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From the Department of Biological Sciences, Kent State University, Kent, Ohio.
| Correspondence to: Dr Srinivasan Vijayaraghavan, Department of Biological Sciences, Kent State University, Kent, OH 44242 (e-mail: svijayar{at}kent.edu). |
| Received for publication July 16, 2003; accepted for publication February 4, 2004. |
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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(GSK-3), is
present in spermatozoa. In somatic cells, GSK-3 is regulated by serine and
tyrosine phosphorylation. In this report, we document that both GSK-3
and GSK-ß isoforms are present in spermatozoa, with GSK-3 being
the predominant isoform. The relationship between GSK-3 serine phosphorylation
and motility was investigated. Serine phosphorylation of GSK-3 increases
significantly in spermatozoa during their passage through the epididymis.
Initiation and stimulation of motility in vitro by isobutyl-methyl-xanthine,
2-chloro-2'-deoxy-adenosine, and calyculin A lead to a dramatic increase
in GSK-3 serine phosphorylation. The concentration-dependent induction of
motility by calyculin A is closely associated with GSK-3 serine
phosphorylation. Immunoprecipitation of GSK-3 and GSK-3ß shows
that both of the GSK-3 isoforms are more active in caput than in caudal
spermatozoa. Calyculin A treatment decreased the activity of both isoforms.
Column chromatography was used to purify inactive GSK-3 from the caudal
sperm extracts. This GSK-3 species was phosphorylated at amino acid
residues serine 21 and tyrosine 214. Inactive GSK-3 is present in
caudal but not in caput epididymal spermatozoa. The enzymes protein kinase B
(PKB; also known as cAkt) and phosphoinositide 3-kinase (PI3-kinase), the
upstream signaling proteins involved in GSK-3 phosphorylation, are both
present in spermatozoa. Fluorescence immunocytochemistry showed that GSK-3 is
present in the head and tail regions of sperm. Our work suggests a novel role
for the signaling system involving GSK-3 in the regulation of sperm
motility.
Key words: Epididymis, protein kinase B, phosphoinositide 3-kinase
(GSK-3) (Vijayaraghavan et al,
2000).
GSK-3 is a signaling enzyme involved in the biochemical pathways mediating
insulin and growth hormone action (Hughes
et al, 1993). GSK-3 has also been implicated in cell survival and
apoptosis (Wang et al, 1994b;
Welsh et al, 1994). Two
isoforms of GSK-3, and ß, encoded by 2 independent genes, are
present in mammalian cells (Woodgett,
1990). GSK-3 is regulated through phosphorylation at its tyrosine
214 amino acid residue and also at its serine 21 (serine 9 in GSK-3ß)
amino acid residue (Hughes et al,
1993). In somatic cells, phosphoinositide 3-kinase (PI3-kinase)
and cAkt are the upstream regulators of GSK-3
(Hemmings, 1997). In somatic
cells, constitutively active GSK-3 is tyrosine phosphorylated. Most of the
studies of somatic cells that are focused on GSK-3ß show that the enzyme
becomes serine phosphorylated and partially inactivated in response to
external signals (Woodgett,
2001).
In this study, we show that both isoforms of GSK-3 are present in
spermatozoa, with GSK-3 being the predominant isoform. Our results also
show that sperm GSK-3 and GSK-3ß are serine phosphorylated in
direct proportion to motility. The serine phosphorylation of GSK-3, measured
by immunoreactivity to an antibody specific for phosphorylated GSK-3, is low
in immotile compared to motile bovine epididymal spermatozoa. This
relationship between sperm motility and GSK-3 serine phosphorylation could
also be demonstrated in vitro using motility inhibitors and stimulators.
Immotile caput spermatozoa, when exposed to a motility stimulator such as
calyculin A, become active, with a concomitant increase in the serine
phosphorylation of GSK-3. Increased serine phosphorylation of GSK-3 resulted
in lower catalytic activity of the enzyme. We also used column chromatography
techniques to confirm our observation that the higher-molecular-weight species
of GSK-3 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) is the tyrosine- and serine-phosphorylated GSK-3 isoform.
This form of GSK-3, phosphorylated at 2 residues, is present in motile caudal
but not immotile caput epididymal spermatozoa. The upstream signaling enzymes,
cAkt and PI3-kinase, involved in the regulation of GSK-3 serine
phosphorylation are present in spermatozoa. This report suggests a novel role
in sperm motility for a signaling system known to be involved in the
regulation of growth and survival of somatic cells.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Sperm Extracts
After incubation, the sperm suspensions, with treatments such as
isobutyl-methyl-xanthine (IBMX; Sigma Chemical Co, St Louis, Mo),
2-chloro-2'-deoxy-adenosine (CdA) (Sigma), calyculin A (Upstate
Biotechnologies, Lake Placid, NY), and sHT31 (Promega, Madison, Wis), were
pelleted by centrifugation at 600 x g for 5 minutes at 4°C.
