FERTILITY AND STERILITY威
VOL. 77, NO. 2, FEBRUARY 2002
Received February 28,
2001; revised and
accepted August 24, 2001.
Supported by the World
Health Organization (grant
97175), the Agencia
Nacional de Promoción de
Ciencia y Tecnologı́a
(PICT97 00207), and the
Consejo Nacional de
Investigaciones Cientı́ficas
y Técnicas (CONICET) of
Argentina.
Presented at the 44th
Annual Meeting of the
Argentine Society of
Clinical Research, Mar del
Plata, Argentina, November
17–20, 1999.
Reprint requests: Mónica
H. Vazquez-Levin, Ph.D.,
Instituto de Biologı́a y
Medicina Experimental,
Vuelta de Obligado 2490,
(1428) Buenos Aires,
Argentina (FAX: 54-11
4786-2564; E-mail:
mhvaz@dna.uba.ar).
0015-0282/02/$22.00
PII S0015-0282(01)02982-X
252
Copyright ©2002 American Society for Reproductive Medicine
Published by Elsevier Science Inc.
Printed on acid-free paper in U.S.A.
Effect of incubating human sperm at room
temperature on capacitation-related events
Clara I. Marı́n-Briggiler, Ph.D., Jorge G. Tezón, Ph.D., Patricia V. Miranda, Ph.D., and
Mónica H. Vazquez-Levin, Ph.D.
Instituto de Biologı́a y Medicina Experimental, CONICET-UBA, Buenos Aires, Argentina
Objective: To determine the effect of human sperm incubation at room temperature (20°C) upon capacita-
tion-related events.
Design: Prospective study.
Setting: Basic research laboratory.
Patient(s): Semen samples were obtained from normozoospermic volunteers. Human follicular fluid (hFF)
was collected from women undergoing assisted reproductive treatment.
Intervention(s): Spermatozoa were incubated for up to 18 hours at 20°C and/or 37°C.
Main Outcome Measure(s): Protein tyrosine phosphorylation patterns, development of hyperactivated
motility, and induction of acrosome reaction (AR) in response to hFF.
Result(s): Spermatozoa incubated for 18 hours at 20°C showed an array of tyrosine phosphorylated proteins
similar to noncapacitated cells. After incubation at 20°C, the percentage of spermatozoa displaying hyperac-
tivated motility and undergoing acrosomal loss in response to hFF was significantly lower when compared
with cells kept the same time at 37°C. Conversely, spermatozoa incubated overnight at 37°C could respond
to hFF, either at 37°C or 20°C. When preincubation at 20°C was followed by sperm exposure to 37°C,
capacitation-related events could be activated. In capacitated cells (16 hours at 37°C), 2-hour incubation at
20°C led to a significant decrease in acrosome reaction inducibility, suggesting sperm decapacitation.
Conclusion(s): Human sperm incubation at room temperature does not allow capacitation, although it does
not affect hFF-induced acrosome reaction in capacitated cells. The blocking effect is overcome when
spermatozoa are exposed to 37°C. (Fertil Steril威 2002;77:252–9. ©2002 by American Society for Reproduc-
tive Medicine.)
Key Words: Acrosome reaction, human follicular fluid, human spermatozoa, sperm capacitation,
temperature
Mammalian spermatozoa cannot fertilize an as homologous zona pellucida (ZP), progester-
oocyte immediately after ejaculation. To be- one, and follicular fluid (2).
come fertilization-competent, the male gamete
Capacitation and AR can be achieved in
must undergo several metabolic and structural
vitro by placing spermatozoa under defined
changes, collectively known as capacitation.
conditions, which include incubation at body
Sperm capacitation is a poorly understood pro-
temperature. Previous studies have shown that
cess that has been associated with modifica-
the incubation temperature has a modulatory
tions in sperm plasma membrane composition
effect on sperm capacitation and AR in animals
and fluidity, alterations in intracellular ion con-
(3– 6). Regarding human spermatozoa, some
centrations, and changes in the oxidative me-
reports have described how temperature influ-
tabolism. In addition, a relationship between
ences cell motility, spontaneous AR, and
sperm capacitation and phosphorylation on ty-
sperm’s ability to penetrate the ZP-free ham-
rosine residues of many sperm proteins has
ster egg (7–9). However, the effect of the in-
been reported in several species, including hu-
cubation temperature on human sperm capaci-
mans (1). As a result of capacitation, sperma-
tation has not been completely characterized.
tozoa develop hyperactivated motility and can
undergo acrosomal exocytosis or acrosome re- Processing of human spermatozoa during
action (AR) in response to certain stimuli, such diagnostic and/or therapeutic procedures usu-
ally involves cell exposure to room temperature for different
periods of time. Little is known about the effect of this
treatment on the sperm’s fertilizing ability in vitro.
The present study was designed to determine [1] whether
incubation at room temperature allows human sperm capac-
itation-related events; [2] whether temperature variations
(exposure to 37°C followed by room temperature or vice
versa) affect sperm function. For this, spermatozoa were
incubated for up to 18 hours at 37°C and/or 20°C. Protein
tyrosine phosphorylation, development of hyperactivated
motility, and AR inducibility in response to human follicular
fluid were evaluated.
MATERIALS AND METHODS
Culture Media
Human sperm medium (HSM) (10) was used throughout
the study. It consisted of 117.5 mM of NaCl, 0.3 mM of
NaH2PO4, 8.6 mM of KCl, 2.5 mM of CaCl2, 0.49 mM of
MgCl2, 2 mM of glucose, 19 mM of sodium lactate, 25 mM
of NaHCO3, 0.25 mM of sodium pyruvate, 50 ␮g/mL of
penicillin, and 75 ␮g/mL of streptomycin.
Semen Samples and Sperm Processing
Semen samples were obtained from normozoospermic
donors according to World Health Organization standards
(11). All samples were obtained with the donor’s written
consent, and the study protocol was approved by a local
institutional review board.
