From the Center for Research on Reproduction & Women's Health, University of Pennsylvania Medical Center,
Philadelphia, Pennsylvania 19104-6080
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
Sperm capacitation in vitro is highly
correlated with an increase in protein tyrosine phosphorylation that is
regulated by cAMP through a unique mode of signal transduction
cross-talk. The activation of this signaling pathway, as well as
capacitation, requires bovine serum albumin (BSA) in the incubation
medium. BSA is hypothesized to modulate capacitation through its
ability to remove cholesterol from the sperm plasma membrane.
Here we demonstrate that the cholesterol-binding heptasaccharides,
methyl- Immediately after ejaculation, mammalian sperm do not possess the
ability to fertilize the egg. They acquire the ability to fertilize
during transit through the female tract in a poorly understood process
known as capacitation (1-4). Capacitation is defined as the
time-dependent acquisition of fertilization competence, and
is generally assessed as the ability of the acrosome-intact sperm to
undergo an acrosome reaction in response to physiological inducers such
as the ZP1 or progesterone
(5, 6), or by the ability of the sperm to fertilize eggs (7).
Capacitation can be mimicked in vitro by incubating
epididymal or ejaculated sperm in a defined medium containing
appropriate concentrations of electrolytes, metabolic energy sources,
Ca2+, HCO3 Previously, we and others have demonstrated that sperm capacitation in
several species is correlated with an increase in tyrosine phosphorylation of a subset of proteins (15-18). Our laboratory has
determined that both protein tyrosine phosphorylation and capacitation
are regulated by cAMP at the level of protein kinase A (PKA), and
requires the presence of Ca2+,
HCO3 In the present study, we tested the hypothesis that BSA is acting
through the removal of cholesterol from the sperm plasma membrane by
analyzing if Materials--
2-OH-propyl- Culture Media--
The basic medium used for the studies with
mouse sperm was a modified Krebs-Ringer bicarbonate medium (HMB-Hepes
buffered), as described by Lee and Storey (23). This medium was first
prepared in the absence of Ca2+, BSA, and pyruvate,
sterilized by passage through a 0.20-µm filter (Nalgene), and frozen
at
A modified Tyrode's Hepes-buffered medium (SpTALPH) was used for the
experiments with bull sperm, as described by Parrish et al.
(24). This medium was modified in the following manner: BSA was
replaced by 1 mg/ml polyvinyl alcohol (PVA) to reduce the ability of
SpTALPH to capacitate sperm (15) and 0.4 mM EDTA was added.
The medium was first prepared in the absence of Ca2+ and
pyruvate and frozen at Preparation of Mouse Sperm--
Caudal epididymal mouse sperm
were collected from CD1 retired breeder males by placing one minced
cauda epididymis in 0.5 ml of medium HMB without BSA at 30 °C. After
5 min the sperm in suspension were washed in 10 ml of the same medium
by centrifugation at 800 × g for 10 min at room
temperature (24 °C). The sperm were then resuspended to a final
concentration of 5-10 × 106 cells/ml and diluted 10 times in the appropriate medium depending on the experiment performed.
After incubation for 1.5 h, except where indicated, the sperm were
concentrated by centrifugation at 5,000 × g for 1 min
(room temperature), the sperm pellet washed in 1 ml of
phosphate-buffered saline, centrifuged again, and the resultant pellet
resuspended in sample buffer (25) without mercaptoethanol and boiled
for 5 min. After centrifugation at 5,000 × g for 3 min, the supernatant was removed, 2-mercaptoethanol was added to a
final concentration of 5% (v/v), the samples boiled for 5 min, and
then subjected to SDS-PAGE as described below.
Preparation of Bovine Sperm--
Ejaculated bovine sperm
(Bos taurus, Holstein), collected by artificial vagina, was
generously provided by the Hofmann Center at New Bolton Center,
University of Pennsylvania School of Veterinary Medicine (Kennett
Square, PA). The sperm were immediately assessed for motility by light
microscopy and then, at 10-20 min following collection, were diluted
1:5 into SpTALPH. These samples were then subjected to two washes by
centrifugation at 375 × g for 10 min each in SpTALPH
to remove seminal plasma, as described previously (17, 24). Sperm were
kept at 22-24 °C in SpTALPH following the washes for transport to
the laboratory (1-2 h), and then subjected to a final wash in SpTALPH.
The concentration of sperm in the SpTALPH solution was determined by
hemocytometer and adjusted to 5 × 107cells/ml
with SpTALPH before initiating the capacitation experiments.
For capacitation, 100-µl aliquots of the sperm suspension (5 × 106 cells), as well as the appropriate test reagents (1-3
µl), were incubated in capped 1.5-ml polypropylene microcentrifuge
tubes at 39 °C in a Thermolyne 37900 culture as described previously (17, 24). Additions to the sperm suspension were made immediately before initiation of the incubation period. Following an incubation period of 4 h, the sperm were concentrated by centrifugation at 10,000 × g for 3 min at room temperature, washed in 1 ml of phosphate-buffered saline containing 0.2 mM
Na3VO4 at room temperature, and the sperm pellet then resuspended in sample buffer (25) without mercaptoethanol and boiled for 5 min. After centrifuging at 10,000 × g
for 3 min, the supernatant was removed, boiled in the presence of 5%
2-mercaptoethanol for 5 min, and then subjected to SDS-PAGE as
described below. For all experiments, sperm from two to three different
mature Holstein bulls were assayed to control for individual variation.