Sperm pellets were suspended in homogenization buffer (10 mmol of Tris per
liter [pH 7.2] containing 1 mmol of EDTA per liter and 1 mmol of EGTA
[ethyleneglycoltetraacetic acid] per liter) supplemented with proteolytic
inhibitors (10 mmol of benzamidine per liter, 0.1 mmol of
N-tosyl-L-lysyl chloromethyl ketone [TPCK] per liter, 0.1%
ß-mercaptoethanol, and 1 mmol of phenylmethylsulfonyl fluoride [PMSF] per
liter), phosphatase inhibitors (1 mmol of sodium vanadate per liter and 1 nmol
of calyculin A per liter), and 1% Triton X-100
(Vijayaraghavan et al, 1996). The sperm suspensions were then centrifuged at 16 000 x g for
15 minutes at 4°C. The supernatants thus obtained were stored at -20°C
until further use for biochemical studies.
SDS-PAGE and Western Blot Analysis
Sperm extracts (50 µg) were separated through 12% polyacrylamide slab
gels (Laemli, 1970). After gel
electrophoresis, proteins were electrophoretically transferred to a
polyvinylidine fluoride membrane (Immobilon-P; Millipore Corp, Bedford, Mass).
Nonspecific protein binding sites on the membrane were blocked with 5% nonfat
dry milk in Tris-buffered saline (TBS; 25 mmol of Tris-HCl per liter, pH 7.4,
and 150 mmol of NaCl per liter). The blots were washed twice for 15 minutes
with TBS containing 0.1% Tween 20 (TTBS) and then incubated overnight with
anti-phosphoserine GSK-3 antibody (Upstate Biotechnologies, Waltham, Mass, or
Cell Signaling Technology, Beverly, Mass) or anti-phosphotyrosine GSK-3
antibody (Biosource, Camarillo, Calif) at a 1:1000 dilution in 5% milk in
TTBS. After washing, the blots were incubated with rabbit anti-sheep (for
anti-phosphoserine GSK-3 antibody; Upstate Biotechnologies) or with
anti-rabbit (for phosphotyrosine) secondary antibody. The blots were then
washed twice (15 minutes each) and 4 times (5 minutes each) before development
with an enhanced chemiluminescence system (Amersham Pharmacia, Piscataway, NJ)
according to the manufacturer's instructions. Parallel blots were developed
with anti–GSK-3 primary antibody (Zymed, San Francisco, Calif) to
ensure equal protein loading. Similar protocols were used for the detection of
PI3-kinase (Upstate Biotechnologies) and cAkt (New England Biolabs, Beverly,
Mass) on Western blots.
GSK-3 Kinase Assay
GSK-3 activity was measured by counting the amount of 32P
transferred from [32P]-
-adenosine triphosphate
([32P]--ATP) to phospho-cyclic adenosine monophosphate
(cAMP)-responsive element binding protein (CREB; Lerner Research Institute,
Cleveland, Ohio). Briefly, sperm extracts were added to phospho-CREB (1 mg/mL)
along with the ATP cocktail mix (200 mmol of HEPES per liter, pH 7.5, 50 mmol
of MgCl2 per liter, 8 mmol of dithiothreitol per liter, 1 mmol of
ß-glycerol phosphate per liter, 400 mmol of ATP per liter, and 0.4 µCi
of [-32] ATP per liter [10 mCi/mL; 3000 Ci/mmol]). The mixture was
incubated at 30°C for 5 minutes. GSK-3 activity was also measured in the
presence of 67 mmol of LiCl per liter. At the end of the incubation, a
12-µL aliquot of the reaction mixture was applied to a phosphocellulose
cation exchanger (P81; Whatman Inc, Clifton, NJ) paper cut into 1.5-x
1.5-cm squares and washed with 0.5% (vol/vol) phosphoric acid. After 3 washes
(10 minutes each) in phosphoric acid, the squares were placed into
scintillation vials with 2 mL of distilled water and counted in a
scintillation counter. The LiCl-sensitive protein kinase activity was assumed
to have originated from GSK-3 (Ryves et
al, 1998). Assays were conducted in duplicate, and means of 2 or
more separate experiments are shown.
Purification of GSK-3
Caudal sperm extracts (50 mL prepared from 5 x 1010
spermatozoa in homogenizing buffer) were passed through a diethylaminoethyl
(DEAE)-cellulose (0.5 x 13 cm) column preequilibrated with homogenizing
buffer with additional 0.05 M KCl (buffer A). The column was washed with 20 mL
of buffer A, and the 70-mL flow through containing all of the GSK-3 in the
original extract was concentrated and applied to a Mono S column (1 mL,
prepacked, high-resolution FPLC; Amersham Pharmacia). The column was washed
with 5 mL of buffer A, which was followed by a linear gradient of
0.05–0.65 M KCl in homogenizing buffer (buffer B). Immunoreactive
fractions containing GSK-3 were pooled and concentrated to 0.5 mL. The same
protocol was used for the purification of GSK-3 from caput spermatozoa.
