}t

al, 1987).

lonophore Studies

The sample was split into 2 equal volumes and thesperm cells were sedimented by centrifugation at 600 X g

for 10 minutes. One sperm pellet was overlayed with 1 or2 ml BWW medium and A23187 was added to a finalconcentration of 10 AM from a 10-mM stock solution indimethylformamide, giving a content of 0.1% (v/v). Theother pellet (control) was overlayed with the same volume

of medium containing no ionophore and 0.1% dimethyl-formamide (vlv). After a 30-minute swim-up, each of thetwo samples was again split into four; two acted as controlwithout ethanol, whereas the other two were the experi-mental samples with 250 mM ethanol. This point was

taken as zero time for the ethanol incubations. The firstsets of samples for analysis of percent acrosome-reactedspermatozoa were taken after 0.25 hour and the second

sets after 3 hours of this incubation regime. From each

sample was taken 5 A1 for the chlortetracyciine fluores-cence assay. The remainder of the sample was used for theindirect immunofluorescence assay.

Chlortetracycline Fluorescence Assay

The chlortetracycline fluorescence assay of the acro-somal status of the spermatozoa was carried out exactly asdescribed by Lee et al (1987). The technique utilizes 0.05%

glutaraldehyde to “freeze” the fluorescence patterns (Ward

and Storey, 1984), so that groups of samples then can becollected at various time points and scored together. Theslides were randomized and scored blind by two scorers. Atotal of 100 spermatozoa were scored for each determina-tion. Acrosomeless spermatozoa were scored by the

absence of fluorescence on the head contrasted to fluores-cence on the midpiece; acrosome-intact spermatozoa havebright fluorescence patterns on the heads.

Indirect 1mm u nofluorescence Assay

The monoclonal antibody HS19 was shown by Wolf et

al (1985) to react with the anterior head portion ofacrosome-intact but not with acrosome-reacted human

spermatozoa. The antibody from the culture was diluted1:9 in Dulbecco’s phosphate-buffered saline (Gibco Labor-

atories, Grand Island, NY) containing 0.01% NaN3. Theassay procedure was that described by Wolf et al (1985)

and Suarez et al (1986), using 100 cells for each determina-tion. Spermatozoa with fluorescence restricted to theacrosomal cap region were scored as acrosome-intact; cells

with no fluorescence over the acrosomal cap region were

scored as acrosomeless.

Ultrastructural Studies

Sperm pellets from samples incubated in the presence orabsence of ethanol were fixed in half-strength Karnov-sky’s solution (Karnovsky, 1965) buffered at pH 7.0 with0.1 M sodium cacodylate. After postfixation in 1% osmium

tetroxide, the specimens were dehydrated in gradedethanol solutions and embedded in Epon. Thin sections

were double stained with uranyl acetate and lead citrate

and examined at 60 kV with a Hitachi 600-C electron

microscope (Nissei Sangyo America, Rolling Meadows, IL)as described previously by lozzo (1984). Electron micro-

graphs were taken at 15,000 to 20,000 X, a magnificationsuitable for detailed study of the sperm head. Quantita-

tion of the acrosomal status was done on 150 cells fromtwo samples with original concentrations of 8 and lOX 106

cells/mi.

Cholesterol Determination

The cholesterol content of both the sperm cells incu-

bated in the presence and absence of ethanol and of the

corresponding suspending medium was determined by anew thin layer chromatography method. After termina-

tion of the incubations at the chosen times, 0.3-mi aliquotsof spermatozoa were removed and centrifuged at 1800 X gfor 10 minutes. The pellet was resuspended in 300 Al ofdistilled water, and both pellet and supernatant were

extracted 4 times with 1 ml of a mixture of chloroform-methanol-ethyl acetate (1:2:1, v/v/v). Recovery of knownamounts of cholesterol carried through the extraction was

98%. The extracts were dried under nitrogen dissolved in20 Al of chloroform-methanol (1:1 v/v); 5 and 10-Al au-quots were spotted on the Whatman HPTLC plates. Cho-lesterol standards were applied in 1 to �-A1 aliquots of a0.1-mg/mi solution in ethanol. Plates were predeveloped

twice in chloroform-methanol (1:1) to 1 cm, then devel-oped in hexane-acetone (80:20). After development, the

plates were air-dried, then heated at 170 C for 2 minutes.Cholesterol was detected by spraying with a solution of

3% Cu(CH3COO)z in 8% H3P04 as described by Touch-stone et al (1983). The developed chromatograms werescanned at 440 nm in a Kontes Model 800 fiber optic

scanner (Kontes, Vineland, NJ), and the peak areas wereintegrated with a Hewlett-Packard 3390A integrator

(Hewlett-Packard Co. Palo Alto, CA). Amounts of choles-terol between 0.05 to 1.5 �g gave linear standard curves.