Steroid Measurements--
Mouse sperm (5 × 106) were incubated in 500 µl of capacitation medium in
either the absence or presence of 3 mg/ml BSA or with the appropriate
SDS-PAGE and Immunoblotting--
SDS-PAGE (25) was performed in
8 or 10% gels. Electrophoretic transfer of proteins to Immobilon P
(Millipore) in all experiments was carried out according to the method
of Towbin et al. (27) at 70 V (constant) for 2 h at
4 °C. Immunodetection was carried out at room temperature as
described previously (28) using a monoclonal antibody against
anti-phosphotyrosine (clone 4G10; UBI) and blots developed using
enhanced chemiluminescence detection with an ECL kit (Amersham Corp.),
according to the manufacturer's instructions.
Coomassie Blue Assay for the Acrosome Reaction--
As one end
point of capacitation, we analyzed the ZP-induced acrosome reaction in
mouse sperm, based on the premise that only capacitated sperm would
undergo exocytosis in response to ZP. The percentage of acrosome
reactions was measured using Coomassie Blue G-250 as described by
Thaler and Cardullo (29). Briefly, sperm were incubated under the
desired experimental conditions for 1.5 h, followed by the
addition of 5 ZP/µl, 5 µM A23187, or buffer for an
additional 30 min. An equal volume of 2 × fixative solution
(7.5% formaldehyde in phosphate-buffered saline) was then added to
each tube. After 10 min, the sperm were centrifuged for 2 min at
10,000 × g and resuspended in 0.1 M
ammonium acetate (pH 9). After centrifugation, the sperm pellet was
resuspended in 20-50 µl of the same buffer, spread on to
poly-L-lysine-coated slides, and air dried. The slides were
then stained with 0.04% (w/v) Coomassie Blue G-250 in 3.5% (v/v)
perchloric acid for 10 min, gently rinsed with deionized
H2O until they appeared blue, air dried, and then mounted
with Permount. To calculate the percentage of acrosome reactions, at
least 200 sperm were counted per experimental condition. The data
presented are the average of at least three different experiments.
In Vitro Fertilization--
In vitro fertilization of
metaphase II-arrested mouse eggs was performed as described previously
(30), with modifications as described in Visconti et al.
(15). PVA was used to maintain the isotonic conditions when sperm were
cultured in media devoid of BSA prior to insemination. When
Release of Cholesterol from the Sperm Plasma Membrane in Media
containing BSA, 2-OH-propyl- Effects of 2-OH-propyl-
Previously, we demonstrated that the increase in protein tyrosine
phosphorylation in capacitation medium containing 3 mg/ml BSA occurred
in a time-dependent manner, increasing significantly at 45 min and reaching maximal levels at 90 min (21). This maximal level of protein tyrosine phosphorylation was correlated with the onset
of the capacitated state in the population of sperm. When 3 mM 2-OH-p- Effects of Cholesterol Sulfate Incubation with Effects of
Although the ZP-induced acrosome reaction is one end point of
capacitation, fertilization still represents the most definitive end
point of this maturational process (5, 7). We examined whether sperm
incubated in media devoid of BSA but containing 2-OH-p- Role of Bicarbonate and Protein Kinase A in Mediating the
Effects of 2-OH-propyl- Although capacitation was initially described approximately 50 years ago, the molecular basis of this extratesticular maturational event is still poorly understood. Nevertheless, recent work by several
laboratories is starting to lead to a unified hypothesis of how this
event is controlled. Capacitation can occur spontaneously in
vitro in defined media without the addition of biological fluids, suggesting that this process can be controlled by the sperm itself. It
is clear that some components of the medium play essential roles in the
regulation of capacitation. Although serum albumin (usually BSA) is
believed to function as a sink for the removal of cholesterol from the
sperm plasma membrane (9-14), it is not known whether cholesterol
removal represents the only function of BSA in the capacitation process.
In attempts to understand the signaling pathways that regulate
capacitation, our laboratory has recently demonstrated that capacitation of mouse, human, and bovine sperm is associated with an
increase in protein tyrosine phosphorylation (15-17), and other laboratories have corroborated these results (18, 33-35). In the
present work, we further studied the mechanisms by which these capacitation-associated changes in protein tyrosine phosphorylation are
regulated, with specific attention being given to the role of
cholesterol efflux in regulating this process. Previously, we
demonstrated that protein tyrosine phosphorylation and capacitation were both dependent on the presence of BSA in the incubation medium (15). Our working hypothesis is that BSA, by acting as a sink for
cholesterol efflux from the sperm plasma membrane, induces an increase
in the permeability of sperm to HCO3 In our previous work, we demonstrated that the capacitation process and
an increase in protein tyrosine phosphorylation were tightly
correlated. The goal of the experiments in this paper was to analyze
how the BSA present in the capacitation medium might regulate the
increase in protein tyrosine phosphorylation and the capacitation
process. Several authors have suggested that BSA is necessary in the
capacitation medium for removal of cholesterol from the plasma membrane
(9-14). Our results demonstrate that substitution of the BSA in the
media with This work is the first to describe the regulation of a transmembrane
signal transduction pathway by cholesterol removal. Normally, the
initiation of signal transduction pathways that result in the
activation of tyrosine kinases involves plasma membrane receptors; such
receptors could be tyrosine kinases or could be associated with
tyrosine kinases. Since sperm present a unique case in which the
increase in protein tyrosine phosphorylation is regulated through a
cAMP pathway, it is possible that the removal of cholesterol would be
necessary for those events leading to the synthesis of cAMP. How
cholesterol removal regulates this pathway is still not known. It is
clear from numerous studies that cholesterol alters the bulk
biophysical properties of biological membranes. For example, this
steroid can increase the orientation order of the membrane lipid
hydrocarbon chains and, as a consequence, can reduce the ability of
membrane proteins to undergo conformational changes that may control
their functions, due to the fact that the membrane is less fluid.