Immunoprecipitation
Immunoprecipitation of GSK-3 from sperm extracts was performed
following the protocol of Fang et al
(2000) with modifications.
Sperm extracts (from 1 x 108 sperm in 100 µL) were diluted
1:1 in immunoprecipitation buffer A (50 mmol of Tris per liter, pH 7.2, 1 mmol
of EGTA per liter, 1 mmol of EDTA per liter, 150 mmol of NaCl per liter, 10%
glycerol [vol/vol], 1% Triton X-100 [vol/vol], 10 mmol of benzamidine per
liter, 0.1 mmol of TPCK per liter, 0.1% ß-mercaptoethanol [vol/vol], 1
mmol of PMSF per liter, 1 mmol of sodium vanadate per liter, and 1 nmol of
calyculin A per liter) and incubated for 2 hours at 4°C by rocking with 5
µg of rabbit GSK-3 (Zymed). Protein G-sepharose beads (40 µL)
were washed 2 times with 500 µL of distilled water and once with buffer A.
Washed beads were then incubated for approximately 1 hour with 100 µL of
buffer B (buffer A with 10% bovine serum albumin [BSA]). The beads, complexed
with the GSK-3 antibody, were pelleted and washed 3 times with 250
µL of buffer A and then mixed with the sperm extracts, which had been
pretreated with DEAE-cellulose. After further incubation for 30 minutes, this
mixture was centrifuged to collect the supernatant. The pellet was washed 2
times in buffer A and resuspended in a final volume of 100 µL of buffer A.
The crude extracts, the supernatants, and the immunoprecipitated pellets were
then used for the GSK-3 assay.
Immunocytochemistry
Spermatozoa were isolated as described above, washed twice, and resuspended
in phosphate-buffered saline (PBS). Approximately 1 x 108
sperm were added to 5 mL of 4% formaldehyde in PBS (pH 7.0) and left on ice
for 30 minutes, which was followed by a 30-minute incubation with 5 mL of a
PBS/4% formaldehyde mixture containing 0.2% Triton X-100. Fifty microliters of
the mixture was then layered onto a polylysine-coated coverslip and allowed to
air dry. The coverslips were washed in TTBS 3 times for 10 minutes each. To
block nonspecific binding, 200 µL of BSA/TTBS solution was layered onto the
coverslips and further incubated in a humidified chamber at room temperature
for 3 hours. The blocking solution was poured off, and the primary antibody,
anti–GSK-3 (Zymed), diluted 1:500 in the BSA/TTBS/goat serum
solution, was added to the coverslips. The coverslips were then left to
incubate overnight in a humidified chamber at 4°C. After three 10-minute
washes in TTBS, goat anti-rabbit conjugated to indocarbocyanine (CY3)
secondary antibody (Jackson Laboratories, West Grove, Pa) was layered onto the
coverslips at a 1:500 dilution in BSA/TTBS/goat serum solution. The secondary
antibody was incubated in a humidified chamber at room temperature for 2 hours
and covered with aluminum foil to shield it from light. After another 5 washes
in TTBS for 10 minutes each (shielded from light), the coverslips were air
dried, mounted to slides using mounting solution, and pressed between tissue
paper to remove excess fluid. The edges were sealed using clear nail polish to
prevent the coverslip from drying. Finally, the coverslips were viewed using a
fluorescence microscope.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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(Vijayaraghavan et al, 2000). However, it is not known whether the GSK-3ß isoform is also present in
spermatozoa and what the relative levels of and ß GSK-3 isoforms
are. Western blot shown in Figure
1 (lane 1) was developed with an antibody raised against the
catalytic domain (KQLLHGEPNVSYICSRYY) of Drosophila GSK-3. The catalytic
domain is virtually identical in GSK-3 from diverse organisms and in the
GSK-3 and GSK-3ß isoforms of the enzyme. The antibody reacts with
both GSK-3 and GSK-3ß. The antibody reacts with 3 protein bands in
Western blot analysis of caudal sperm extracts
(Figure 1, lane 1). The most
prominent band at 51 kd corresponds to GSK-3. The band of lower
intensity is a doublet. The lowermost band (47 kd) of this doublet corresponds
to GSK-3ß, and the identity of the middle band is not known. This
assignment is based on a comparison of lane 1 with lanes 2 and 3.
Figure 1, lane 2, was developed
using an affinity-purified antibody raised against the unique carboxy terminus
of GSK-3. This GSK-3–specific antibody shows 1
immunoreactive protein at 51 kd. Development of the blot (lane 3) with a mouse
monoclonal antibody specific for GSK-3ß showed 1 band at 47 kd.