The sperm cholesterol in the developed chromatogramswas further analyzed for impurities by gas chromatog-

raphy/mass spectrometry on a Hewlett-Packard 50890

GC/5970 MSD using a 50% phenyl-methyl-silicon column

with 0.17-Am film thickness, operated with temperatureprogramming from 130 C to 200 C at 14 C per minute.Identification was based on retention time and fragmenta-

tion patterns with cholesterol standards run under thesame conditions. Cholesterol was identified as the onlycomponent present, with a detection limit of 1 ng.

Preparation of Cholesterol Microdispersions

Cholesterol microdispersions were prepared by sus-pending cholesterol in BWW medium containing 3% HSA,resulting in the spontaneous formation of a hydratedliquid-crystalline phase. Sonication for 30 minutes resulted

in a slightly turbid dispersion of cholesterol that becamean optically clear solution after filtration through a Milli-pore 0.45-Am membrane. The concentration of choles-terol was determined by HPTLC as described above.

Acetaldehyde Determination

After incubation was terminated, sperm suspensionswere centrifuged at 1800 X g for 10 minutes. Then, 200-Al

(0

-J - (I)

flITlfl.l 0E -�o I11�,., -

0III IT1�\S E

0(0

* 0

C)()‘.lR(>t

Fig. 1. Percent acrosomal loss observed in human spermatozoaas a function of incubation time and ethanol concentrations,using the chlortetracycine fluorescence assay. Sperm cell concen-trations ranged between 10 and 20 X 106 cells/mI. Each point

represents the mean ± SD of 10 determinations.

Incubation Time in Hours

(1 125 250

[Ethanol] mM

Fig. 2. Percent acrosomal loss after 0.25 and 3-hour incuba-

tions observed in human spermatozoa, plotted as a function ofethanol concentration in the medium, using the data from Fig. 1.

Open diamonds: 0.25 hour; closed circles: 3 hours.

360 Journal of Andrology . November/December 1988 Vol. 9

aliquots of the supernatant were inserted in sealed vialscontaining n-propanol as internal standard. Head space

GLC analysis was performed using a Perkin-Elmer GC3920 (Perkin-Elmer Corp, Norwalk, CT), provided with aflame ionization detector and CF 60/80 Carbopack B/5%Carbowax 20M clinical packing 1-1766 (Supelco, Inc. Bel-lefonte, PA). Analysis was carried out under isothermicconditions at a flow rate of 23 ml per minute. The detec-tion limit of acetaldehyde concentration was 0.4 AM.

Statistical Analysis

Statistical significance of the data was determined by

the paired Student’s t test using the computer programStatworks (Heyden and Sons, Inc. Philadelphia, PA). Avalue of P < 0.05 was taken as statistically significant.

Results

Effects of Ethanol Concentration

The percentage of acrosomal loss in human sper-

matozoa, as determined by the chlortetracycline

fluorescence assay, increased as the ethanol concen-

tration of the incubation medium was increased (Fig.

1). In the absence of ethanol, less than 10% of the

spermatozoa showed loss of acrosomes after 6 hours

of incubation, in agreement with the results of Byrd

and Wolf (1986) and Lee et al (1987) obtained under

comparable conditions. In contrast, ethanol concen-

trations as low as 5 mM produced a marked increase

in the percentage of acrosomal loss at this time point.

At higher ethanol concentrations, acrosomeless sper-

matozoa were observed at the earliest time point

with 25% of the sperm population in this state at 250

mM. The effect of ethanol on the observed percent

loss of acrosomes is shown as a function of ethanol

concentration in the incubation medium for the 0.25

and 6-hour time points in Fig. 2. Assessment of viabil-

ity and motility at these time points showed no dif-

ferences between control samples and those incu-

bated at ethanol concentrations up to 250 mM.

Accelerated acrosomal loss in the presence of ethanol

does not cause loss of either viability or motility.

The chlortetracycline assay (Lee et al, 1987) for the

human sperm acrosome reaction has been calibrated

against both the indirect immunofluorescence assay

(Wolf et al, 1985; Suarez et al, 1986) and the triple-

stain assay (Talbot and Chacon, 1981) under condi-

tions of unperturbed incubation and acrosome reac-

tion induction with either ionophore A23187 (Wolf

et al, 1985; Byrd and Wolf, 1986) or with high con-

centrations of acid-solubilized mouse zona pellucida

protein. These conditions appear to induce physio-

logic acrosome reactions. The solvent used with the

ionophore was dimethylformamide, which was

shown to have no effect on this reaction by itself,

compared with appropriate controls. In our study, the

chiortetracycline and indirect immunofluorescence

assays were compared in the absence and presence of

250 mM ethanol at 0.25 and 3-hour incubation times

No. 6 ETHANOL AND ACROSOMAL LOSS . Alvarez et al 361

(Table 1). Essentially identical percentages of acro-

somal loss were obtained by the two assays under the

same conditions. The chlortetracycline assay may

therefore be taken to be valid for assaying acrosome-

intact and acrosomeless spermatozoa in the presence

of up to 250 mM ethanol. As pointed out below, the

loss of the acrosome in the presence of ethanol does

not appear to correspond to the physiologic acrosome

reaction; the terms acrosomal loss and acrosomeless

are therefore used instead of acrosome reaction and

acrosome-reacted.