The semen parameters profile of the ejaculates used in the
present study was as follows: [1] Semen volume, 3.3 mL (1.4
to 7.0) as the mean value (maximum to minimum values);
[2] concentration, 79 ⫻ 106 cells/mL (30 to 126); [3] mo-
tility (grade a ⫹ b), 84% (63 to 95); and normal sperm
morphology by Kruger criteria, 26% (20 to 37). All ejacu-
lates contained less than 1 ⫻ 106 round cells/mL. In each
experiment, ejaculates from different donors were used.
After complete liquefaction, samples were subjected to
sperm selection using glass wool columns (Microfibre Man-
ville, Denver, CO) (12), and highly motile cells were resus-
pended in HSM supplemented with 2.6% bovine serum
albumin (BSA, cat #A7030; Sigma Chemical Co., St. Louis,
MO) as the capacitating medium. Sperm concentration was
adjusted to 1.5 ⫻ 106 cells/mL, and 2-mL aliquots were
incubated either at 37°C or at 20°C, 5% CO2 in air, for
different periods of time (see below). We selected 20°C as
the standard laboratory room temperature, and those incuba-
tions were done using a heated bath. Motility after incuba-
tion was determined by observation under light microscopy
(⫻400 magnification; Alphaphot-2 YS2, Nikon, Tokyo, Ja-
pan). Only samples with more than 75% progressive motile
cells were included in the study. Sperm vitality was assessed
using 0.5% eosin Y in aqueous sodium chloride solution as
described elsewhere (11).
FERTILITY & STERILITY威
Detection of Tyrosine-Phosphorylated
Proteins
Proteins from spermatozoa that were incubated under
different experimental conditions were analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and
Western immunoblotting, using a monoclonal antiphospho-
tyrosine antibody (cat # 05-321; Upstate Biotechnology Inc.,
Lake Placid, NY) as previously described (13).
Hyperactivation Analysis
Aliquots of 5 ␮L of spermatozoa (10 ⫻ 106 cells/mL)
incubated for 4 hours at 37°C or at 20°C were placed in a
MicroCell chamber (Conception Technologies, San Diego,
CA) and were analyzed with the use of an IVOS V10.9
CASA instrument (Hamilton-Thorne Research, Beverly,
MA). The settings used during the analysis were: number of
frames analyzed per second ⫽ 30, frame rate ⫽ 60 Hz,
minimum contrast ⫽ 80, minimum cell size ⫽ 3 pixels, and
minimum static contrast ⫽ 30. The kinematic values for at
least 200 motile spermatozoa were analyzed in each sample,
and cells with curvilinear velocity (VCL) ⱖ 100 ␮m/sec,
lateral head displacement (ALH) ⱖ 5 ␮m, and linearity
(LIN) ⱕ 60% were considered hyperactivated (14).
Follicular Fluid Preparation
Human follicular fluid (hFF) was obtained, with patient
consent, from women undergoing assisted fertilization fol-
lowing an ovulation-induction protocol previously described
(15). Follicles were aspirated, and the fluids were centri-
fuged for 15 minutes at 1500 ⫻ g to remove cellular debris.
Aliquots from 10 hFF samples were pooled and stored at
⫺20°C until further use. Throughout the study two different
pools of hFF were used, which proved to be equally capable
of inducing a significant increase in human sperm AR.
Acrosome Reaction Induction
To determine the effect of incubation at room temperature
on human sperm capacitation and hFF-induced AR, two sets
of experiments were conducted. First, aliquots of 0.75 ⫻ 106
spermatozoa were incubated for 18 hours at either 37°C or
20°C and then were exposed to 10% human follicular fluid
(hFF-induced AR) or buffer (spontaneous AR) for 45 min-
utes, 5% CO2 in air.
Second, to distinguish whether incubation at 20°C was
affecting the AR itself and/or the capacitation process, sperm
suspensions were incubated for 18 hours at either 37°C or
20°C, divided into two aliquots and exposed to hFF or buffer
at 37°C or 20°C. Thus, four experimental conditions were
obtained: 37/37, 37/20, 20/37, and 20/20 (temperature of the
overnight incubation/temperature of the AR-induction peri-
od). To achieve temperature stabilization before adding the
hFF or the buffer, spermatozoa were preincubated for 30
minutes at the temperature used during the AR induction
period.
To analyze whether variations in the incubation temper-
ature had any effect on sperm capacitation, cells were incu-
253
bated for 18 hours under different conditions (see below) and
then were exposed to hFF or buffer. In this case, AR induc- FIGURE 1
tion in all aliquots was performed at 37°C. Protein tyrosine phosphorylation patterns in human sperma-
At the end of the AR-induction procedure, sperm suspen- tozoa incubated at 37°C vs. 20°C. Motile spermatozoa were
sions were processed and the acrosomal status was evaluated resuspended in HSM and incubated for 0 hours (lane A) and
18 hours at 37°C (lane B) or at 20°C (lane C). Sperm protein
as described (13). patterns were analyzed by Western immunoblot using a
monoclonal antiphosphotyrosine antibody. Molecular weight
Statistical Analysis standards (Mr ⫻ 10⫺3) are indicated on the left. A typical
Data were expressed as mean ⫾ SE. To assume normal experiment is shown. This experiment was performed three
distribution, percentages were converted to ratios and sub- times obtaining similar results.
jected to the arc sine square root transformation. Acrosome
reaction results were expressed as the percentage of acro-
some-reacted spermatozoa or AR inducibility (% hFF-in-
duced AR minus % spontaneous AR). These results, the
percentages of motile and live cells, as well as the kinematic
values and percentage of hyperactivation of spermatozoa
incubated under different conditions, were compared by
Student’s t-test or one-way ANOVA and Student-Newman-
Keuls multiple comparison test. Statistical analyses were
done with an IBM-compatible computer using the GraphPad
InStat program (GraphPad Software, San Diego, CA).