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Higher Levels of Serine-Phosphorylated GSK-3 in Caput Than in Caudal Epididymal Spermatozoa
In a previous study, we showed that the tyrosine phosphorylation of GSK-3
increased in direct proportion to sperm motility
(Vijayaraghavan et al, 2000). In somatic cells, GSK-3 is also serine phosphorylated
(Wang et al, 1994a). The
purpose of the following experiments was to examine whether sperm GSK-3 was
serine phosphorylated and whether this phosphorylation was related to
motility. We used an antibody specific to serine-phosphorylated GSK-3.
Western blot analysis shows that GSK-3 is serine phosphorylated in
bovine epididymal spermatozoa and that its phosphorylation is significantly
higher in motile caudal than in immotile caput spermatozoa
(Figure 2, panel A, lane 1,
compared to lane 2). The difference in intensities of immunoreactive serine
phosphorylation of GSK-3 is not due to higher amounts of GSK-3 in caudal sperm
extracts. A duplicate Western blot developed with an antibody against
GSK-3 shows roughly equal amounts of GSK-3 in caput and caudal
epididymal sperm extracts (Figure
2, panel A, lanes 3 and 4). Note that GSK-3 in caudal sperm
(Figure 2A, lanes 1 and 3) has
an apparently higher molecular weight than its counterpart in caput sperm
(Figure 2B, lanes 2 and 4). We
also used an antibody for serine-phosphorylated GSK-3 that reacts against
serine-phosphorylated GSK-3 and GSK-3ß. This antibody shows 2
bands corresponding to the 2 isoforms.
Figure 2 (panel B) also shows
that both GSK-3 and GSK-ß in caudal spermatozoa are phosphorylated
to a greater extent than in caput epididymal spermatozoa
(Figure 2, panel B, lane 1,
compared to lane 2). Figure 2,
panel B, lanes 3 and 4, shows equal amounts of GSK-3 and GSK-3ß in
the extracts. Once again, note that GSK-3 is at a slightly higher
molecular weight in caudal than in caput sperm
(Figure 2B, lanes 1 and 3
compared to lanes 2 and 4). Western blot probed with anti-phosphotyrosine
GSK-3 antibody showed that GSK-3 in both caudal and caput sperm is
tyrosine phosphorylated (Figure
2, panel C).
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Serine Phosphorylation Varies in Direct Proportion to Motility
Note that in somatic cells, GSK-3 is serine phosphorylated in response to
external signals. It was therefore surprising that GSK-3 in spermatozoa was
serine phosphorylated in the apparent absence of any external signaling
molecules. Since GSK-3 was serine phosphorylated to a greater extent in motile
caudal spermatozoa, we next examined whether GSK-3 serine phosphorylation
could be altered in vitro when the motility status of spermatozoa was altered
by pharmacological means. The relationship between GSK-3 serine
phosphorylation and sperm motility was investigated using 2 motility
stimulators, IBMX and CdA, and a motility inhibitor, sHT31
(Vijayaraghavan et al, 1996).
Immotile caput spermatozoa were treated with 0.5 mmol of IBMX per liter, 10
µmol of CdA per liter, or 10 µmol of sHT31 per liter. Extracts from
control and treated sperm were subjected to Western blot analysis with
antibodies against serine-phosphorylated GSK-3. Data in
Figure 3, panel A, show that
the motility stimulators caused an increase in GSK-3 serine phosphorylation
(lanes 2 and 3), whereas treatment with the motility inhibitor sHT31 (lane 4)
caused a virtual elimination of serine phosphorylation when compared to
control sperm (lane 1). Duplicate blots probed with GSK-3 antibody show
that equal amounts of protein are present in extracts from control and treated
spermatozoa (Figure 3, panel
B).
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Purification of GSK-3 From Caudal and Caput Sperm Extracts
Next, we wanted to further confirm the identity and serine phosphorylation
status of the GSK-3 species present in sperm extracts using column
chromatography. We used DEAE-cellulose and Mono S columns for partial
purification of GSK-3 from caudal and caput sperm extracts. Column fractions
were analyzed for GSK-3 immunoreactivity and catalytic activity. Specific
activity of GSK-3 in the pooled caudal sperm extracts was 9.18 U of protein
per milligram. A summary of the purification steps is provided in
Table 1. The extracts were
first passed through a DEAE-cellulose column. All of the GSK-3 in the extracts
was present in the flow-through fraction (specific activity, 14.4 U of protein
per milligram). The flow-through fraction was concentrated and passed through
a Mono S column. GSK-3 immunoreactivity was present in the flow-through and
gradient (0.125–0.165 M KCl) fractions.
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The enzyme from caput sperm extracts was purified using the same protocol.
The extracts with a specific activity of 10.7 U of protein per milligram were
passed through DEAE-cellulose column. As with caudal sperm extracts, the
flow-through fraction contained all of the immunoreactive GSK-3. The
concentrated DEAE-cellulose flow through (specific activity, 15.6 U of protein
per milligram) was applied to a Mono S column, and GSK-3 ( and ß)
was obtained in the Mono S gradient (0.125–0.165M KCl) with a specific
activity of 64.08 U of protein per milligram
(Table 2).