From the results presented in Figs. 1 and 2, it is

apparent that ethanol does not inhibit, but rather

accelerates, acrosomal loss in human spermatozoa.

The ultrastructure of the sperm head was investi-

gated in parallel samples treated without or with 250

mM ethanol for 0.25 hour. Control acrosome-intact

and ethanol-treated acrosomeless spermatozoa are

shown in Figs. 3A and 3B, respectively. When 150

cells from the ethanol-treated population were exam-

ined, 23% had lost acrosomes, in good agreement

with the values obtained with the other two assays

(Table 1). Among these nonintact spermatozoa,

attempts were made to find cells that were undergo-

ing the acrosome reaction in an intermediate state,

with the characteristic vesiculation observed when

the plasma and outer acrosomal membranes fuse

(Barros et al, 1967; Bedford, 1968; reviews: Bedford

and Cooper, 1978; Meizel, 1984). Such vesiculation

was observed by Suarez et al (1986) when acrosome

reactions were induced by human follicular fluid.

This condition presumably approximates the physio-

logic one, as the equatorial segment remained intact.

In the presence of ethanol, however, no vesiculated

specimens were found; most of the spermatozoa that

were not intact had lost not only their acrosomes and

acrosomal contents, but also the plasma membrane

over the equatorial segment (Fig. 3B). The other two

morphologic forms observed in the ethanol-treated

sample are shown in Figs. 3C and 3D; these forms

were not seen in the control samples. The expanded

plasma membrane seen in the specimen shown in Fig.

3C accounted for 8 to 10 cells observed in the group.

Variations in the degree of expansion made the scor-

ing somewhat uncertain. A similar form was also

reported by Suarez et al (1986). The unique form

depicted in Fig. 3D showed an arrangement of mem-

branes around the sperm head that was the nearest

analog observed to the vesiculation seen in the

ethanol-treated sample. The loss of plasma mem-

brane from this midpiece indicates that it is not vesic-

ulation, however, but membrane fragmentation.

TABLE 1. Comparison of Chlortetracycline and IndirectImmunofluorescence Assays for the Percent Acrosomal

Loss in Human Spermatozoa under Control andInduction Conditions

Additionst

.

IncubatIonTime (h)

Percent Acros omal Loss

CTC hF

None 0.253.0

<15±4

06±3

Dimethylforma-

mide (DMF), 0.25 5 ± 2 5 ± 1

O.1%(v/v) 3.0 7±3 7±2

250 mM Ethanol 0.25

3.0

26 ± 3

38±2

25 ± 8

37±3

250 mM Ethanol + 0.25 24 ± 4 25 ± 4

0.1%(v/v)DMF 3.0 37±2 38±3

10 MM A23187, 0.25 3 ± 2 4 ± 2

0.1% (v/v) DMF 3.0 50 ± 2 48 ± 2

250 mM Ethanol + 0.25 44 ± 7 45 ± 5

10UMA23187, 3.0 49±7 48±7

0.1% (v/v) DMF

‘Values are means ± SD for 10 separate samples from five

donors for the chlortetracycline (CTC) assay and for threeseparate samples from three donors for the indirect immuno-fluorescence assay (IIF). Differences between the two assaysfor a given experimental condition were not significant (P>

0.1).

tConditions for inductions of the acrosomal loss were 250mM ethanol, 250mM ethanol plus inophore A23187, after both

0.25 and 3.0-hour incubations, and ionophore A23187 aloneafter the 3.0-hour incubation. Sperm cell concentrations rangedfrom 10 to 25 X 10� cells/mi. The differences between percentloss under induction conditions and control conditions (includ-ing A23187 at 0.25 hours) were highly significant (P <0.001).Differences among treatments with A23187 with and withoutethanol at 3.0 hours, A23187 with ethanol at 0.25 hours, andethanol at 3.0 hours were not significant (P>0.05). Difference

among these values and those for ethanol at 0.25 hour were

significant (P <0.05).

