Theriogenology 57 (2002) 1493±1501
Capacitation and acrosomal exocytosis are enhanced
by incubation of stallion spermatozoa in a
commercial semen extender
Angela C. Pommer, Jennifer J. Linfor, Stuart A. Meyers*
Sperm Biology Laboratory, Department of Anatomy, Physiology, and Cell Biology, School of Veterinary
Medicine, University of California, One Shields Avn., Davis, CA 95616, USA
Received 19 December 2000; accepted 31 August 2001
Abstract
Preserved stallion semen often has decreased spermatozoal motility and fertility that can vary
signi®cantly between individual stallions. It is not known whether the medium used for extending
equine sperm contributes to these decreases by inducing premature capacitation during storage. If
spermatozoa undergo capacitation or acrosome reaction prior to insemination, this could result in a
diminished capacity to penetrate the cumulus mass and fertilize the egg. We hypothesized that skim
milk-based semen extenders, similar to those used in cooled storage, stabilize sperm membranes and
prolong sperm motility and longevity. However, this could decrease the ef®ciency of sperm to
undergo subsequent capacitation in vivo. This study was designed to evaluate the effects from two
media on sperm function. Spermatozoal motility was analyzed, intracellular calcium was measured,
and the ability of sperm to undergo acrosome reaction was compared after incubation in a skim milk
extender (SME) and Tyrode's medium containing albumin, lactate, and pyruvate (TALP) at 37 8C.
Results suggest that the SME facilitated capacitation as detected by an increase in both intracellular
calcium and acrosome reactions, and a decrease in motility, as compared to TALP. Our data support a
shortened functional lifespan for equine sperm in skim milk extender, which indicates that further
re®nements in cooled semen preservation are required to improve fertility of transported equine
semen. # 2002 Elsevier Science Inc. All rights reserved.
Keywords: Spermatozoa; Stallion; Capacitation; Acrosome; Extender
1. Introduction
It has been demonstrated that sperm must be able to access the female upper genital tract,
enter the uterine tubes, bind to oviductal epithelium, capacitate, and undergo the acrosome
*
Corresponding author. Tel.: ‡1-530-752-9511; fax: ‡1-530-752-7690.
E-mail address: [email protected] (S.A. Meyers).
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved.
PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 0 6 5 9 - 3
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A.C. Pommer et al. / Theriogenology 57 (2002) 1493±1501
reaction in order to fertilize an egg [1]. Capacitation of sperm has been described as a
complex of poorly de®ned cellular events that occur in situ within the female reproductive
tract that are obligatory for the acrosome reaction and fertilization to progress [1±3].
Capacitation involves sperm plasma membrane events that lead to an increased
cellular calcium in¯ux, fusion and vesiculation of the plasma and outer acrosomal
membranes, and loss of the acrosomal protein matrix in a process termed the acrosome
reaction, an exocytotic event [1]. When acrosomal exocytosis occurs, sperm release
hydrolytic enzymes and proteases including hyaluronidase and acrosin, respectively,
which allow the sperm to digest their way through the cumulus extracellular matrix
(ECM) and zona pellucida surrounding the egg. If sperm prematurely capacitate or
acrosome react, they could have a diminished capacity to penetrate the cumulus mass
and fertilize the egg.
Stored semen often has decreased spermatozoal motility and fertility that can vary
signi®cantly between individual stallions [4,5]. Preserving sperm function following
cooled or frozen storage is essential to maintain optimum fertility. Bovine and equine
sperm have been demonstrated to exhibit signs of premature capacitation associated with
cryopreservation [6,7]; however, the mechanism of the action is undetermined. It is
plausible, therefore, that cell longevity is compromised by premature capacitation-related
changes associated with storage of sperm in different media. If so, sperm would then
display a decreased ability to fertilize oocytes at the natural site of fertilization in the
oviductal ampulla following insemination.
This study was designed to compare the ability of sperm to undergo acrosome reactions
after induction of capacitation by incubating the sperm for 3 h at 37 8C in two different
media. This temperature (37 8C) was selected in order to approximate the intra-uterine
environment. The two media utilized were a commercial skim milk-based semen extender
(SME) and Tyrode's medium containing albumin, lactate, and pyruvate (TALP), a
chemically de®ned medium used to induce sperm capacitation in vitro [8,9]. We hypothesized that skim milk-based semen extenders stabilize sperm membranes and prolong sperm
motility and longevity but, as such, render the sperm ineffective at undergoing subsequent
capacitation in vivo. The results of this study will be used to modify semen storage
methods.