RESULTS
Effect of Sperm Incubation at 20°C on
Protein Tyrosine Phosphorylation
After 18-hours of incubation at 37°C, an increase in the
levels of sperm protein tyrosine phosphorylation was ob-
served in comparison with noncapacitated cells (Fig. 1;
compare lanes A and B), as previously reported (for a list of
references, see [13]). In contrast, no enhancement in tyrosine
phosphorylation was seen when spermatozoa were incubated
for 18 hours at 20°C (see Fig. 1, lane C). Phosphorylation
was specific for tyrosine residues, as immunoreactivity was
completely abolished when the antibody was previously
blocked with O-phosphotyrosine (data not shown).
Effect of Sperm Incubation at 20°C on the
Development of Hyperactivated Motility
When spermatozoa were incubated for 4 hours at 37°C or Marı́n-Briggiler. Temperature and human sperm capacitation. Fertil Steril 2002.

20°C, similar percentages of progressive motility were ob-

tained (93% ⫾ 2% vs. 92% ⫾ 3%, respectively). However,
only cells kept at 37°C were able to develop hyperactivated
motility (16% ⫾ 2% at 37°C vs. 5% ⫾ 2% at 20°C;
P⬍.001). As shown in Table 1, the kinematic parameters
found in spermatozoa subjected to both conditions were
significantly different.
Effect of Sperm Incubation at 20°C on
Acrosome Reaction Induced With Human
Follicular Fluid
Spermatozoa maintained for 18 hours at 37°C and then
exposed to hFF at the same temperature showed a signifi-
cantly higher percentage of AR than did cells incubated in
the absence of the inducer (38% ⫾ 3% for hFF-induced AR
vs. 14% ⫾ 2% for spontaneous AR; P⬍.001) (Fig. 2, left
panel). Conversely, when spermatozoa were kept overnight
at 20°C, no response to hFF was observed (6% ⫾ 1% for
hFF-induced AR vs. 5% ⫾ 1% for spontaneous AR) (see
Fig. 2, right panel). It is worth noting that the percentage of
spontaneous AR was also significantly diminished under this
condition (see Fig. 2). This difference could not be attributed
to an effect of the incubation temperature on sperm vitality
or motility, because results were similar after both treatments
(Table 2).
Spermatozoa are able to undergo the AR in response to
hFF only if they have completed the capacitation process
(12). To determine whether the incubation at 20°C was
affecting the AR itself and/or capacitation, spermatozoa
were kept for 18 hours at 37°C or 20°C, and incubated at