Calcium Effects

Because the presence of Ca2� in the medium has

been shown to be essential for the occurrence of the

acrosome reaction in all mammalian spermatozoa

studied to date (Yanagimachi and Usui, 1974; Yana-

gimachi, 1981; Meizel, 1984), the effect of this cation

on the ethanol-enhanced acrosomal loss in human

spermatozoa was examined. For this purpose, the

0.25-hour time point with 250 mM ethanol was2+chosen. In the absence of added Ca , less than 1%

acrosomal loss was observed by the chlortetracycline

assay. In the presence of Ca2�, a maximum of 25%

was observed, with Ca2� giving half-maximal effect

362 Journal of Andrology November/December 1988

q�

Vol. 9

F ,�

.9

,1

The possibility that the ethanol effect could be due

- -. ...�

5’-

- --,. ..� -

1’#{149}

Fig. 3. Transmission electron micrographs of human spermatozoa after 0.25 hour incubation in the absence (A) and presence (B, C, and

D) of 250 mM ethanol. The vast majority of the acrosomeless spermatozoa (compromising 23% of the 150 cells examined) exhibited

features identical to those shown in panel B, with loss of plasma and outer acrosomal membrane, acrosomal contents, and equatorial

segment. Acrosome-intact spermatozoa (comprising 70% of 150 cells examined) had the structure shown in panel A with intact plasma and

outer acrosomal membranes. A small percentage (7%) of the ethanol-treated cells exhibited the swollen acrosome seen in panel C to

varying degrees. The cell in panel D, unique to the ethanol-treated sample, showed multiple continuous layers of membranes, indicated by

the arrowheads. Panels A and B (X 15,000); panel C (X 20,000); panel D (X 18,000).

at about 0.5 mM (Fig. 4). The effects of 2 mM ECTA

and 0.2 mM verapamil were also examined under

these same conditions in the presence of 1.7 mM

Ca2�, the normal concentration of this cation in BWW

medium, with the results shown in Table 2. As

expected from the result obtained in the absence of

added Ca2�, ECTA, as well as verapamil, blocked the

ethanol-induced acrosomal loss.

Alcohol Specificity

The question of whether the accelerated acro-

somal loss seen with ethanol is specific to that alcohol

was addressed in the experiment reported in Table 3.

Three concentrations of the normal alcohols, C1 to

C4, were tested. The higher alcohols were more

effective in the acute acceleration after 0.25 hour of

incubation (Table 3). The potency of the alcohols

correlates qualitatively with their partition coeffi-

cients between water and dipalmitoyl phosphatidyl-

choline as either bulk phase (Hill, 1975) or in the form

of vesicles (Kamaya et al, 1981) (Table 3).

Acetaldehyde and Lipid Peroxidation

50

25

(0a,0

-J

E0

0I-

0

-- TABLE 2. Effect of EGTA and Verapamil on the- - Percent Acros’omal Loss in Human Spermatozoa Observed in

the Presence of 250 mM Ethanol after 0.25 Hours Incubation’

0 2.5

No.6 ETHANOL AND ACROSOMAL LOSS . Alvarez et al 363

PercentAcrosomal

Inhibitor Loss’

None 27±4EGTA (2 mM) 1±2Verapamil (0.2 mM)t 1 ± 1

‘Values are the mean ± SD of 10 samples from five differentdonors obtained with the chiortetracychine assay. For the sam-pies treated with EGTA, the range was 0 to 3%, and for thosetreated with verapamil, the range was 0 to 2%. Sperm cellconcentrations ranged between 10 and 25 X 108 cells/mI.

tVerapamil was added as an 80-mM stock solution in ace-tone, giving a final acetone content of 0.25% (v/v). Acetonealone at this concentration in the absence of ethanol gave 1 ±

1% acrosome reactions (range 0 to 2%).

[Ca++] mM

Fig. 4. Dependence of ethanol-induced acrosomal loss inhuman spermatozoa on the calcium concentration in the medium.Incubations were for 0.25 hour in the presence of 250 mM

ethanol. Sperm cell concentrations ranged between 8 and 18 X

106 cells/mi. Calcium concentrations were 0.5, 1.0, 2.0, 3.0, 4.0

and 5.0 mM. Each point represents the mean ± SD of 10 determi-nations with the chlortetracycline fluorescence assay.

to its oxidation product, acetaldehyde, was ruled out

by analysis for this compound after incubation. Even

in the samples containing 250 mM ethanol, acetalde-

hyde could not be detected after incubations up to 6

hours. The detection limit was 0.4 AM, well below

the 2 to 8-AM levels found by Anderson et al (1985)

in the testis of mice fed ethanol, which was shown by

them to have no effect on the mouse spermatozoa.

The possibility that lipid peroxidation in human

spermatozoa (Alvarez et al, 1987) could be enhanced

by ethanol, and so accelerate acrosomal loss, was also

ruled out. Determination of the rate of malonalde-

hyde production, the index used for assessing lipid

peroxidation (Alvarez and Storey, 1982), from 10

samples in the absence or presence of 250 mM

ethanol showed no difference in rates for the same

san-ple. Different samples showed different rates,

but all showed the same lipoperoxidative lethal end-

point of 0.1 nmol/108 cells observed in the earlier

study (Alvarez et al, 1987).