2. Materials and methods
2.1. Chemicals and reagents
Fluorescein-conjugated Pisum sativum agglutinin (PSA) was obtained from Vector
Laboratories (Burlingame, CA). Ethidium homodimer-1, Fluo-3 AM, Live-Dead1 Sperm
Viability Kit, and Pluronic F-127 were purchased from Molecular Probes (Eugene, OR). EZ Mixin-``Cool-Store/Transport'' equine semen extender containing amikacin was purchased from Animal Reproduction Systems (Chino, CA). Dulbecco's phosphate-buffered
saline (DPBS) was obtained from Gibco BRL (Grand Island, NY). All other chemicals
were obtained from Sigma Chemical Company (St. Louis, MO).
A.C. Pommer et al. / Theriogenology 57 (2002) 1493±1501
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2.2. Animals and semen
Semen was obtained from ®ve fertile stallions individually housed at the Veterinary
Medical Teaching Hospital and the Animal Science Horse Barn located at the University of
California, Davis. Stallions were maintained on a diet of mixed grass hay and Omolene
200, with fresh water ad libitum and daily exercise according to Institutional Animal Care
and Use Committee protocols at the University of California. Semen was collected using an
arti®cial vagina and a phantom mare. A nylon mesh ®lter was used to eliminate the gel
fraction, and only allow the sperm-rich fraction of the ejaculate to enter into the collection
bottle. Half of the gel-free semen was immediately diluted 1:1 (v/v) into SME (E-Z Mixin)
and the other half was diluted 1:1 (v/v) into TALP capacitation medium [9]. Both media
had been warmed to 37 8C prior to semen collection, and the diluted semen was transported
to the laboratory within 5 min of collection.
2.3. Sperm processing for capacitation and acrosome reactions
Upon arrival at the laboratory, the TALP semen sample was centrifuged at 100 g for
5 min to sediment debris. Sperm were capacitated as previously described [9]. Brie¯y, the
supernatant (3 ml) was layered over two Percoll-TALP gradients, each consisting of an
84% lower layer and a 42% upper layer. After centrifugation at 300 g for 20 min the
sperm pellets were collected and placed in 4 ml TALP. The sperm suspension was
centrifuged for 10 min at 300 g and the resulting sperm pellet was resuspended in
1 ml TALP. The sperm concentration was determined using a hemacytometer and diluted
to 20 106 /ml. The concentration of sperm in the SME was also determined and diluted to
20 106 /ml. One milliliter of each sperm suspension was divided into two siliconized
microcentrifuge tubes. One tube of each served as a control, and acrosome reactions were
induced in the other tube by using 1 mM calcium ionophore A23187. The samples were
incubated for 3 h at 37 8C in a humidi®ed 95% air and 5% CO2 atmosphere incubator. A
10 ml aliquot was taken from each sample and placed on a warmed glass slide, coverslipped, and placed on a stage warmer (Minitube; Verona, WI). Total and progressive
motility were estimated and recorded by a single observer using an Olympus BX-60
microscope with differential interference contrast optics at 0 h, and following the 3 h
incubation period.
After in vitro capacitation (3 h at 37 8C), sperm suspensions from all treatments were
®xed for 10 min in 2% paraformaldehyde and washed by centrifugation and resuspension
with DPBS. The samples were permeablized using 95% ethanol ( 20 8C) for 10 min and
then washed with DPBS. The samples were incubated for 10 min in DPBS containing 5%
bovine serum albumin (BSA, blocking solution) and double labeled with ¯uorescein±PSA
and ethidium homodimer-1 for 10 min in the dark. The cells were washed with, and
resuspended in, 500 ml DPBS. A drop of a ¯uorescence enhancer was added (Vectashield1;
Vector) to preserve cell ¯uorescence. Sperm cell samples were placed on glass microscope
slides with coverslips and ¯uorescence of 200 cells was visualized using oil immersion at
magni®cation 1000 with an Olympus BX-60 ¯uorescence microscope using a ¯uorescein
®lter with excitation at wavelength 480/30 and emission at wavelength 535/40. Viability
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was determined in duplicate cell suspensions in all treatments by observing 200 cells using
the Live-Dead1 Sperm Viability Kit that utilizes Sybr1-14 and propidium iodide
¯uorescence stains for differential labeling. Sperm cells were not simultaneously evaluated
for viability and acrosomal status because the ¯uorescence spectral overlap of the ethidium
homodimer and propidium iodide in the two methods would have interfered with detection
of cell viability in acrosome-reacted cells. Consequently, the viability status of individual
acrosome-reacted or -intact cells could not be determined.
