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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.
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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.