Effect of Cholesterol

In a number of studies, loss of cholesterol from the

sperm plasma membrane has been implicated as

being a concomitant of capacitation (Davis, 1980,

1981; Davis et al, 1980; Go and Wolf, 1985). A

detailed theory of capacitation and the acrosome

reaction, based on the postulate of cholesterol and

cholesterol sulfate efflux from the plasma membrane

overlying the acrosome, has been proposed (Langlais

and Roberts, 1985). The possibility that ethanol

accelerated acrosomal loss in human spermatozoa by

enhancing cholesterol efflux was therefore exam-

ined. The time course of cholesterol efflux in the

presence of different ethanol concentrations is shown

in Fig. 5. There was a dose-dependent decrease in the

cholesterol content of the spermatozoa with time.

TABLE 3. Comparison of the Effects of n-C1 to n-C4Alcohols on the Percent Acrosomal Loss in Human

Spermatozoa with their Partition Coefficients between

Diphosphatidyl Choline (DPPC) and Water

Alcohol

Percent Acrosom al Loss’Partition

Coefficientt

DPPC/H2025 mM 125 mM 250 mM

CH3OHC2H5OHn-C3H7OHn-C4H9OH

<1<15 ± 2

23 ± 3

<210± 2

30 ± 448 ± 5

5±225 ± 3

45 ± 549 ± 5

0.7 -

4.3 2.5

22.7 19.0114 61.6

‘Values are means ± SD for three separate samples from

three donors, determined with the chlortetracychine assay after0.25 hour of incubation. Sperm cell concentrations rangedbetween 12 and 21 X 108 cells/mI.

tValues in the left-hand column are the measured coetf i-cients for partition between liquid DPPC and water (Hill, 1975);those in the right-hand column are for partition between DPPCvesicles and water (Kamaya et al, 1981).

71)

35

0

TABLE 4. Effect of Cholesterol Microdispersions on the

Percent Acrosomal Loss and Cholesterol Content ofHuman Spermatozoa during Incubation in the Presence

and Absence of Ethanol*

Incubation time in hours

364 Journal of Andrology November/December 1988 Vol. 9

C,)

0)C)

a,

0‘-�-

0)�.

.-

0I-

0)-C,)

a)0

.c0

0 2 4

Fig. 5. Ethanol-induced changes in cholesterol concentration

in human spermatozoa as a function of incubation time at differ-ent ethanol concentrations. Sperm cell concentrations ranged

between 10 and 20 X 106 cells/mI. Each point represents the mean

± SD of 10 determinations. Ethanol concentrations are as fol-lows: closed circles, 0 mM; open triangles, 25 mM; open circles, 50mM; open squares, 125 mM; open diamonds, 250 mM.

Analysis of the cholesterol content of the medium

showed an increase with time that accounted quan-

titatively for the loss of cholesterol from the cells.

Comparison of the cholesterol loss at 4 hours (Fig. 5)

with the percentage acrosomal loss at the same time

point (Fig. 1) at the different ethanol concentrations

suggests a good correlation. In order to separate cho-

lesterol loss effects from other ethanol effects, the

experiment was repeated at 25 and 250 mM ethanol

in the presence of cholesterol microdispersions to

maintain saturation of the medium by cholesterol.

The results are shown in Table 4. The cholesterol

content of the sperm cells remained the same during

the 4-hour incubation, while the percentage of acro-

somal loss was the same at a given ethanol concentra-

tion as in the absence of the microdispersions. This

result indicates that ethanol acceleration of acro-

somal loss operates through a mechanism that does

not involve net cell loss of cholesterol.

Discussion

The results of this study show that ethanol in

concentrations up to 250 mM accelerates the loss of

acrosomes from human spermatozoa in a dose-

dependent manner. Both the percentage of cells

induced and the rate of acrosomal loss are increased.

IncubationTime (h)

EthanolAdded (mM)

CholesterolContent

�g/1O8 Cells

PercenttAcrosomal

Loss

0.252.04.0

000

70±677±181±1

<2<2

6±1

0.252.04.0

252525

70±675±178±1

<210±116±1

0.252.04.0

250250250

71±574±177±1

23±334±1

41±1

*Sperm cell concentrations ranged between 10 and 18 X 108

cells/mI. Cholesterol microdispersions were prepared as de-scribed in Materials and Methods. The cholesterol concentra-tion was adjusted to maintain a constant cholesterol/cell ratio

of 64 ± 4 Mg/i 08 cells (mean ± SD for the nine experimentsshown).

tValues given are means ± SD for 10 samples from fivedifferent donors obtained with the chlortetracychine assay.Values <2 ranged from 0% to 2% acrosome reactions. Thevalues for the cholesterol content do not differ significantlyfrom each other (P>0.1)

The reaction leading to acrosomal loss must be rapid,

since no intermediate vesiculation was observed, and

appears to involve not only loss of the acrosomal

contents but also of the equatorial segment (Fig. 3B).