254 Marı́n-Briggiler et al. Temperature and human sperm capacitation Vol. 77, No. 2, February 2002
 TABLE 1
Kinematic characteristics of spermatozoa incubated for 4 hours at 37°C or at 20°C.
Parameter
VAP
VSL
VCL
ALH
BCF
STR
LIN
Note: Results are expressed as mean ⫾ SE, n ⫽ 9.
Abbreviations: VAP ⫽ average path velocity, VSL ⫽ straight-line velocity, VCL ⫽ curvilinear velocity, ALH ⫽ amplitude of lateral head displacement,
BCF ⫽ beat/cross frequency, STR ⫽ straightness, LIN ⫽ linearity.
a
P⬍.01 vs. values obtained for spermatozoa incubated at 37°C.
Marı́n-Briggiler. Temperature and human sperm capacitation. Fertil Steril 2002.
either 37°C or 20°C during the AR-induction period. The
results of AR inducibility obtained for each treatment are
shown in Figure 3. The inducibility of AR in cells incubated
for 18 hours at 37°C was similar when exposure to hFF was
done either at 20°C or 37°C (18% ⫾ 2% for 37/20 condition
vs. 24% ⫾ 3% for 37/37 condition). On the other hand, AR
was significantly reduced in spermatozoa subjected to 20/37
and 20/20 conditions (4% ⫾ 1% or 2% ⫾ 1% vs. 24% ⫾ 3%,
respectively; P⬍.001). Altogether, these results indicate that
FIGURE 2
Acrosome reaction in human spermatozoa incubated at 37°C
vs. 20°C. After 18-hours incubation in HSM at 37°C (left
panel) or at 20°C (right panel), spermatozoa were exposed to
10% human follicular fluid (hFF) or buffer (spontaneous; Spt)
for 45 minutes at the same temperature. The percentage of
acrosome-reacted spermatozoa was determined by Pisum
sativum agglutinin staining. Results are expressed as
mean ⫾ SE, n ⫽ 15. aP⬍.001 vs. spontaneous AR at 37°C.
b
P⬍.001 vs. hFF AR at 37°C.
Marı́n-Briggiler. Temperature and human sperm capacitation. Fertil Steril 2002.
FERTILITY & STERILITY威
37°C 20°C
70 ⫾ 3 50 ⫾ 3a
63 ⫾ 3 43 ⫾ 3a
107 ⫾ 4 78 ⫾ 4a
5⫾1 4 ⫾ 1a
28 ⫾ 1 24 ⫾ 1a
87 ⫾ 1 83 ⫾ 2a
58 ⫾ 1 53 ⫾ 1a
human sperm capacitation does not take place at 20°C. In
contrast, acrosomal exocytosis in response to hFF can occur
at this temperature in previously capacitated cells.
Effect of Temperature Changes on Events
Related to Human Sperm Capacitation
To evaluate whether temperature variations (sequential
exposure to 20°C and 37°C or vice versa) during in vitro
sperm handling affect capacitation, cells were incubated for
an 18-hour period with changes at the initial or the last 2
hours. Under these conditions, protein tyrosine phosphory-
lation patterns as well as AR inducibility in response to hFF
were evaluated, and the results were compared with those
obtained for cells incubated the whole period at 37°C or
20°C.
When a short exposure (2 hours) at 20°C was preceded or
followed by a long sperm incubation at body temperature
(conditions 2 hours at 20°C ⫹ 16 hours at 37°C, and 16
hours at 37°C ⫹ 2 hours at 20°C), the protein tyrosine
phosphorylation patterns were similar to those obtained after
18 hours at 37°C (Fig. 4, lanes D vs. B, and E vs. B).
To determine whether the low level of protein phosphor-
ylation found in spermatozoa incubated overnight at 20°C
(see Figs. 1 and 4, lane C) could be modified by cell
exposure to body temperature, cells were placed at 37°C for
2 hours before or after a 16-hour incubation at 20°C. Sperm
incubation at body temperature for the first 2 hours did not
induce noticeable changes in the levels of protein tyrosine
phosphorylation (see Fig. 4, lanes F vs. C). In contrast,
sperm exposure to body temperature in the last 2 hours led to
a discrete increase in protein tyrosine phosphorylation (see
Fig. 4, lanes G vs. I and G vs. C), and the signal was similar
to that obtained for cells incubated for only 2 hours at 37°C
(data not shown). These results suggest that the phosphory-
lation on tyrosine residues of spermatozoa incubated at 20°C
can be activated when cells are moved to 37°C.
Figure 5 shows the results of AR inducibility in response
255
 TABLE 2

Percentage of human sperm motility and vitality after incubation for a total 18 hours under different conditions.

Incubation conditions % motility % vitality

18 h at 37°C 92 ⫾ 1 94 ⫾ 2
18 h at 20°C 93 ⫾ 1 94 ⫾ 3
2 h at 20°C ⫹ 16 h at 37°C 90 ⫾ 2 92 ⫾ 1
16 h at 37°C ⫹ 2 h at 20°C 84 ⫾ 3a 92 ⫾ 1
2 h at 37°C ⫹ 16 h at 20°C 92 ⫾ 1 92 ⫾ 3
16 h at 20°C ⫹ 2 h at 37°C 93 ⫾ 1 94 ⫾ 1
Note: Results are expressed as mean ⫾ SE, n ⫽ 12.
a
P⬍.01 vs. values in the same column.
Marı́n-Briggiler. Temperature and human sperm capacitation. Fertil Steril 2002.