Therefore, high concentrations of cholesterol in the membrane might
inhibit membrane protein function. This "indirect" effect of
cholesterol on membrane protein function might stabilize those membrane
and transmembrane events that are part of the "intrinsic"
regulatory nature of capacitation. Cholesterol has also been
demonstrated to have "direct" effects by binding to and regulating
membrane protein function; such binding may serve to exert a positive
or negative modulatory effect on the protein in question. In fact,
studies of several membrane-associated ion transporters
(e.g. Na+,K+-ATPase and GABA
transporter) by cholesterol supports the idea that both direct
and indirect effects of this steroid on the regulation of enzyme/ion
channel activity could be invoked (36, 37). In the case of capaciation,
it is possible that the removal of cholesterol from the sperm plasma
membrane could increase the permeability of the sperm to certain ions,
such as HCO3 In capacitation in vitro, BSA is acting as an extracellular
acceptor for cholesterol. The efflux of cholesterol to extracellular acceptors in somatic cells is normally mediated by high density lipoproteins or specific subpopulations of high density lipoproteins (38-40). It is generally accepted that cholesterol efflux occurs by an
aqueous diffusion mechanism in which the cholesterol molecules de-adsorb from the plasma membrane into the aqueous phase, diffuse, and
are solubilized by an acceptor particle (39, 41). In this work we
demonstrated that -cyclodextrin and OH-propyl-
-cyclodextrin, promote
the release of cholesterol from the mouse sperm plasma membrane in
media devoid of BSA. Both of these
-cyclodextrins were also
demonstrated to increase protein tyrosine phosphorylation in the
absence of BSA in both mouse and bull sperm, and the patterns of
phosphorylation were similar to those induced by media containing BSA.
The potency of the different
-cyclodextrins to increase protein
tyrosine phosphorylation in sperm was correlated with their cholesterol
binding efficiencies, and preincubation of the
-cyclodextrins with
cholesterol-SO4
to saturate their
cholesterol-binding sites blocked the ability of these compounds to
stimulate protein tyrosine phosphorylation. The
-cyclodextrin effect
on protein tyrosine phosphorylation was both NaHCO3 and
protein kinase A-dependent. The
-cyclodextrins were also
able to capacitate mouse sperm in the absence of BSA, as measured by
the ability of the zona pellucida to induce the acrosome
reaction and by successful fertilization in vitro. In summary,
-cyclodextrins can completely replace BSA in media to support signal transduction leading to capacitation. These data further
support the coupling of cholesterol efflux to the activation of
membrane and transmembrane signaling events leading to the activation
of a unique signaling pathway involving the cross-talk between cAMP and
tyrosine kinase second messenger systems, thus defining a new mode of
cellular signal transduction initiated by cholesterol release.
INTRODUCTION
Top
Abstract
Introduction
References
, and a protein
source (usually bovine serum albumin;BSA) (7). Capacitation has also
been correlated with changes in sperm intracellular ion concentrations,
metabolism, and motility (7, 8). Although these changes have been known
for many years to accompany this maturational event, the molecular
basis underlying these events is poorly understood. Remarkably,
capacitation in vitro can occur in the absence of any
specific external stimulus, suggesting that specific aspects of the
capacitation process can be initiated and controlled intrinsically by
the sperm itself, and that certain minimal environmental requirements
must be met. This intrinsic nature of the capacitation process is of
great interest from a cell regulation standpoint. It is possible that a
controlling factor(s) intrinsic to the sperm plasma membrane may
regulate changes in the properties of the membrane and that a
de-repression of a set of preprogrammed cellular events must ultimately
occur to promote the development of the capacitated state. The
requirement for BSA in regulating capacitation is thought to be due to
its ability to remove cholesterol from the sperm plasma membrane
(9-14). It is proposed that cholesterol efflux then leads to changes
in membrane architecture and fluidity that give rise to the capacitated state. Cholesterol efflux, therefore, may represent an integral part of
this intrinsic regulatory property of sperm to undergo capacitation.
, and BSA in the medium (19). This
mode of signal transduction cross-talk is, to date, unique to sperm.
Although BSA is believed to be required for capacitation as a
consequence of its ability to serve as a cholesterol-binding molecule,
it is still not known if the only action of BSA is through the removal
of cholesterol.
-Cyclodextrins are cyclic heptasaccharides consisting of
(1-4)-glucopyranose units (20). These compounds are water-soluble, are able to effectively solubilize non-polar substances, and because of
these properties, have been used to deliver hydrophobic drugs (20).
These compounds are also able to promote cholesterol efflux from a
variety of somatic cells (21, 22). Yancey et al. (21) demonstrated that the order of potency in accepting cholesterol is
methyl-
-cyclodextrin (M-
-CD) > OH-propyl-
-cyclodextrin
(2-OH-p-
-CD) >
-cyclodextrin. These studies also showed that
there is an initial rapid efflux of cholesterol from the plasma
membrane of mouse L-cells and human fibroblasts in response to
-cyclodextrins (21).
-cyclodextrins are able to replace BSA in the medium to
up-regulate signal transduction pathways leading to protein tyrosine
phosphorylation as well as capacitation. We demonstrate that both
M-
-CD and 2-OH-p-
-CD promote the release of cholesterol from the
mouse sperm plasma membrane in the absence of BSA. Addition of these
-cyclodextrins to the medium also increased protein tyrosine
phosphorylation in the absence of BSA in mouse and bull sperm, and the
patterns of phosphorylation were similar to those patterns seen in
media containing BSA. These effects of
-cyclodextrins were likely
due to their ability to capture cholesterol from the sperm plasma
membrane. The
-cyclodextrin effect on protein tyrosine
phosphorylation was dependent on the presence of NaHCO3 in
the capacitation medium and the effect was cAMP-dependent.