2.4. Calcium measurements
Separate aliquots of sperm, processed as described above, were analyzed for intracellular calcium. Cells in either SME or TALP, at a concentration of 20 106 /ml, were placed
into siliconized microcentrifuge tubes (500 ml per tube, two tubes per treatment) and
incubated at 37 8C in 5% CO2 in air for 1.5 or 3 h. The calcium indicator Fluo-3 AM
(5 mM) containing 0.1% Pluronic F-127 in dimethyl sulfoxide (®nal concentrations) was
loaded into cells for the last 30 min of incubation. After incubation, excess dye was
removed from cells by centrifugation and resuspended in fresh medium to the original
500 ml volume. For calcium measurements, 1 ml (2 500 ml) of cell suspension was
placed in a methacrylate cuvette (Fisher; Pittsburgh, PA) containing a micro stir bar and
read in a Hitachi F-2000 spectro¯uorometer. The ¯uorescence response level was 1 s, with
an excitation of 488 nm and an emission of 530 nm. The bandpass widths for both
excitation and emission were 10 nm. Samples were maintained at 37 8C in a Lauda heating
circulator water bath, which also controlled the temperature of the magnetic stirring turret
in the spectro¯uorometer.
For both the 1.5 and 3 h incubations, Fluo-3 ¯uorescence intensity was determined for a
minimum of 60 s (base line) before treatment with 1 mM (®nal concentration) calcium
ionophore A23187. Changes in intracellular calcium ([Ca2‡]i) were monitored for
10 0:5 min, due to sample variation. Maximum ¯uorescence was obtained by adding
1 mM digitonin, and minimum ¯uorescence was obtained by subsequent addition of either
50 mM EGTA/50 mM Tris or 12.5 mM EGTA/12.5 mM Tris to SME and TALP samples,
respectively. All values are listed at ®nal, working concentrations. The measurements
described above were used to calculate [Ca2‡]i with the equation ‰Ca2‡ Ši ˆ K d …F F min †/
(F max F), where Kd is the dissociation constant of Fluo-3, F the ¯uorescence intensity,
Fmin the minimum Fluo-3 ¯uorescence, and Fmax is the maximum Fluo-3 ¯uorescence
[10]. The Kd used for Fluo-3 was 316 nM [11,12].
To ensure there were no effects from medium on cellular uptake of Fluo-3 AM, cells
were incubated and loaded in SME or TALP as described above and evaluated on the
spectro¯uorometer in either the same media used for loading or the opposite media. Cellfree and dye-free media was also read on the spectro¯uorometer under the same experimental conditions and the optical densities of the two media observed.
2.5. Statistical analysis
Data were analyzed using ANOVA techniques with Minitab1 statistical software
(Minitab Inc., State College, PA). A two-way analysis of variance was used to evaluate
A.C. Pommer et al. / Theriogenology 57 (2002) 1493±1501
1497
media and stallion or treatment and stallion. Changes in [Ca2‡]i were analyzed using oneway ANOVA to compare differences between media.
3. Results
3.1. Acrosome reactions
Baseline acrosomal status (e.g. without ionophore treatment) was not different between
sperm samples incubated for 3 h in TALP or SME media. Induction of acrosome reactions
using calcium ionophore (1 mM) during the 3 h incubation period resulted in a greater
percentage of acrosome reacted cells (P < 0:05) when compared to cells incubated in their
respective medium containing no ionophore (Fig. 1).
The ionophore-treated SME cells had a signi®cantly greater percentage of acrosome
reactions than the ionophore-treated TALP cells (P < 0:05) as detected by ¯uoresceinated
lectin staining. There were no signi®cant differences between stallions in response to
calcium ionophore and no stallion by ionophore treatment interaction was detected.
3.2. Sperm motility
Total and progressive sperm motility was decreased at the end of the 3 h incubation at
37 8C in both SME and TALP medium (P < 0:05). At 3 h, both total and progressive
motility were not different between sperm incubated in the two media. In both SME and
TALP, total (Table 1) and progressive (Table 2) sperm motility after 3 h incubation was
Fig. 1. Acrosome reactions following calcium ionophore (A23187) treatment. Percentage of acrosome reacted
cells as detected using FITC-PSA after 3 h incubation of calcium ionophore-treated (1 mM) and -untreated
(0 mM) sperm. Samples were incubated in either TALP or SME medium at 37 8C in humidi®ed 95% air and 5%
CO2 atmosphere. Values are expressed as mean percents S:E:M:, n ˆ 5. Superscripts (a, b, c) denote
signi®cance (P < 0:05) within and between media treatments.