to hFF in spermatozoa subjected to the conditions described
above. Initial incubation at 20°C did not alter sperm ability
to respond to hFF (AR inducibility: 19% ⫾ 3% for 2 hours
at 20°C ⫹ 16 hours at 37°C vs. 22% ⫾ 4% for 18 hours at
37°C), suggesting that initial sperm exposure at 20°C does
not permanently inhibit the capacitation process. In contrast,
when spermatozoa were exposed to 20°C at the end of the
incubation period, a statistically significant decrease in the
AR inducibility was observed (5% ⫾ 1% for 16 hours at
FIGURE 3
Inducibility of AR in human spermatozoa incubated under
different temperature conditions. Motile spermatozoa were
maintained for 18 hours in HSM at 37°C or 20°C and exposed
to 10% human follicular fluid (hFF-induced AR) or buffer
(spontaneous AR) at 37°C or 20°C. Four experimental con-
ditions were then obtained, as indicated beneath the bars.
AR inducibility was calculated as % hFF-induced AR minus
% spontaneous AR. Results are expressed as mean ⫾ SE,
n ⫽ 15. aP⬍.001 vs. 37/37 and 37/20.
37°C ⫹ 2 hours at 20°C vs. 22% ⫾ 4% for 18 hours at 37°C;
P⬍.001). Because cells maintained for 16 hours at body
temperature can undergo AR in response to hFF (AR induc-
ibility: 22% ⫾ 3% for 16 hours at 37°C vs. 22% ⫾ 6% for
18 hours at 37°C; n ⫽ 4), the 2-hour exposure of capacitated
spermatozoa to 20°C may be conducive to sperm decapaci-
tation.
We then analyzed whether the impairment in capacitation
observed in spermatozoa maintained for long periods of time
at 20°C (see Figs. 2 and 3) could be overcome by a short
incubation at 37°C. Spermatozoa placed for 2 hours at body
temperature before or after 16 hours at 20°C were unable to
respond to hFF (AR inducibility: 2% ⫾ 1% and 1% ⫾ 1%,
respectively). These results show that, in spermatozoa kept
overnight at room temperature, the additional 2-hour incu-
bation at 37°C is insufficient to promote the capacitation-
related changes required for AR in response to a physiologic
stimulus.
Finally, we examined whether these changes in the incu-
bation temperature had any effect on sperm motility. Similar
percentages of motile spermatozoa were obtained in all eval-
uated conditions, with the exception of cells incubated for 16
hours at 37°C ⫹ 2 hours at 20°C (see Table 2). Under this
condition, there was a statistically significant decrease
(P⬍.01) in the progressive motility, which was not attributed
to sperm death (see Table 2). The reduction in the percentage
of motile cells was accompanied by a change in the type of
movement, with most spermatozoa displaying slow motion
(data not shown).

DISCUSSION
The results from the present study suggest that an ade-
quate incubation temperature is required for the expression
of human sperm fertilizing ability in vitro. Although com-
pletion of capacitation does not occur at 20°C, acrosomal
loss in response to hFF can take place at 20°C in previously
capacitated cells.
Marı́n-Briggiler. Temperature and human sperm capacitation. Fertil Steril 2002. It is well established that an increase in protein tyrosine

256 Marı́n-Briggiler et al. Temperature and human sperm capacitation Vol. 77, No. 2, February 2002
 FIGURE 4

Protein tyrosine phosphorylation patterns in human spermatozoa subjected to variations in the incubation temperature. Motile
spermatozoa were resuspended in HSM and incubated for 0 hours (lane A), for 18 hours at 37°C (lane B), for 18 hours at 20°C
(lane C), for a total period of 18 hours with combined incubations at 37°C and 20°C (lanes D to G), for 16 hours at 37°C (lane
H), or for 16 hours at 20°C (lane I). Sperm protein patterns were analyzed by Western immunoblot using a monoclonal
antiphosphotyrosine antibody. Molecular weight standards (Mr ⫻ 10⫺3) are indicated on the left. A typical experiment is shown.
This experiment was performed four times obtaining similar results.

Marı́n-Briggiler. Temperature and human sperm capacitation. Fertil Steril 2002.