-Cyclodextrins were also able to capacitate mouse sperm in the
absence of BSA, as measured by the ability of the ZP to induce the
acrosome reaction and by successful fertilization in vitro.
This work is the first to demonstrate that a chemically defined
non-protein constituent can substitute in capacitation media for
protein cholesterol acceptors. These data further support the idea that
cholesterol efflux is, in some way, coupled to the activation of
membrane and transmembrane signaling events leading to the activation
of a unique cross-talk between cAMP and tyrosine kinase second
messenger systems, and thus defines a new mode of cellular signal transduction.
EXPERIMENTAL PROCEDURES
-cyclodextrin,
methyl-
-cyclodextrin, and BSA (Fraction V, Sigma) were purchased
from Sigma. Anti-phosphotyrosine antibody (clone 4G10) was from UBI,
Lake Placid, NY. Solvents were from EM Science (chromatographic grade).
Analtech diphasic plates (Unibond RP18/silica gel, 10 × 12 cm, 250 µm thickness) were obtained from Analtech Inc. (Newark,
DE). Whatman HP-K silica gel plates (10 × 10 cm, 250 µm
thickness) were purchased from Whatman Inc. (Clifton, NJ).
20 °C in aliquots for single use. Working "complete" media
were prepared by adding Ca2+ (1.7 mM), pyruvate
(1 mM), and BSA (3 mg/ml), followed by gassing with 5%
CO2 in air to pH 7.3.
20 °C in aliquots for single use. Working
complete SpTALPH medium was prepared by adding Ca2+ (2 mM), pyruvate (1 mM), NaHCO3 (10 mM), PVA (1 mg/ml), and gentamycin (50 mg/ml). The pH of
SpTALPH was adjusted to 7.4 following equilibration for 1 h at
39 °C in room air, and sterilized by passage through a 0.20-µm
filter (Nalgene, Fisher, Pittsburgh, PA).
-cyclodextrins for 1.5 h. After this period, each aliquot was
centrifuged for 10 min at 10,000 × g and cholesterol, desmosterol, and cholesterol-SO4
were
measured in the sperm pellet and in the resultant medium supernatant as
described previously (26). Briefly, sperm pellets were extracted with
20 volumes of chloroform/methanol (1:1, v/v), vortexed for 10 s,
centrifuged at 800 × g for 3 min, and the supernatant evaporated to dryness under N2. The resultant supernatants
following the initial centrifugation were then extracted with 6 volumes of chloroform/methanol (2:1, v/v), vortexed for 10 s, centrifuged at 800 × g for 10 s, and the lower organic phase
aspirated and evaporated to dryness. Both the sperm pellet and medium
supernatant extracts were dissolved in 20 µl of chloroform/methanol
(1:1, v/v), and 4-µl aliquots applied to silver nitrate-impregnated Whatman HP-K silica gel microplates (Whatman Inc., Clifton, NJ) (5 × 5 cm, 250 µm thickness). Aliquots (4 µl) of cholesterol, desmosterol, cholesterol sulfate, and desmosterol (Sigma) at a concentration of 0.1 mg/ml, were applied on separate lanes as reference
standards. The plates were pre-developed in chloroform/methanol (1:1,
v/v) to 1 cm from the lower edge of the plate. This pre-development step was used to minimize eddy diffusion which results in band broadening and lower resolution. Following pre-development, the plates
were thoroughly dried and then developed in chloroform/acetone (95:5,
v/v) in the same dimension. Following development, the plates were
thoroughly dried, dipped in a 10% solution of copper sulfate (in 8%
phosphoric acid), and placed on a CAMAG Plate Heater III at 185 °C
for 5 min. The resulting bands were scanned at 400 nm in the
reflectance mode using a Shimadzu CS-9000 spectrodensitometer (Shimadzu
Scientific Instruments, Columbia, MD). The integrated areas obtained
for the unknowns were interpolated with the standard curves obtained
for cholesterol, desmosterol, and cholesterol sulfate standards, and
the values expressed as nanograms/106 cells.
-cyclodextrins were included in the capacitation media, the medium
containing the capacitated sperm were diluted 100-fold prior to
insemination of the eggs.
-Cyclodextrins were also present in the
insemination media in some of the experiments. All in vitro
fertilizations were performed using metaphase-arrested eggs retrieved
at approximately 14 h post-human chorionic gonadotropin injection.
Phase-contrast optics were used to evaluate fertilization by looking
for the presence of the second polar body and the formation of both the
male and female pronuclei.
RESULTS
-cyclodextrin, or
Methyl-
-cyclodextrin--
Previously, we demonstrated in mouse
sperm that both protein tyrosine phosphorylation and capacitation
required the presence of BSA in the incubation medium (15). The role of
BSA in regulating signal transduction leading to capacitation has been
postulated to involve the removal of cholesterol from the sperm plasma
membrane (9-14). This hypothesis would predict that other cholesterol
binding compounds that also remove cholesterol from the sperm plasma
membrane could substitute for BSA in media to regulate signal
transduction leading to capacitation. To test this model we first
determined if cholesterol,
cholesterol-SO4
, and desmosterol were
removed from the sperm plasma membrane by 2-OH-p-
-CD and M-
-CD
under our experimental conditions. This was done by directly measuring
the cholesterol released into the incubation medium, as well as that
remaining associated with the sperm after incubating the sperm in the
absence or presence of these compounds. Cauda epididymal sperm were
incubated in HMB medium in the absence or presence of 3 mg/ml BSA, 1 and 3 mM 2-OH-p-
-CD, or 1 and 3 mM M-
-CD.