It therefore is different in a major way from the

acrosome reaction induced by follicular fluid, during

which vesiculation is observed and the equatorial

segment remains intact (Suarez et al, 1986). These

differences would explain the observation of Rogers

et al (1987) that ethanol inhibited the penetration of

zona-free hamster eggs by human spermatozoa in

just the concentration range examined in this study.

The opposite result would have been expected if

ethanol had enhanced the percentage of normal

acrosome-reacted cells, which are the ones with the

capability of penetrating zona-free eggs (Yanagima-

chi, 1981). The loss of the equatorial segment plo sma

membrane in the presence of ethanol would eAfec-

tively prevent sperm-egg fusion (Bedford �d

Cooper, 1978). This aspect of ethanol-induced acro-

somal loss would not be picked up by either the

chlortetracycline or the indirect immunofluorescence

assay, since both assays depend only on the presence

or absence of the outer acrosomal and overlying

plasma membranes (Wolf et al, 1985; Lee et al, 1987).

No.6 ETHANOL AND ACROSOMAL LOSS . Alvarez et al 365

One aspect of the ethanol-induced acrosornal loss

that is shared with the physiologic acrosome reaction

is the requirement for Ca2�. Addition of sufficient

EGTA to chelate the 1.7 mM Ca2� in the BWW

medium prevents acrosomal loss after the 0.25-hour

incubation in the presence of 250 mM ethanol (Table

2). The calcium channel blocker verapamil also has an

inhibitory effect at 0.2 mM. This observation is in

contrast to that of Roldan et al (1986), who found

that 0.1 mM verapamil accelerated the onset of the

acrosome reaction in guinea pig spermatozoa and did

not affect the reaction in hamster spermatozoa.

Ethanol was absent in these experiments. Roldan et

a! (1986) were using conditions that have been care-

fully worked out to provide maximal spontaneous

acrosome reactions in the spermatozoa of these two

species. Comparable conditions have not been found

for human spermatozoa, so that the effect of vera-

pamil on the percentage of acrosome reactions in the

absence of ethanol cannot be assessed accurately. At

present, the inhibitory effect of verapamil has been

observed only in the presence of ethanol. This effect

may eventually provide a way to differentiate be-

tween ethanol-accelerated acrosomal loss and the

physiologic acrosome reaction in human spermato-

zoa.

The effect of ethanol is not specific to this particu-

lar compound (Table 3); it does, however, appear to

be quite specific to the straight-chain alcohols. Ace-

tone, used as the solvent for verapamil and tested

separately in this study (Table 2), had no effect at 43

mM (250 mg %), and neither did dimethylformamide

at 14 mM (100mg %) (Table 2). Anderson et al (1982)

tested t-butanol with negative results, whereas n-

butanol (Table 3) is more potent than ethanol. The

qualitative correlation of the potency of the normal

alcohols in accelerating acrosomal loss with their par-

tition coefficient in dipalmitoyl phosphatidylcholine

(Table 3) indicates that alcohols with straight chains

may distribute into the plasma membrane more

effectively than branched molecules. This supposi-

tion remains to be tested.

The induction of acrosomal loss is observed at

ethanol concentrations low enough to be encoun-

tered during social imbibition of alcoholic beverages.

The concentration of 25 mM corresponds to 115

mg %; in many states, the legal limit for blood alcohol

in defining intoxication while driving is 100 mg % or

22 mM (Mason and Dubowski, 1974). It is therefore

conceivable that nonphysiologic acrosomal loss may

occur at an accelerated rate due to periodic exposure

of spermatozoa to low levels of ethanol in either the

male or the female reproductive tract, with adverse

effects on fertility. The samples used in this study

were from healthy, fertile donors. The question of

whether congenital factors, illness, malnutrition, or

prolonged and elevated ethanol consumption may

render human spermatozoa more sensitive to this

effect remains to be answered by appropriate clinical

tests. This study does suggest that testing the sper-

matozoa of male patients with unexplained infertility

for sensitivity to ethanol acceleration of acrosomal

loss may prove to be a useful diagnostic tool.

Acknowledgments

The authors wish to thank Dr. Kathleen Bechtol of Genentech,Inc for a generous supply of monoclonal antibody HS-19 and Dr.

Gabor Huszar of Yale University for consultation and advice.They are also grateful to Dr. Melitza Ausmanas of the Andrology

Laboratory, Department of Obstetrics and Gynecology, for aid inscheduling normal donors of semen samples, and to Ms. DebbieCoffin and Ms. Barbara Brewer for expert secretarial assistance.

References

Alvarez JG, Storey BT. Spontaneous lipid peroxidation in rabbit

epididymal spermatozoa. Biol Reprod 1982; 27:1102-1108.