phosphorylation accompanies the capacitation process. Sev-
eral studies have dealt with the absolute requirement of some
medium components for tyrosine phosphorylation of human
sperm proteins (13, 16 –18). Our study is the first to describe
the temperature dependence of this phenomenon in humans;
the results reveal that spermatozoa incubated at 20°C
showed protein tyrosine phosphorylation patterns similar to
noncapacitated cells. This result is in agreement with recent
findings in hamsters (19).
Protein phosphorylation on tyrosine residues is a complex
event that involves the participation of several transmem-
brane and intracellular signaling pathways (20), which may
be affected by the incubation temperature. In this regard, a
modification in the temperature of incubation has been
shown to affect membrane lipid diffusibility and peroxida-
tion (21–25), as well as antigen distribution on sperm plasma
membrane (26). Alterations in lipids and membrane fluidity
can cause changes in ion permeability (especially Ca2⫹ and
HCO3⫺) and in the activity of membrane-bound enzymes
(27–30).
Considering that protein phosphorylation is the result of a
phosphorylation/dephosphorylation equilibrium, it is possi-
ble that the temperature would directly affect the balance,
although inadequate functioning of other related enzymes
cannot be ruled out. Cholesterol efflux has been associated
with the activation of these transduction mechanisms
(18, 31), so it can also be speculated that, in cells incubated
at 20°C, protein tyrosine phosphorylation can be inhibited by
a blockage in the removal of cholesterol or other decapaci-
tating factors.
Regarding sperm motility, similar percentages of progres-
sively motile cells were obtained after incubation either at
37°C or 20°C. However, with the use of a CASA system it
was found that spermatozoa incubated for 4 hours at 20°C
were unable to develop hyperactivation, and showed differ-
ences in motion parameters when compared with cells ca-
pacitated at 37°C. In other species, previous reports have
shown that the development of hyperactivated motility is
highly dependent on the incubation temperature (3, 6). In
hamster spermatozoa, the mechanism by which temperature