As shown in Table I, a majority of the measurable cholesterol,
cholesterol-SO4
, and desmosterol
remained associated with the sperm following a 1.5-h incubation in
media devoid of BSA. This is in contrast to sperm incubated in media
containing 3 mg/ml BSA for the same period of time, where a significant
release of these sterols into the media was observed. 2-OH-p-
-CD,
which is a very effective cholesterol binding heptasaccharide, mediated
steroid release from sperm in a concentration-dependent
manner in the absence of BSA (Table I). This
-cyclodextrin was more
effective than BSA in mediating steroid release from the sperm, as
evidenced by the significantly higher steroid concentrations observed
in the media following incubation. M-
-CD, likewise, mediated steroid release from sperm in a concentration-dependent manner, but
was more potent than 2-OH-p-
-CD, consistent with the fact that
M-
-CD is the most potent
-cyclodextrin with respect to its
affinity for cholesterol binding (21).
Effect of media containing BSA, 2-OH-propyl--cyclodextrin, or
methyl-
-cyclodextrin on the release of cholesterol, desmosterol and
cholesterol-SC4 from mouse sperm
-CD, or 1 or 3 mM M-
-CD. After 1.5 h, the suspension was
centrifuged and the different steroids in the sperm pellet and in the
media supernatant were measured. Medium alone, either in the presence
or absence of BSA, contains no detectable levels of these steroids (not
shown). In the presence of 3 mg/ml BSA, 1 or 3 mM
2-OH-p-
-CD, and 1 or 3 mM of M-
-CD, there
was an increase in cholesterol and desmosterol released from the sperm
which was recovered in the media. This release was significant
(P < 0.01) when compared to the control (minus BSA or
minus
-cyclodextrin), as assessed using the Student t
test (42). The following abbreviations were used: C, cholesterol; D,
desmosterol; CSO4; cholesterol-SO4; ND, not detectable.
Values are expressed as nanograms of steroid/106 sperm,
n = 6.
-cyclodextrin and Methyl-
-cyclodextrin
on Sperm Protein Tyrosine Phosphorylation in Media Devoid of
BSA--
As mentioned above, BSA is proposed to act through its
ability to serve as an acceptor for cholesterol and by promoting
cholesterol efflux from the sperm plasma membrane. Since the presence
of BSA in the capacitation medium was required for the
capacitation-associated increase in protein tyrosine phosphorylation
(15), it was hypothesized that BSA could work through its ability to
sequester cholesterol. Therefore, we examined whether cholesterol
removal by
-cyclodextrins in medium devoid of BSA was sufficient to
activate the signal transduction pathways that lead to an increase in
protein tyrosine phosphorylation. Cauda epididymal sperm were incubated
for 1.5 h in BSA-free medium in the absence or presence of various
concentrations of 2-OH-p-
-CD and M-
-CD. As shown in Fig.
1, both
-cyclodextrins were able to
induce an increase in protein tyrosine phosphorylation in a
concentration-dependent manner, with IC50
values of 0.5 and 0.1 mM for 2-OH-p-
-CD and M-
-CD,
respectively (Fig. 1). The differences in the effective concentrations
of these compounds in supporting protein tyrosine phosphorylation is in
agreement with their reported efficiencies as cholesterol-binding
molecules (21). It should be noted that at concentrations of M-
-CD
greater than 1 mM sperm viability started to decline, as
assessed by a decrease in sperm motility. This decrease in viability
likely accounts for the reduction in protein tyrosine phosphorylation seen at these higher concentrations of M-
-CD (Fig. 1). 2-OH-p-
-CD also had a deleterious effect on sperm, but this was observed at
concentrations higher than 20 mM (data not shown).
View larger version (77K):
[in a new window]
Fig. 1.
Concentration-dependent effects
of 2-OH-propyl- -cyclodextrin and
methyl-
-cyclodextrin on protein tyrosine
phosphorylation in mouse sperm. Cauda epididymal sperm were
incubated in media HMB devoid of BSA and in the absence or presence of
the indicated concentrations of the
-cyclodextrins for a period of
1.5 h. Sperm extracts were then prepared, proteins subjected to
SDS-PAGE, transferred to Immobilon P, and probed with an
anti-phosphotyrosine antibody as described under "Experimental
Procedures." A representative experiment is shown. Similar results
were obtained in at least two other experiments.
-CD was substituted for BSA in the incubation medium, we observed an increase in protein tyrosine phosphorylation following 5 min of incubation, and this phosphorylation reached a
maximal level at 45 min (Fig. 2).
View larger version (76K):
[in a new window]
Fig. 2.
Effects of
2-OH-propyl- -cyclodextrin on the time course
of protein tyrosine phosphorylation in mouse sperm. Sperm were
incubated in media HMB devoid of BSA and in the presence of the
indicated concentrations of 2-OH-propyl-
-cyclodextrin for the
periods of time indicated. Protein tyrosine phosphorylation was
assessed as described in the legend to Fig. 1. A representative
experiment is shown. Similar results were obtained in at least two
other experiments.
-Cyclodextrins on
Their Ability to Stimulate Protein Tyrosine Phosphorylation--
As
stated earlier,
-cyclodextrins such as M-
-CD and 2-OH-p-
-CD
both possess the ability to serve as cholesterol acceptors. If the
-cyclodextrin effects observed on protein tyrosine phosphorylation were coupled to their ability to sequester cholesterol, it would be
predicted that these compounds would lose their effects on protein
tyrosine phosphorylation if their steroid binding capacity was
abrogated by preincubation with
cholesterol-SO4
. As shown in Fig.