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Table 1
Percentage of total motility at 0 h, and after 3 h incubation, of calcium ionophore-treated (1 mM) and -untreated
(0 mM) sperm
Medium
Calcium ionophore (mM)
0h
TALP
TALP
SME
SME
0
1
0
1
91
91
82
82
3h
3
3
2
2
68
43
68
2
Decrease (%)
6
9
4
2,c
26
53
17
97
Samples were incubated in either TALP or SME medium at 37 8C in humidi®ed 95% air and 5% CO2
atmosphere. Values are expressed as mean S:E:M:, n ˆ 5. The symbol () in superscript denotes signi®cance
(P < 0:05) between calcium ionophore treatments within medium. The symbol (c) in superscript denotes
signi®cance (P < 0:05) between media.
lower in ionophore-treated cells when compared to control cells, in their respective
medium, without ionophore (P < 0:05).
After the 3 h incubation with ionophore, there was signi®cantly lower total and
progressive motility in the SME cells as compared to the TALP cells (P < 0:05). Although
sperm motility declined to very low levels in ionophore-treated samples, the population
viability percentages were not different between sperm treated with 1 mM ionophore and
control vehicle only (78 3 and 83 1, respectively).
3.3. Intracellular calcium measurements
Cells incubated in SME exhibited signi®cantly higher changes in intracellular calcium
(P < 0:05), when challenged with calcium ionophore A23187, than cells incubated in
TALP at both the 1.5 and 3 h time points (Table 3). Results from experiments to evaluate
Fluo-3 AM cell loading (data not shown) indicated that neither TALP nor SME had an
effect on loading of Fluo-3 AM dye in equine sperm. Calcium measurement and response
of cells to ionophore were consistent with other experiments presented, regardless of the
media in which they were loaded and evaluated. Results from cell-free media (not shown)
con®rmed that the optical density of the SME was two-fold higher than that of TALP.
Results are expressed as percent change in [Ca2‡]i from base line.
Table 2
Percentage of progressive motility at 0 h, and after 3 h incubation, of calcium ionophore-treated (1 mM) and untreated (0 mM) sperm
Medium
Calcium ionophore (mM)
0h
TALP
TALP
SME
SME
0
1
0
1
87
87
77
77
3h
4
4
2
2
62
33
54
0
Decrease (%)
7
12
12
0c
29
62
30
100
Samples were incubated in either TALP or SME medium at 37 8C in humidi®ed 95% air and 5% CO2
atmosphere. Values are expressed as mean S:E:M:, n ˆ 5. The symbol () in superscript denotes signi®cance
(P < 0:05) between calcium ionophore treatments within medium. The symbol (c) in superscript denotes
signi®cance (P < 0:05) between media.
A.C. Pommer et al. / Theriogenology 57 (2002) 1493±1501
1499
Table 3
Percent changes in intracellular calcium, from baseline, after 1.5 or 3 h of incubation of calcium ionophoretreated (1 mM) and -untreated (0 mM) sperm
Media
Time of incubation (h)
Change (%) in [Ca2‡]i from baseline
TALP
TALP
SME
SME
1.5
3
1.5
3
205
232
348
347
24
20
25
33
Samples were incubated in either TALP or SME medium at 37 8C in humidi®ed 95% air and 5% CO2
atmosphere. Values are expressed as the means of ®ve replicates ‡ = S:E:M: The symbol () in superscript
denotes signi®cance (P < 0:05) between the two media.
4. Discussion
Transport of cooled equine semen for use in arti®cial insemination most commonly
involves dilution of the ejaculate in a nonfat dry skim milk-based semen extender
containing an antibiotic supplement which is based on the formula originally published
by Kenney and coworkers [13]. This type of extender contains glucose as an energy source
for the sperm, as well as a variety of milk-based proteins and carbohydrates that assist in
physiological buffering and cellular protection. Although pregnancy rates may be
decreased when mares are inseminated with cooled, stored semen as compared to fresh,
extended semen [14] there are inherent, and often signi®cant, stallion differences in the
fertility of preserved sperm [15]. Reasons for this decreased fertility could include
premature capacitation-like changes which would decrease sperm longevity by activation
of cellular metabolic processes.