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Figure 4 shows the Western
blot data of the column fractions. Figure
4, panel A, shows blots probed with GSK-3 antibodies, while
panel B shows duplicate blots probed with GSK-3ß antibody. The notable
difference between caudal and caput sperm is that a portion of GSK-3 in
caudal sperm extracts is present in the Mono S flow-through fraction, whereas
there is no detectable GSK-3 in the corresponding fraction in caput sperm
extracts (lane 4 in Figure 4,
panels A and B). The Mono S gradient fractions of caput and caudal sperm
extracts contained both GSK-3 and GSK-3ß (lane 3 in
Figure 4, panels A and B).
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Blots probed with serine-phosphorylated GSK-3/ß antibody showed
that both GSK-3 and GSK-3ß were phosphorylated at their serine
positions in caudal and caput sperm extracts
(Figure 4, panel C). Individual
lanes show DEAE-cellulose flow through (lane 1), Mono S gradient (lane 2), and
Mono S flow through (lane 3). Significantly, in caudal sperm, most of the
serine-phosphorylated GSK-3 was present in the Mono S flow-through
fraction as a high-molecular-weight form with a low specific activity of 7.8 U
of protein per milligram. The enzyme is absent in the Mono S flow-through
fraction of caput sperm extracts (Figure
4, panel C, lane 3).
Caput and Caudal Spermatozoa Incubated With Calyculin A
We further examined the relationship between motility and GSK-3
phosphorylation using the motility stimulator calyculin A. Calyculin A is a
protein phosphatase inhibitor that initiates or stimulates motility at
nanomolar concentrations (Vijayaraghavan
et al, 1996). Caput spermatozoa were treated with 50 nmol of
calyculin A per liter and extracts of control and treated spermatozoa were
analyzed for GSK-3 serine phosphorylation. Western blots in panel A were
developed with antibodies specific for GSK-3. The data show that
calyculin treatment increases GSK-3 serine phosphorylation (lane 4 compared to
lane 3 in panel A). Figure 5, lanes 1 and 2, shows that extracts from control and calyculin-treated sperm
contain equal amounts of GSK-3. Blots in
Figure 5, panel B, were
developed with antibodies that react against both GSK-3 and
GSK-3ß. These data show that the serine phosphorylation of both
GSK-3 and GSK-ß is increased by calyculin treatment (lane 4
compared to lane 3 in panel B, Figure
5). Figure 5, lanes
1 and 2, shows that equal amounts of protein were loaded. Visual evaluation
confirmed our earlier reports (Vijayaraghavan et al,
1996,
2000) that this concentration
of calyculin A induced motility in immotile caput spermatozoa.
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Next, we examined whether there was a dose-dependent increase in serine
phosphorylation and an increase in the molecular weight of GSK-3 in
sperm treated with calyculin A. Caput epididymal spermatozoa were incubated
with concentrations of calyculin A that varied from 0 to 50 nmol/L. Western
blot analysis of extracts of control and treated sperm showed that calyculin A
caused a dose-dependent increase in GSK-3 serine phosphorylation at
concentrations between 10 and 50 nmol/L
(Figure 6, panel A). Panel B
shows that equal amounts of GSK-3 are present. Note that there is an increase
in the apparent molecular weight of GSK-3 in SDS-PAGE with increased
serine phosphorylation. We have previously shown that increasing the calyculin
A concentration caused a dose-dependent increase in the motility of immotile
caput epididymal spermatozoa, with a maximum motility effect observed at
concentrations of calyculin A that varied from 15 to 25 nmol/L
(Vijayaraghavan et al,
2000).
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Relationship Between GSK-3 Serine Phosphorylation and Catalytic Activity
In somatic cells, serine phosphorylation is known to decrease the catalytic
activity of GSK-3. We next determined if this expected relationship between
GSK-3 phosphorylation and its catalytic activity also held true in
spermatozoa. We have previously shown that GSK-3 activity is high in caput
compared to caudal epididymal spermatozoa. This measurement was determined by
an indirect assay based on the ability of GSK-3 to activate the inactive
protein phosphatase (PP1) and inhibitor I2 complex. The measurement was also
complicated by the fact that sperm extracts had high endogenous protein
phosphatase activity. A direct assay using a synthetic prephosphorylated amino
acid sequence domain in CREB was used in this study
(Wang et al, 1994b). Protein
kinase activity sensitive to lithium was thought to have originated from GSK-3
(Stambolic et al, 1996).
Measurement of GSK-3 in soluble sperm extracts, using this assay, showed that
immotile caput spermatozoa have about 2.5-fold higher catalytic activity than
do caudal epididymal spermatozoa (Figure
7).