3, preincubation of either M-
-CD or
2-OH-p-
-CD with increasing concentrations of
cholesterol-SO4
inhibited the ability
of the
-cyclodextrins to support sperm protein tyrosine
phosphorylation. A similar inhibitory effect on protein tyrosine
phosphorylation was observed following preincubation of 2-OH-p-
-CD
with cholesterol (Fig. 3). Taken together, these data suggest that the
-cyclodextrins are inducing an increase in sperm protein tyrosine
phosphorylation by their ability to function as cholesterol
acceptors.
View larger version (74K):
[in a new window]
Fig. 3.
Effects of preloading
2-OH-propyl- -cyclodextrin and methyl-
-cyclodextrin with various
concentrations of cholesterol-SO4
or saturating
2-OH-propyl-
-cyclodextrin with cholesterol on the
capacitation-associated increase in protein tyrosine
phosphorylation. Cauda epididymal mouse sperm were collected as
described under "Experimental Procedures." They were then incubated
in HMB medium in the absence or presence of 0.5 mM M-
-CD
or 1 mM 2-OH-p-
-CD and in the absence or presence of
increasing concentrations of
cholesterol-SO4
or saturating
cholesterol as shown in the figure. After 1.5 h of incubation, the
pattern of protein tyrosine phosphorylation was analyzed by PAGE and
immunoblotting using anti-phosphotyrosine antibodies. This experiment
was performed at least three times with similar results. A
representative experiment is shown.
-Cyclodextrins on Sperm Capacitation in Media Devoid
of BSA--
We have previously used three independent methods to
assess the capacitation status of mouse sperm, and have demonstrated that protein tyrosine phosphorylation is highly correlated with the
onset of the capacitated state. These methods include: 1) the onset of
the B pattern of chlortetracycline fluorescence (31); 2) the ability of
the sperm to undergo a ZP-induced acrosome reaction; and 3) the ability
to fertilize eggs in vitro. We have utilized the latter two
methods to determine whether medium devoid of BSA, but supplemented
with 2-OH-p-
-CD, supports capacitation of mouse sperm. We did not
use M-
-CD in these experiments because of the deleterious effect of
this compound on sperm viability. Fig. 4 demonstrates that sperm incubated in media devoid of either BSA or
2-OH-p-
-CD did not undergo capacitation, as assessed by their inability to undergo a ZP-induced acrosome reaction. In contrast, significant percentages of these sperm underwent an acrosome reaction when incubated with the cation ionophore A23187, consistent with previous observations that this agent can induce acrosomal exocytosis in a non-regulated, capacitation-independent fashion (5, 7). Following
incubation in media containing 3 mg/ml BSA the sperm became
capacitated, as evidenced by their ability to undergo a ZP-induced
acrosome reaction (Fig. 4). Incubation of sperm in BSA-free medium
containing various concentrations of 2-OH-p-
-CD, likewise,
stimulated capacitation. It is interesting to note that incubation of
sperm in media containing 2-OH-p-
-CD resulted in both an increase in
percentages of both spontaneous and A23187-induced acrosome reactions
(Fig. 4). This increase in the percentage of spontaneous acrosome
reactions increased dramatically at concentrations of 2-OH-p-
-CD
above 1 mM (Fig. 5),
suggesting that higher concentrations of this
-cyclodextrin may
render these sperm extremely labile so that they undergo exocytosis
spontaneously; this would also be consistent with the toxic effects of
these compounds at higher concentrations. Likewise, M-
-CD displayed
a concentration-dependent ability to increase the
percentages of spontaneous acrosome reactions (data not shown).
These effects were more potent than those observed with 2-OH-p-
-CD,
consistent with the difference in potency of these two
-cyclodextrins to bind cholesterol.
View larger version (27K):
[in a new window]
Fig. 4.
Effects of bovine serum albumin and
2-OH-propyl- -cyclodextrin on the percentage of spontaneous,
zona pellucida- and A23187-induced acrosome reactions of
mouse sperm. Sperm were incubated in medium HMB devoid of BSA,
medium containing 3 mg/ml BSA, or medium containing either 0.5 or 1 mM 2-OH-propyl-
-cyclodextrin for 1.5 h prior to
assessing acrosomal exocytosis following the addition of ZP or A23187
using the Coomassie G-250 method (see "Experimental Procedures").
Open bars, spontaneous acrosome reactions. Hatched
bars, acrosome reactions induced by 5 ZP/µl. Filled
bars, acrosome reactions induced by 5 µM A23187.
Results presented represent the mean ± S.E. and represent results
from three independent experiments (n = 3).
Asterisk (*) indicates significant difference
(p < 0.01) between the ZP-induced acrosome reaction
and the corresponding spontaneous acrosome reaction as determined using
the t test of the arcsin transformation as described by Zar
(42).
View larger version (17K):
[in a new window]
Fig. 5.
Concentration-dependent effects
2-OH-propyl- -cyclodextrin on the percentage spontaneous acrosome
reactions of mouse sperm. Sperm were incubated in medium HMB
containing the concentrations of 2-OH-propyl-
-cyclodextrin indicated
for a period of 1.5 h. Control experiments were performed using
sperm incubated in medium HMB containing 3 mg/ml BSA. Acrosome
reactions were assessed using the Coomassie G-250 method (see
"Experimental Procedures"). Results presented represent the
mean ± S.E. and represent results from three independent
experiments.