FERTILITY & STERILITY威 257


FIGURE 5
Inducibility of AR in human spermatozoa subjected to varia-
tions in the incubation temperature. Motile spermatozoa
were incubated for a total period of 18 hours in HSM as
indicated beneath the bars and exposed to 10% human
follicular fluid (hFF-induced AR) or buffer (spontaneous AR) at
37°C. The AR inducibility was calculated as % hFF-induced
AR minus % spontaneous AR. Results are expressed as
mean ⫾ SE, n ⫽ 7. aP⬍.001 vs. 18 hours 37°C and 2 hours
20°C ⫹ 16 hours 37°C.
Marı́n-Briggiler. Temperature and human sperm capacitation. Fertil Steril 2002.
would regulate hyperactivation involves phosphorylation of
an 80-kd protein (19), a putative homologue to the AKAP-
82. Human AKAP-82 and its precursor have been localized
in the flagellum, and found to be tyrosine phosphorylated in
a capacitation-dependent manner (32, 33). Whether human
spermatozoa possess a temperature-regulated mechanism
similar to that seen in hamsters will require further investi-
gation.
Our results indicate that capacitated spermatozoa undergo
acrosomal exocytosis in response to hFF at 20°C. The hFF-
induced AR involves the interaction of specific agonist mol-
ecules (34 –36) with sperm plasma membrane receptors. This
leads to the activation of several intracellular pathways,
resulting in the release of the acrosomal content. As this
phenomenon occurs with similar efficiency either at 37°C or
at 20°C, the underlying mechanisms may not have a strict
temperature requirement. However, the complete under-
standing of the temperature effect on these signal transduc-
tion processes requires further analysis.
Concerning the spontaneous acrosomal loss, the present
study has shown a low percentage of acrosome-reacted sper-
matozoa after overnight incubation at 20°C. Because sperm
ability to undergo AR in the absence of an inducer has been
related to the membrane cholesterol content (37– 40), it
could be proposed that these reduced AR levels would be, in
258 Marı́n-Briggiler et al. Temperature and human sperm capacitation
part, a result of a failure in cholesterol efflux and/or attrib-
uted to alterations in membrane dynamics.
Considering that sperm capacitation is a temperature-
dependent phenomenon, variations in the incubation temper-
ature could cause alterations in some events associated with
this process. In spermatozoa incubated for a long period of
time at 20°C, the exposure to 37°C during the last 2 hours
resulted in a slight increase in protein tyrosine phosphoryla-
tion, in agreement with a previous report in the hamster (19).
Nevertheless, these cells failed to undergo acrosomal exo-
cytosis in response to hFF. An 8-hour incubation period at
body temperature has been shown to be necessary to reach
maximal hFF-induced AR (12); however, the period of time
at 37°C required for spermatozoa preincubated at 20°C to
respond to hFF is still unknown.
Our results show that cell exposure to 20°C before a long
incubation at body temperature did not affect either the
occurrence of protein tyrosine phosphorylation or AR induc-
ibility. In contrast, sperm incubation at 20°C during the last
2 hours of the capacitation period caused a failure in cell
response to hFF and a reduction in sperm motility. Never-
theless, no significant decrease in protein tyrosine phosphor-
ylation was noticeable, suggesting that the incubation of
capacitated spermatozoa at 20°C affects intracellular events
that are independent and/or downstream of protein tyrosine
phosphorylation, and that lead to AR. However, Western
immunoblot analysis may have failed to detect subtle
changes in the phosphorylation pattern. A longer cell expo-
sure to 20°C might be required to reveal the occurrence of
protein dephosphorylation.
In summary, our results show that the incubation temper-
ature regulates cellular mechanisms involved in sperm ca-
pacitation. In particular, sperm incubation at room tempera-
ture (20°C) produces a temporary blockage of capacitation-
related events. When maintained under this condition, cells
are in a “quiescent” state (noncapacitated, with low percent-
ages of spontaneous AR), retaining their functional proper-
ties that can be expressed at body temperature. Besides its
potential contribution in the understanding of the capacita-
tion process, the model of sperm exposure to room temper-
ature may be useful in male infertility diagnosis and/or
treatment. Spermatozoa could be transported at 20°C and
stored at this temperature for delayed inseminations as well
as reinsemination procedures. Incubation at 20°C could also
be beneficial in the so-called “fast capacitators”—patients
whose spermatozoa are characterized by a rapid capacitation
process (41, 42). The complexity and precise timing of fer-
tilization can make such rapid capacitation detrimental.
Acknowledgments: The authors thank Patricia Delcourt (Instituto de Bio-
logı́a y Medicina Experimental, Buenos Aires, Argentina) for her technical
Vol. 77, No. 2, February 2002
assistance, and members of Fertilab, Buenos Aires, Argentina for providing
the hFF samples used in the study.
References
1. Visconti PE, Kopf GS. Regulation of protein phosphorylation during
sperm capacitation. Biol Reprod 1998;59:1– 6.
2. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neill JD, eds.
The physiology of reproduction. New York: Raven Press, 1994:189 –
317.
3. Mahi CA, Yanagimachi R. The effect of temperature, osmolarity and
hydrogen ion concentration on the activation and the acrosome reaction
of golden hamster spermatozoa. J Reprod Fertil 1973;35:55– 66.
4. Lenz RW, Ball GD, Leibfried ML, Ax RL, First NL. In vitro maturation
and fertilization of bovine oocytes are temperature-dependent pro-
cesses. Biol Reprod 1983;29:173–9.
5. Fleming AD, Kuehl TJ. Effects of temperature upon capacitation of
guinea pig spermatozoa. J Exp Zool 1985;233:405–11.
6. Si Y. Temperature-dependent hyperactivated movement of hamster
spermatozoa. Biol Reprod 1997;57:1407–12.
7. White DR, Phillips DM, Bedford JM. Factors affecting the acrosome
reaction in human spermatozoa. J Reprod Fertil 1990;90:71– 80.
8. Cohen J, Fehilly CB, Walters DE. Prolonged storage of human sper-
matozoa at room temperature or in a refrigerator. Fertil Steril 1985;44:
254 – 62.
9. Kraemer M, Fillion C, Martin-Pont B, Auger J. Factors influencing
human sperm kinematic measurements by the Celltrak computer-as-