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Next, we investigated whether increased GSK-3 serine phosphorylation due to
calyculin A treatment in vitro (Figure
4) was also accompanied by a decrease in GSK-3 activity. Data in
Figure 8, panel A, show that in
both caput and caudal sperm extracts, calyculin A treatment resulted in a 50%
reduction in GSK-3 activity compared to controls. Data in Figures
7 and
8 (panel A) are activity
measurements in crude extracts. Activity measurements in crude extracts are
due to both GSK-3 and GSK-3ß and are subject to potential
interference by nonspecific factors present in extracts. We therefore measured
GSK-3 activity following immunoprecipitation with GSK-3 antibodies.
Immunoprecipitation using the GSK-3 antibody completely pulled down
GSK-3 from sperm extracts, leaving behind the GSK-3ß antibody in
the supernatant. This technique, confirmed by Western blotting (figures not
shown), permitted measurement of the individual activities of the 2 isoforms
of GSK-3 in caudal and caput sperm extracts. The activities of both
GSK-3 and GSK-3ß were higher, nearly double in caput vs caudal
sperm extracts. Treatment with 50 nmol of calyculin A per liter decreased
GSK-3 activity in both caput and caudal sperm
(Figure 8, panel A). This
decrease in activity was observed for both GSK-3 isoforms (ie, in GSK-3
immunoprecipitates and in the supernatants of extracts following
immunoprecipitation) (Figure 8,
panels B and C). In caput sperm treated with 50 nmol of calyculin A per liter,
GSK-3 activity was reduced by about 39% (from 96.0 to 59.0 U)
(Figure 8, panel B), while the
activity for the GSK-3ß isoform was reduced by about 48% (from 51.0 to
26.0 U) (Figure 8, panel C).
Activity measurements in crude extracts showed a net inhibition of 46%. In
caudal sperm, with the addition of 50 nmol of calyculin A per liter,
GSK-3 activity was reduced by approximately 68% (from 80.3 to 25.7 U),
while the GSK-3ß activity was reduced by about 52% (from 54.3 to 26.4 U).
Inhibition in crude caudal extracts was 52%
(Figure 8, panel C). Also note
that GSK-3 activity in crude sperm extracts is higher in caput than in caudal
sperm, which further confirms the data in
Figure 7. Enzyme activity
values for extracts shown in Figure
8 are higher than those in
Figure 7, because the activity
assay in Figure 8 was performed
with the flow-through fraction of extracts passed through DEAE-cellulose mini
columns. The DEAE column appears to remove unknown GSK-3 inhibitors from sperm
extracts.
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Western Blot Analysis of PI3-Kinase and cAkt
In somatic cells, GSK-3 is phosphorylated and inactivated by protein kinase
B (PKB; also known as cAkt). cAkt, in turn, is activated by PI3-kinase. We
examined whether these 2 upstream GSK-3–regulating enzymes are present
in spermatozoa. Antibodies against PI3-kinase and cAkt were used in Western
blot analysis of bovine epididymal sperm extracts. PI3-kinase is a heterodimer
composed of an 85-kd regulatory subunit and a 110-kd catalytic subunit.
Antibodies against the 85-kd subunit show that it is present in spermatozoa
(Figure 9, lane 1). Carboxy
terminus antibodies against cAkt, which is a 62-kd protein, show
immunoreactive bands in this expected range in caput and caudal spermatozoa.
Note that the antibody shows 2 closely spaced bands in caudal sperm extracts
(Figure 9, lane 2). It is
possible that the slower migrating band is a phosphorylated form of cAkt.
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GSK-3 Immunocytochemistry
Finally, we examined the localization of GSK-3 in spermatozoa. An antibody
against GSK-3 was used on caput and caudal spermatozoa. No specific
differences could be seen in the localization of GSK-3 between caput
and caudal spermatozoa. Intense staining was observed in the posterior portion
of the head, in the equatorial segment, and along the length of the tail
(Figure 10).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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and GSK-3ß
(Woodgett, 1990). The
-isoform is a 51-kd protein sharing 95% identity with the 47-kd
ß-isoform in the kinase domain. Catalytic activity of GSK-3 is regulated
by the phosphorylation of its tyrosine 214 in both and by its serine 21 and
serine 9 residues in GSK-3 and GSK-3ß, respectively
(Wang et al, 1994a). The
presence of GSK-3 in spermatozoa was first reported by Vijayaraghavan et al
(1996). On the basis of an
indirect assay, its activity was approximately threefold higher in immotile
caput than in motile caudal spermatozoa, which suggests that it is involved in
motility regulation (Smith et al,
1999). Recently, we reported the existence of a
tyrosine-phosphorylated protein, GSK-3 in bovine spermatozoa, and found
that its tyrosine phosphorylation varied directly with motility
(Vijayaraghavan et al,
2000).