-CD supported
capacitation with respect to their ability to fertilize eggs in
vitro. The success of in vitro fertilization was
monitored by the formation of both the male and female pronuclei. As
previously demonstrated, sperm incubated in the absence of BSA were
unable to fertilize eggs (Fig.
6A). When PVA was substituted for BSA in the capacitation and fertilization media to inhibit the
formation of sperm aggregates and prevent sticking of the eggs to
various surfaces, PVA-containing media was not demonstrated to increase
sperm protein tyrosine phosphorylation (data not shown) or support
in vitro fertilization (Fig. 6A). In contrast,
sperm incubated in media containing 0.5 and 1 mM
2-OH-p-
-CD were able to fertilize eggs (Fig. 6A),
demonstrating that this
-cyclodextrin can support capacitation of
the sperm in the absence of BSA. In contrast, in vitro
fertilization performed with sperm incubated in media containing higher
concentrations of 2-OH-p-
-CD (3 mM) displayed reduced
fertilization rates (Fig. 6B), likely due to the increased
incidence of spontaneous acrosome reactions (see Fig. 5); acrosome
reacted sperm will not bind to the ZP and, therefore, are unable to
fertilize ZP-intact eggs.
View larger version (17K):
[in a new window]
Fig. 6.
Effects of 2-OH-propyl- -cyclodextrin
incubation with mouse sperm on the ability of sperm to fertilize eggs
in vitro. All methods are as described under
"Experimental Procedures." Sperm were incubated for 1.5 h in
medium containing 3 mg/ml BSA, 0.1% polyvinyl alcohol or various
concentrations of 2-OH-propyl-
-cyclodextrin to promote capacitation
as indicated in the figure. The sperm were then diluted with media and
incubated with superovulated zona pellucida-intact mouse
eggs. Pronuclei were then scored approximately 18 h following
insemination. A represents experiments performed with 0, 0.5, and 1 mM 2-OH-propyl-
-cyclodextrin. B
represents experiments performed with 0 and 3 mM
2-OH-propyl-
-cyclodextrin. Data represent the results of three
independent experiments where a minimum of 30 eggs were analyzed per
experiment.
-Cyclodextrin Effect on Mouse Sperm Protein Tyrosine
Phosphorylation--
Work from our laboratory (17, 19, 32) and others
(18) has demonstrated that activation of PKA is an important component of the capacitation process. We have also demonstrated that the elevation of cAMP and activation of PKA up-regulates protein tyrosine phosphorylation during capacitation (17, 19). It is hypothesized that
the BSA-mediated removal of cholesterol from the sperm plasma membrane
alters membrane dynamics such that changes in
HCO3
and Ca2+ conductance
occur, leading to an activation of the sperm adenylyl cyclase, an
elevation of cAMP, activation of PKA, and an increase in protein
tyrosine phosphorylation (19, 32). Since
-cyclodextrins can support
protein tyrosine phosphorylation and capacitation, we examined whether
the effects of these compounds on protein tyrosine phosphorylation were
regulated in some manner by HCO3
and
PKA. In contrast to sperm incubated in
HCO3
-containing medium plus 3 mM 2-OH-p-
-CD, incubation of sperm in
HCO3
-free medium containing 3 mM 2-OH-p-
-CD did not display protein tyrosine
phosphorylation (Fig. 7). Moreover H-89,
an inhibitor of PKA, inhibited the protein tyrosine phosphorylation
induced by 3 mM 2-OH-p-
-CD in medium containing
HCO3
in a
concentration-dependent manner with an IC50 of
~1 µM (Fig. 7), consistent with the concentration
needed to block cellular activation of the PKA pathway leading to
protein tyrosine phosphorylation in sperm (19). Taken together, these
data suggest that the effects of 2-OH-p-
-CD on protein tyrosine
phosphorylation require HCO3
and are
mediated through PKA, similar to that seen with BSA (19).
View larger version (69K):
[in a new window]
Fig. 7.
Effects of extracellular NaHCO3
and H-89 on the 2-OH-propyl- -cyclodextrin-induced increase in
protein tyrosine phosphorylation in mouse sperm. Left
panel, sperm were incubated for 1.5 h in medium HM
(
NaHCO3) or medium HMB (+NaHCO3) containing 3 mM 2-OH-propyl-
-cyclodextrin prior to assessment of
protein tyrosine phosphorylation. Right panel, sperm
were incubated for 1.5 h in medium HMB containing 3 mM
2-OH-propyl-
-cyclodextrin in the absence or presence of the
indicated concentrations of H-89 prior to assessment of protein
tyrosine phosphorylation. A representative experiment is shown. Similar
results were obtained in at least two other experiments.