Alvarez JG, Touchstone JC, Blasco L, Storey BT. Spontaneous

lipid peroxidation and production of hydrogen peroxide and

superoxide in human spermatozoa. Superoxide dismutase as

major enzyme protectant against oxygen toxicity. J Androl

1987; 8:338-348.

Alvarez MR. Aneuploidy in male germ cells induced by alcohol

ingestion. Gamete Res 1985; 12:165-170.

Amelar RD. Infertility in men: diagnosis and treatment. Philadel-

phia: Davis, 1966.

Anderson RA Jr. Beyler SA, Zaneveld LJD. Alterations of male

reproduction induced by chronic ingestion of ethanol: devel-

opment of an animal model. Fertil Steril 1978; 30:103-105.Anderson RA Jr, Furby JE, Oswald C, Zaneveld LiD. Teratologi-

cal evaluation of mouse fetuses after paternal alcohol inges-

tion. Neurobehav Toxicol Teratol 1981; 3:117-120.

Anderson RA Jr. Reddy JM, Joyce C, Willis BR, Van der Ven H,

Zaneveld LJD. Inhibition of mouse sperm capacitation by

ethanol. Biol Reprod 1982; 27:833-840.

Anderson RA Jr. Reddy JM, Zaneveld LJD. Prevention of in vitro

fertilizing capacity of mouse spermatozoa by ethanol. JAndrol 1980a; 1:81-82.

Anderson RA Jr. Willis BR, Oswald C, Reddy JM, Beyler SA,

Zaneveld LJD. Hormonal imbalance and alterations in testic-

ular morphology induced by chronic ingestion of ethanol.

Biochem Pharmacol 1980b, 29:1409-1419.Anderson RA Jr. Willis BR. Alcohol and male fertility. BrJ Alcohol

Alcohol 1981; 16:179-185.

Anderson RA Jr, Willis BR, Oswald C, Zaneveld LJD. Male repro-ductive tract sensitivity to ethanol: a critical overview. Phar-

macol Biochem Behav 1983a; 18(suppl 1):305-310.

Anderson RA Jr, Willis BR, Oswald C, Zaneveld LJD. Ethanol-

induced male infertility: impairment of spermatozoa. J Phar-

macol Exp Ther 1983b; 225:479-486.

Anderson RA Jr, Willis BR, Oswald C, Zaneveld LJD. Partial

reversal of ethanol-induced male reproductive pathology fol-

lowing abstinence. Alcohol Alcohol 1985; 20:273-286.

Badr FM, Bartke A. Effect of ethyl alcohol on plasma testosterone

level in mice. Steroids 1974; 23:921-927.

Barros C, Bedford JM, Franklin LE, Austin CR. Membrane vesicu-

366 Journal of Andrology #{149}November/December 1988 Vol. 9

lation as a feature of the mammalian acrosome reaction. J CellBiol 1967; 34:C1-C5.

Bedford JM. Ultrastructural changes in the sperm head during

fertilization in the rabbit. Am J Anat 1968; 123:329-358.

Bedford JM, Cooper GW. Membrane fusion events in the fertili-

zation of vertebrate eggs. In: Poste G, Nicolson GL, eds. Cell

surface reviews; Vol. 5. Membrane fusion. Amsterdam:

Elsevier/North-Holland, 1978; 65-125.

Biggers JD, Whitten WK, Whittingham DC. The culture of

mouse embryos in vitro. In: Daniel JC Jr. ed. Methods in

mammalian embryology. San Francisco: Freeman, 1971;

86-116.

Byrd W, Wolf DP. Acrosomal status in fresh and capacitated

human ejaculated sperm. Biol Reprod 1986; 34:859-869.

Cash MKM, Rogers BJ. The effect of alcohol on fertilization by

human and hamster spermatozoa lAbstr]. Biol Reprod 1981;

24(suppl 1):139A.

Davis BK. Interaction of lipids with the plasma membranes ofsperm cells. I. The antifertilization action of cholesterol. Arch

Androl 1980; 5:249-254.

Davis BK. Timing of fertilization in mammals: sperm cholesterolphospholipid ratio as a determinant of the capacitation inter-

val. Proc NatI Acad Sci USA 1981; 78:7560-7564.

Davis BK, Byrne R, Bedigan K. Studies on the mechanism of

capacitation: albumin-mediated changes in plasma mem-

brane lipids during in vitro incubation of rat sperm cells. Proc

NatI Acad Sci USA 1980; 77:1546-1550.

Florman HM, Bechtol KB, Wassarman PM. Enzymatic dissection

of the functions of mouse egg’s receptor for sperm. Dev Biol1984; 106:243-255.

Go K, Wolf DP. Albumin-mediated changes in sperm sterol con-tent during capacitation. Biol Reprod 1985; 32:145-153.