In the present study, we first examined whether both isoforms of GSK-3 are
present in spermatozoa. Western blots for caudal sperm extracts with
isoform-specific GSK-3 antibodies (Figure
1) showed that both GSK-3 ( and ß) isoforms are
present in bovine spermatozoa. On the basis of the intensity of
immunoreactivity, GSK-3 appears to be the predominant isoform in
spermatozoa. Next, we examined whether sperm GSK-3 is serine and tyrosine
phosphorylated. Western blot analysis with an antibody specific to
serine-phosphorylated GSK-3 showed a dramatic enhancement in GSK-3
serine phosphorylation in motile caudal compared to immotile caput spermatozoa
(Figure 2, panel A), while a
slight increase in the phosphorylation of tyrosine 214 residue was observed
when duplicate blots were used for the anti–phospho-GSK-3 antibody
(Figure 2, panel C).
Antibody-recognizing phosphorylated serine residue in both GSK-3 isoforms
showed that the serine phosphorylation of GSK-3 and GSK-3ß is
increased with motility (Figure
2, panel B). Thus, 2 different phosphorylation site-specific
antibodies confirmed that the serine phosphorylation of GSK-3 is higher in
caudal than in caput spermatozoa. It should be emphasized that in somatic
cells, GSK-3 is serine phosphorylated only in response to external signals.
Remarkably, the serine phosphorylation of GSK-3 is observed in caudal
epididymal spermatozoa even in the apparent absence of any cellular
activator.
To further examine the relationship between sperm motility and GSK-3 serine phosphorylation, we used compounds that stimulate or inhibit motility. Western blot analysis showed that caput and caudal spermatozoa treated with sHT31, a motility inhibitor, resulted in a dramatic decrease in the serine phosphorylation of GSK-3 when compared to control spermatozoa (Figure 3). Motility stimulators such as IBMX and CdA also caused a noticeable increase in GSK-3 serine phosphorylation. We further examined the relationship between motility and GSK-3 serine phosphorylation using the motility activator calyculin A. Sperm suspensions with 1 x 108 sperm per milliliter, incubated for 15 minutes in the presence of calyculin A (with concentrations between 0 and 50 nmol/L), showed a dose-dependent increase in serine phosphorylation. Calyculin A at a concentration of 5 nmol/L increased serine phosphorylation in caput sperm, reaching a maximum at 20 nmol/L. This increase in serine phosphorylation matches the concentration-dependent motility initiation by calyculin A (Vijayaraghavan et al, 1996).
An increase in GSK-3 serine phosphorylation accompanies sperm maturation
and the acquisition of motility in the epididymis. This increased serine
phosphorylation also results in the expected decrease in GSK-3 catalytic
activity. Western blot analysis also showed that in both caput and caudal
sperm, GSK-3 appears to be both serine and tyrosine phosphorylated.
However, GSK-3 in the caudal sperm appears to have a higher molecular
weight in SDS-PAGE analysis than in caput sperm. One explanation for this
observation is that GSK-3 in caput sperm may be either serine or
tyrosine phosphorylated, whereas in caudal sperm, it may be phosphorylated at
both residues. To examine this possibility, we partially purified GSK-3
from sperm extracts. In an anion exchange column (DEAE-cellulose), all of the
GSK-3 in caput and caudal sperm extracts was found in the flow-through
fraction. The enzyme in this flow-through fraction had a higher activity than
the whole extracts, which is probably due to the removal of interfering
proteins from the extracts. When the anion exchange flow-through fraction was
analyzed using the Mono S column (cation exchanger), immunoreactive GSK-3 was
obtained in both the flow-through and gradient fractions
(Table 1) in the caudal sperm,
whereas in the caput sperm, all of the GSK-3 eluted in the gradient fractions
(Table 2). Western blots showed
that, in caudal sperm, the higher-molecular-weight fraction of GSK-3
was present in the Mono S flow-through fraction
(Figure 4, panel A) with a low
specific activity (7.8 U of protein per milligram) compared to the
low-molecular-weight forms, GSK-3 and GSK-3ß in the gradient
fractions (Figure 4, panels A
and B), which had a specific activity of 38.97 U of protein per milligram
(Table 1). In the case of caput
sperm, all the GSK-3 and GSK-ß present in the Mono S gradient had
a specific activity of 64.08 U of protein per milligram. The 2
molecular-weight forms of GSK-3 could be due to differences in
phosphorylation: the low-molecular-weight form may be phosphorylated at one
residue—either at serine 21 or tyrosine 214—while the
higher-molecular-weight form may be phosphorylated at both residues. This
higher-molecular-weight form of GSK-3 is present in caudal but not in
caput epididymal spermatozoa. An alternate possibility for the difference in
molecular weight could be due to glycosylation.
Next, we investigated whether changes in GSK-3 activity accompany
motility-associated changes in serine phosphorylation. We first compared GSK-3
catalytic activity in extracts of cell number–adjusted caput and caudal
spermatozoa. The activity of GSK-3 was significantly higher in caput than in
caudal sperm. Furthermore, calyculin A, which increased the serine
phosphorylation of GSK-3 and GSK-ß and initiated the motility in
immotile caput spermatozoa, inhibited the GSK-3 activity in caput and caudal
spermatozoa by approximately 50%. Measurement of GSK-3 and GSK-3ß
activity following immunoprecipitation showed that both isoforms are less
active in caudal than in caput spermatozoa. In both caput and caudal
spermatozoa, treatment with 50 nmol of calyculin A per liter inhibited the
activities of both isoforms of GSK-3.