In a previous study from our laboratory, we determined that cooled storage (4 8C, 24 h)
and frozen storage ( 196 8C) of stallion sperm rendered acrosomal disruption to treated
sperm [6]. In that study, sperm treated by a short exposure to the calcium ionophore,
A23187, failed to undergo acrosome reaction following 2 h incubation under capacitating
conditions. However, acrosomal damage was apparent whether the cells were treated with
calcium ionophore or not. A higher percentage of incompletely stained acrosomes were
observed in frozen sperm than in fresh sperm. Incompletely stained acrosomes were also
observed more frequently in cooled, extended sperm at a greater concentration than for
fresh extended sperm. Both media used in that study contained skim milk components.
Therefore, we hypothesized that acrosomal function and intracellular calcium ¯ux may be
compromised during cooled storage, although for some stallions, skim milk provides
adequate protection to sperm cells regarding motility and fertility. We designed an
experiment to compare a milk-based diluent with a conventional culture medium.
Although the sperm incubated in TALP medium required washing and Percoll±gradient
centrifugation to remove seminal plasma (treatments not required for SME-treated sperm),
the study was designed to compare a de®ned incubation medium using a laboratory-based
method with a commercial semen extender under ®eld conditions (immediate dilution of
sperm in SME). This experiment was performed at 37 8C in order to evaluate sperm
function under capacitating conditions.
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A.C. Pommer et al. / Theriogenology 57 (2002) 1493±1501
In the present study, total and progressive sperm motility signi®cantly declined over the
3 h incubation period for sperm in both media. Ionophore treatment induced a marked and
signi®cant decrease in motility when compared to control cells. Furthermore, after 3 h
incubation with ionophore, total and progressive motility was signi®cantly lower in SME
incubated cells than TALP incubated cells. Ionophore certainly disrupts sperm membrane
integrity and ¯oods the cell with calcium [16], which would negatively affect sperm
motility and could account for motility differences between ionophore-treated and untreated sperm in the present study.
The calcium ionophore, A23187, induced acrosome reactions at a higher rate than
cells incubated in their respective control media alone. However, when ionophoretreated cells incubated in the two media were compared, there was signi®cantly higher
percentage of acrosome-reacted cells in SME than in TALP. In addition, cells incubated
in SME had signi®cantly higher percent changes in intracellular calcium when challenged
with ionophore than cells incubated in TALP medium. This result was surprising in
that we expected the capacitation medium, TALP, to support more capacitation-related cell
functions such as acrosome reactions and increased intracellular calcium. The greater
percent change in intracellular calcium could be due to the elevated calcium content in
the milk-based extender, nearly six-fold higher than TALP. Clinical chemistry analysis
determined total calcium content in SME at 30.6 mg/dl where as total calcium content
in TALP was 5.7 mg/dl. Consequently, there was more calcium available in SME for
passive or active cellular uptake; thus, a greater magnitude of intracellular calcium
was able to enter the cells at initiation of the acrosome reaction. Since calcium in¯ux
is a prerequisite for the acrosome reaction to occur [17,18], the cells in the SME would
have elevated calcium similar to conditions associated with sperm capacitation.
Cooled semen may not survive optimally in commercial skim milk extenders because
of increased acrosome reactions, lowered motility, and increased intracellular calcium
when compared to cells incubated in a capacitation medium. This suggests that the
sperm are prematurely capacitating in the SME, and upon insemination in the mare may
not fertilize the egg due to compromised function. Capacitation prior to insemination
is detrimental because the sperm may have a diminished ability to reach the ovum,
penetrate the cumulus cells, and bind to the zona pellucida. Our data support a shortened
functional lifespan for equine sperm stored and shipped in skim milk extender, which
indicates that improvements in semen preservation are required to improve fertility
of transported semen for some stallions that display poor sperm function of preserved
semen.
Acknowledgements
The authors gratefully acknowledge the assistance of Dr. Jan Roser, Department of
Animal Science, College of Agriculture and Environmental Sciences, and Drs. Myrthe
Wessel and Dierdra Carver, Ms. Julie Baumber and Dr. Barry A. Ball, Department of
Population Health and Reproduction, School of Veterinary Medicine, for procurement of
semen samples and scienti®c advice. This work was supported by a grant from the USDA
(no. 98-35203-6584).
A.C. Pommer et al. / Theriogenology 57 (2002) 1493±1501
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