Hill MW. Partition coefficients of some anaesthetic-like mole-

cules between water and smectic mesophases of dipalmitoyl

phosphatidylcholine. Biochem Soc Trans 1975; 3:149-152.

lozzo RV. Biosynthesis of heparan sulfate proteoglycan by

human colon carcinoma cells and its localization at the cellsurface. J Cell Biol 1984; 99:403-417.

Joyce C, Oswald C, Anderson RA Jr. Inhibition of fertilization in

vivo by ethanol IAbstrl. Biol Reprod 1981; 24(suppl 1):138A.Kamaya H, Kaneshina S, Issaku U. Partition equilibrium of inha-

lation anesthetics and alcohols between water and mem-

branes of phospholipids with varying chain lengths. Biochim

Biophys Acta 1981; 646:135-142.Karnovsky MJ. A formaldehyde-glutaraldehyde fixative of high

osmolality for use in electron microscopy EAbstri. J Cell Biol1965; 27:137a.

Langlais J, Roberts KD. A molecular model of sperm capacitation

and the acrosome reaction of mammalian spermatozoa.

Gamete Res 1985; 12:183-224.Lee MA, Trucco CS, Bechtol KB, Wummer N, Kopf CS, Blasco L,

Storey BT. Capacitation and acrosome reactions in human

spermatozoa monitored by a chlortetracycline fluorescence

assay. Fertil Steril 1987; 48:649-658.

Mason MF, Dubowski KM. Alcohol, traffic, and chemical testing

in the United States: a resume and some remaining problems.

Clin Chem 1974; 20:126-140.

Meizel S. The importance of hydrolytic enzymes to an exocytotic

event, the mammalian acrosome reaction. Biol Rev 1984;

59:125-157.

Moore HDM, Smith CA, Hartman TD, Bye AP. Visualization and

characterization of the acrosome reaction of human sperma-

tozoa by immunolocalization with monoclonal antibody.

Gamete Res 1987; 17:245-259.

Overstreet JW, Yanagimachi R, Katz DF, Hayashi K, Hanson FW.

Penetration of human spermatozoa into the human zona

pellucida and the zona-free hamster egg: a study of fertile

donors and infertile patients. Fertil Steril 1980; 33:534-542.

Rogers BJ, Cash MKM, Vaughn WK. Ethanol inhibits human and

hamster sperm penetration of eggs. Gamete Res 1987;

16:97-107.

Roldan ERS, Shibata S. Yanagimachi R. Effect of Ca2’ channelantagonists on the acrosome reaction of guinea pig and

golden hamster spermatozoa. Gamete Res 1986; 13:281-292.

Salonen I. Exposure to ethanol during capacitation impairs the

fertilizing ability of human spermatozoa in vitro. mt J Androl1986; 9:259-270.

Suarez SS, Wolf DP, Meizel S. Induction of the acrosome reaction

in human spermatozoa by a fraction of human follicular fluid.

Gamete Res 1986; 14:107-121.Talbot P. Chacon RS. A triple stain technique for evaluating

normal acrosome reactions of human sperm. J Exp Zool 1981;

215:201-208.

Touchstone JC, Levin SS, Dobbins MF, Beers PC. Analysis ofsaturated and nonsaturated phospholipids in biological fluids.

Journal of Liquid Chromatography 1983; 6:179-192.Van Thiel DH, Lester R. Sex and alcohol. N EngI J Med 1974;

291:251-253.Ward CR, Storey BT. Determination of the time course of capaci-

tation in mouse spermatozoa using a chlortetracycline fluo-

rescence assay. Dev Biol 1984; 104:287-296.

Weathersbee PS, Lodge JR. A review of ethanol’s effects on the

reproductive process. J Reprod Med 1978; 21:63-68.Willis BR, Anderson RA Jr. Oswald C, Zaneveld LJD. Ethanol-

induced male reproductive tract pathology as a function of

ethanol dose and duration of exposure. J Pharmacol Exp Ther

1983; 225:470-478.

Wolf DP, Boldt J, Byrd W, Bechtol KB. Acrosomal status evalua-

tion in human ejaculated sperm with monoclonal antibodies.

Biol Reprod 1985; 32:1157-1162.

Yanagimachi R. Mechanism of fertilization in mammals. In: Mas-troianni L, Biggers JD, eds. Fertilization and early develop-

ment in vitro. New York: Plenum, 1981:81-182.

Yanagimachi R, Usui N. Calcium dependence of the acrosomereaction and activation of guinea pig spermatozoa. Exp Cell

Res 1974; 89:161-164.

Postdoctoral Position

A postdoctoral position to study the biochemistry and physiology of ABP is currently available. A

suitable person would have a background in current biochemical and immunological techniques.

Purified proteins, monoclonal and polyclonal antibodies, and the reagents needed for these studies

are already on hand. Contact: Benjamin J. Danzo, PhD, Department of OB/GYN, Vanderbilt

University, Nashville, TN 37232. Telephone: (615) 322-4433.