-cyclodextrin and Methyl-
-cyclodextrin
on Protein Tyrosine Phosphorylation in Bovine Sperm--
A
cAMP-dependent increase in protein tyrosine phosphorylation
has been demonstrated to accompany sperm capacitation in other species,
including the human (18) and bull (17). We also examined whether
cyclodextrins could modulate protein tyrosine phosphorylation in bull
sperm. Incubation of ejaculated bull sperm in media devoid of heparin,
which would not support protein tyrosine phosphorylations or promote
capacitation (17), and in the presence of various concentrations of
2-OH-p-
-CD (Fig. 8A) or
M-
-CD (Fig. 8E) had no effects or moderate effects at
higher concentrations, respectively, on protein tyrosine
phosphorylation. In contrast, when similar experiments were performed
in media containing heparin (Fig. 8, B and F),
both
-cyclodextrins displayed concentration-dependent increases in protein tyrosine phosphorylation, with M-
-CD being the
greatly more effective compound on a molar basis. This difference in
potency of the two
-cyclodextrins is consistent with their relative
potencies in binding cholesterol. Likewise, the addition of
Bt2cAMP plus IBMX to media devoid of heparin (Fig. 8,
C and G) also supported
-cyclodextrin-induced
protein tyrosine phosphorylation. It is interesting to note that the
concentration response curves with both
-cyclodextrins were shifted
to the left when compared with experiments performed in
heparin-containing media alone (compare Fig. 8, C with
B, and G with F). Finally, the
addition of both
-cyclodextrins to bovine sperm incubated in media
containing both heparin and Bt2cAMP plus IBMX (Fig. 8,
D and H) resulted in an even more potent
-cyclodextrin effect on protein tyrosine phosphorylation than in
media containing heparin alone or in media containing
Bt2cAMP plus IBMX. These data suggest that
-cyclodextrins can replace BSA to increase protein tyrosine
phosphorylation in bull sperm. These experiments suggest that, similar
to the mouse, the presence of proteins or non-protein compounds that
possess the ability to bind bovine sperm membrane cholesterol are
essential to initiate signal transduction leading to an increase in
protein tyrosine phosphorylation.
View larger version (94K):
[in a new window]
Fig. 8.
Concentration-dependent effects of
2-OH-propyl- -cyclodextrin and methyl-
-cyclodextrin on protein
tyrosine phosphorylation in ejaculated bull sperm incubated in media
supplemented with heparin, dibutyryl cAMP/IBMX, and heparin plus
dibutyryl cAMP/IBMX. Ejaculated sperm were obtained, washed, and
incubated as described under "Experimental Procedures." Sperm were
then incubated for 4 h in either the absence or presence of
various concentrations of 2-OH-propyl-
-cyclodextrin
(A-D) or methyl-
-cyclodextrin (E-H) in
media containing no additions (A and E), 10 µg/ml heparin (B and F), 1 mM
Bt2cAMP/100 µM IBMX (C and
G), or 10 µg/ml heparin plus 1 mM
Bt2cAMP/100 µM IBMX (D and
H). Representative experiments are shown. The experiments
with 2-OH-propyl-
-cyclodextrin were performed 3 times using the
ejaculates from 3 different bulls. The experiments with
methyl-
-cyclodextrin were performed 2 times using the ejaculates
from 2 different bulls.
DISCUSSION
and Ca2+ ions. These ions are able to stimulate the
activity of the sperm adenylyl cyclase, and in this way increase cAMP
concentrations and PKA activity leading to an up-regulation of protein
tyrosine phosphorylation. We are currently examining several aspects of this hypothesis. For example, the inter-relationship between BSA and
cholesterol movement appears to be important in the regulation of
protein tyrosine phosphorylation and capacitation, since preloading BSA
with cholesterol sulfate to block the ability of BSA to sequester sperm
plasma membrane cholesterol inhibits protein tyrosine phosphorylation and capacitation.2
-cyclodextrins, heptasaccharides with high affinities for
cholesterol, dramatically reduced the sperm membrane-associated
cholesterol, induced protein tyrosine phosphorylation, and resulted in
sperm capacitation.
and/or Ca2+,
which are capable of stimulating the sperm adenylyl cyclase. This
remains to be tested experimentally.
-cyclodextrins induce the increase in protein
tyrosine phosphorylation through a cAMP/PKA pathway and support
capacitation. Moreover, the effects of different
-cyclodextrins to
initiate signaling and bring about functional capacitation are directly
related to their affinities for capturing cholesterol. Aside from the
fact that these data highlight a new and important role for cholesterol
in initiating transmembrane signal transduction, these data also
suggest that completely defined media devoid of protein could be
utilized in the various assisted reproductive technologies to obtain
successful fertilization in vitro. It is also interesting to
note that these
-cyclodextrins (specifically M-
-CD) are very
effective in inducing spontaneous acrosome reactions, as well as
reducing sperm motility, thus defining a possible use for these
compounds as contraceptive agents.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. David Purdon (National Institutes of Health) for insightful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants HD 06274 and HD 22732 (to G. S. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by the Rockefeller Foundation and National Institutes of Health Grant HD06274.
¶ Supported by United States Department of Agriculture Grant 9502560.
Supported by National Institutes of Health Grant HD06274.
Current address: Wyeth Ayerst Laboratories, Princeton, NJ.
** Supported by National Institutes of Health Grant HD22732. Current address: Dept. of Pathology and Laboratory Medicine, Allegheny University Health System, Philadelphia, PA.
Supported by a Fogarty International Training Grant.
§§ Supported by the Bournat Foundation.
¶¶ Supported by National Institutes of Health Grant IH HD-36146 and a grant from the Cystic Fibrosis Foundation. Current address: Dept. of Obstetrics and Gynecology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215.
|| To whom correspondence should be addressed: Center for Research on Reproduction and Women's Health, Rm. 313, John Morgan Bldg., University of Pennsylvania Medical Center, Philadelphia, PA 19104-6080. Tel.: 215-662-6071; Fax: 215-349-5118 or 215-573-4337; E-mail: kopf{at}mail.med.upenn.edu.
The abbreviations used are:
ZP, zona pellucida; BSA, bovine serum albumin; PKA, protein kinase A; M--CD, methyl-
-cyclodextrin; 2-OH-p-
-CD, OH-propyl-
-cyclodextrin; PVA, polyvinyl alcohol; PAGE, polyacrylamide gel electrophoresis; IBMX, isobutylmethylxanthine; Bt2cAMP, dibutyryl cAMP.
2 P. E. Visconti and G. S. Kopf, unpublished data.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|