sisted sperm analysis. Human Reprod 1998;13:611–9.
10. Suarez SS, Wolf DP, Meizel S. Induction of the acrosome reaction in
human spermatozoa by a fraction of human follicular fluid. Gamete Res
1986;14:107–21.
11. World Health Organization. WHO laboratory manual for the examina-
tion of human semen and sperm-cervical mucus interaction. 4th ed.
New York: Cambridge University Press, 1999.
12. Calvo L, Vantman D, Banks SM, Tezón JG, Koukoulis G, Dennison L,
et al. Follicular fluid-induced acrosome reaction distinguishes a sub-
group of men with unexplained infertility not identified by semen
analysis. Fertil Steril 1989;52:1048 –54.
13. Marı́n-Briggiler CI, Vazquez-Levin M, Gonzalez Echeverria F, Bla-
quier JA, Tezón JG, Miranda PV. Strontium supports human sperm
capacitation but not follicular fluid-induced acrosome reaction. Biol
Reprod 1999;61:673– 80.
14. Mortimer ST, Mortimer D. Kinematics of human spermatozoa incu-
bated under capacitating conditions. J Androl 1990;11:195–203.
15. Meldrum DR, Wisot A, Hamilton F, Gutlay AL, Huynh D, Kempton
W. Timing of initiation and dose schedule of leuprolide influence the
time course of ovarian suppression. Fertil Steril 1988;50:400 –2.
16. Luconi M, Krausz C, Forti G, Baldi E. Extracellular calcium negatively
modulates tyrosine phosphorylation and tyrosine kinase activity during
capacitation of human spermatozoa. Biol Reprod 1996;55:207–16.
17. Emiliozzi C, Fenichel P. Protein tyrosine phosphorylation is associated
with capacitation of human sperm in vitro but is not sufficient for its
completion. Biol Reprod 1997;56:674 –9.
18. Osheroff JE, Visconti PE, Valenzuela JP, Travis AJ, Alvarez J, Kopf
GS. Regulation of human sperm capacitation by a cholesterol efflux-
stimulated signal transduction pathway leading to protein kinase A-me-
diated up-regulation of protein tyrosine phosphorylation. Mol Hum
Reprod 1999;5:1017–26.
19. Si Y. Hyperactivation of hamster sperm motility by temperature-de-
pendent tyrosine phosphorylation of an 80-kDa protein. Biol Reprod
1999;61:247–52.
20. Visconti PE, Galantino-Homer HL, Moore GD, Bailey JL, Ning X,
Fornes M, et al. The molecular basis of sperm capacitation. J Androl
1998;19:242– 8.
21. Alvarez JG, Storey BT. Spontaneous lipid peroxidation in rabbit and
mouse epididymal spermatozoa: dependence of rate on temperature and
oxygen concentration. Biol Reprod 1985;32:342–51.
FERTILITY & STERILITY威
22. Canvin AT, Buhr MM. Effect of temperature on the fluidity of boar
sperm membranes. J Reprod Fertil 1989;85:533– 40.
23. Harrison RA, Ashworth PJC, Miller NGA. Bicarbonate/CO2, an effec-
tor of capacitation, induces a rapid and reversible change in the lipid
architecture of boar sperm plasma membranes. Mol Reprod Dev 1996;
45:378 –91.
24. Ladha S, James PS, Clark DC, Howes EA, Jones R. Lateral mobility of
plasma membrane lipids in bull spermatozoa: heterogeneity between
surface domains and rigidification following cell death. J Cell Sci
1997;110:1041–50.
25. Wolfe CA, James PS, Mackie AR, Ladha S, Jones R. Regionalized lipid
diffusion in the plasma membrane of mammalian spermatozoa. Biol
Reprod 1998;59:1506 –14.
26. Villarroya S, Scholler R. Lateral diffusion of a human sperm-head
antigen during incubation in a capacitation medium and induction of the
acrosome reaction in vitro. J Reprod Fertil 1987;80:545– 62.
27. Holt WV, North RD. Thermotropic phase transitions in the plasma
membrane of spermatozoa. J Reprod Fertil 1986;78:447–57.
28. Babcock DF, Singh JP, Lardy H. Alteration of membrane permeability
to calcium ions during maturation of bovine spermatozoa. Dev Biol
1979;69:85–93.
29. Tocanne JF, Dupou-Cezanne L, Lopez A. Lipid lateral diffusion and
membrane organization. FEBS Lett 1989;257:10 –5.
30. Harrison RAP, Gadella BM. Membrane changes during capacitation
with special reference to lipid architecture. In: Fenichel P, Parinaud J,
eds. Human sperm acrosome reaction. Montrouge/Paris, France: John
Libbey Eurotext, Les editions INSERM, 1995:45– 65.
31. Visconti PE, Ning XP, Fornes MW, Alvarez JG, Stein P, Connors SA,
et al. Cholesterol efflux-mediated signal transduction in mammalian
sperm: cholesterol release signals an increase in protein tyrosine phos-
phorylation during mouse sperm capacitation. Dev Biol 1999;214:429 –
43.
32. Carrera A, Moos J, Ning XP, Gerton GL, Tesarik J, Kopf GS, et al.
Regulation of protein tyrosine phosphorylation in human sperm by a
calcium/calmodulin-dependent mechanism: identification of a kinase
anchor proteins as major substrates for tyrosine phosphorylation. Dev
Biol 1996;180:284 –96.
33. Turner RMO, Eriksson RLM, Gerton GL, Moss SB. Relationship
between sperm motility and the processing and tyrosine phosphoryla-
tion of two human sperm fibrous sheath proteins, pro-hAKAP82 and
hAKAP82. Mol Hum Reprod 1999;5:816 –24.
34. Osman RA, Andria JL, Jones AD, Meizel S. Steroid induced exocyto-
sis: the human sperm acrosome reaction. Biochem Biophys Res Com-
mun 1989;160:828 –33.
35. Blackmore PF, Beebe SJ, Danforth DR, Alexander N. Progesterone and
17␣-hydroxyprogesterone. Novel stimulators of calcium influx in hu-
man sperm. J Biol Chem 1990;265:1376 – 80.
36. Saragüeta P, Lanuza G, Miranda PV, Tezón JG, Barañao JL. Immuno-
globulins from human follicular fluid induce the acrosome reaction in
human sperm. Mol Reprod Dev 1994;39:280 – 8.
37. Byrd W, Tsu J, Wolf DP. Kinetics of spontaneous and induced acro-
somal loss in human sperm incubated under capacitating and nonca-
pacitating conditions. Gamete Res 1989;22:109 –22.
38. Cheetham JJ, Chen RJ, Epand RM. Interaction of calcium and choles-
terol sulphate induces membrane destabilization and fusion: implica-
tions for the acrosome reaction. Biochim Biophys Acta 1990;1024:367–
72.
39. Benoff S, Hurley I, Cooper GW, Mandel FS, Rosenfeld DL, Hershlag
A. Head-specific mannose-ligand receptor expression in human sper-
matozoa is dependent on capacitation-associated membrane cholesterol
loss. Hum Reprod 1993;8:2141–54.
40. Iborra A, Compayó M, Martı́nez P, Morros A. Cholesterol efflux
promotes acrosome reaction in goat spermatozoa. Biol Reprod 2000;
62:378 – 83.
41. Perreault SD, Rogers BJ. Capacitation pattern of human spermatozoa.
Fertil Steril 1982;38:258 – 60.
42. Stock CE, Fraser LR. The acrosome reaction in human sperm from men
of proven fertility. Hum Reprod 1987;2:109 –19.
259