Immunocytochemistry using the GSK-3 antibody showed that GSK-3 is
located at the equatorial segment, on the postacrosomal region, and on the
principal piece of the tail in both caudal and caput spermatozoa. Antibodies
detecting both GSK-3 and GSK-3ß and detecting GSK-3ß only
also were used for immunocytochemistry (data not shown). These antibodies
exhibited the same pattern of localization, suggesting that GSK-3 and
GSK-3ß reside in the same location. Their localization would be
consistent with their role in sperm motility and fertilization.
Spermatozoa are terminally differentiated cells with little protein
synthesis (Toshimori, 1998).
Therefore, the phosphorylation and dephosphorylation of the proteins already
present must be important mechanisms for regulating protein and cellular
function. The increase in the phosphorylation of GSK-3 and GSK-3ß
upon treatments with motility stimulators suggests that a protein kinase
responsible for its phosphorylation is present in spermatozoa.
To date, 2 enzymes are known to phosphorylate GSK-3—protein kinase A (PKA) (Fang et al, 2000) and PKB/cAkt (Hemmings, 1997; Brazil and Hemmings, 2001). cAkt is regulated by PI3-kinase. Upon cellular stimulation, PI3-kinases recruited to the cell surface generate inositol lipids such as PtdIns(3)P, PtdIns(3,4)P2, Ptd-Ins(3,5)P2, and PtdIns(3,4,5)P3 (Leevers et al, 1999; Vanhaesebroeck and Waterfield, 1999; Cantrell, 2001), which bind and activate PH-domain–containing enzymes. One of these is phosphoinositide-dependent kinase 1 (PDK1), which, when activated, phosphorylates Akt (Hemmings, 1997; Brazil and Hemmings, 2001). Serine phosphorylation and inhibition of GSK-3 is primarily mediated by Akt (Gold et al, 2000; Krasilnikov, 2000). We have shown that both cAkt and PI3-kinase are present in spermatozoa. Western blot analysis showed that an antibody against PI3-kinase could detect a protein at 85-kd, while the antibody against cAkt detected 2 protein bands at 62 kd. GSK-3 was first identified as a rate-limiting enzyme in glycogen synthesis (Embi et al, 1980). Recent studies have identified a number of new roles and substrates for GSK-3 (Woodgett, 2001). Two major pathways involving GSK-3 are Wnt/wingless signaling and a PI3-kinase pathway involved in apoptosis and cell survival (Woodgett, 2001). The identification of PI3-kinase and cAkt in sperm suggests a new role for GSK-3 representing a novel role for this signaling mechanism in terminally differentiated spermatozoa.
An important question raised by our studies concerns the external signal responsible for the changes in sperm GSK-3 in vivo. In somatic cells, changes in GSK-3 phosphorylation are induced by external signals. It is not known what external signals, if any, are involved in epididymal sperm maturation and motility initiation. It is also intriguing that a variety of treatments in vitro such as IBMX, 2-chloroadenosine, and calyculin A alter GSK-3 phosphorylation. This suggests that the net phosphorylation of GSK-3 is not static but undergoes active turnover in spermatozoa suspended in simple salt buffers in vitro. The biological significance of this turnover is not known. It is possible that a paracrine factor, perhaps adenosine (Vijayaraghavan and Hoskins, 1986) secreted from spermatozoa, is one of the signaling factors responsible for controlling GSK-3 phosphorylation.
Another question raised by our studies concerns the physiological significance of GSK-3 phosphorylation. Since we have not yet identified the protein substrates of GSK-3, it is difficult to speculate how GSK-3 acts in spermatozoa. We emphasize that the present study showing a relationship between GSK-3 activity, serine phosphorylation, and sperm motility is correlative. One of the limitations in designing further studies that might shed light on the exact role of GSK-3 in sperm function is, as noted, the lack of knowledge of external signaling molecules that may activate spermatozoa. It is therefore not possible to experimentally manipulate the signaling pathway involving sperm GSK-3 to examine the physiological consequences by altering GSK-3 activity. Another limitation is our inability to specifically inactivate GSK-3 by pharmacological means. We are currently evaluating the use of a number of inhibitors, commercially available, claimed to be specific for GSK-3. Preliminary results suggest that the effects of these inhibitors on GSK-3 activity in sperm are variable. That GSK-3 is conserved in sperm from a variety of species (eg, avian, rodent, bovine, crustacean, echinoderm) suggests that the enzyme is involved in some essential function within spermatozoa. Studies in our laboratory are currently focused on identifying substrates for sperm GSK-3 and on examining the activity of the enzyme in sea urchin compared to mammalian spermatozoa. The studies may shed further light on the physiological role of the enzyme in spermatozoa.
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