First published online 26 March 2003
doi: 10.1242/jcs.00396
Calmodulin antagonists differentially affect capacitation-associated protein tyrosine phosphorylation of mouse sperm components
Hai-Tao Zeng* and
Daulat R. P. Tulsiani
Departments of Obstetrics and Gynecology and Cell Biology, Vanderbilt
University School of Medicine, Room D-3243 MCN, Nashville, TN 37232-2633,
USA
* Present address: Research Center of Molecular Biology, Central South
University, Xiang Ya School of Medicine, Changsha, Hunan 410078, Peoples
Republic of China
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Fig. 1. Time-dependent protein tyrosine phosphorylation of mouse spermatozoa. Cauda
epididymal spermatozoa were suspended in EKRB medium supplemented with 3 mg
BSA/ml (A) or 1 mM methyl-ß-cyclodextrin (B) and incubated at 37°C
for 60 minutes or 90 minutes as described under Materials and Methods. At the
indicated time intervals, spermatozoa were washed, extracted and subjected to
SDS-PAGE. The resolved components were transferred to a nitrocellulose sheet,
and the tyrosine-phosphorylated bands were revealed. A representative
experiment, out of a total of at least three experiments, is shown. The sharp
band at the position of 116 kDa appears in all lanes and is nonspecific. This
band is also present in all lanes in Figs
2 and
3. Spermatozoa incubated in
EKPB medium (without BSA or methy-ß-cyclodextrin) for 60 minutes or 90
minutes revealed little or no phosphorylation.
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Fig. 2. Effects of CaM antagonists on tyrosine phosphorylation of the mouse sperm
components. Spermatozoa were incubated for 90 minutes at 37°C in EKRB
medium supplemented with 3 mg BSA/ml (A) or 1 mM methyl-ß-cyclodextrin
(B) in the absence or presence of different concentrations (µM) of CaM
antagonists (W7, 25; OA, 20; CZ, 2.5). The lanes with solvent alone (water,
methanol and DMSO) are controls for each antagonist. Tyrosine phosphorylation
of the sperm components was revealed as above. The figure shows results of a
representative experiment. Similar results were obtained in two other
experiments.
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Fig. 3. Effects of CaM antagonists on tyrosine phosphorylation of the mouse sperm
components. Spermatozoa were incubated at 37°C for 90 minutes in EKRB
medium supplemented with 3 mg BSA/ml (A) or 1 mM methyl-ß-cyclodextrin
(B) in the absence (control) or presence of different concentrations (µM)
of CaM antagonists (compound 48/80, 2; W13, 1.58; CBD, 0.16). Tyrosine
phosphorylation was revealed as above. The figure shows data of a
representative experiment. Similar results were obtained in two other
experiments. A slight variation in phosphorylated proteins around 80 kDa in
this figure (compare A versus B) is probably due to different running
conditions.
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Fig. 4. Effect of CaM on protein tyrosine phosphorylation of mouse sperm
components. Cauda epididymal spermatozoa were incubated in EKRB medium
supplemented with 3 mg/ml BSA in the absence or presence of 10 µM CaM.
After 90 minutes at 37°C under 5% CO2 in air, sperm cells were
pelleted, washed, extracted, resolved by SDS-PAGE, and
protein-tyrosine-phosphorylated components were revealed as above. Bands from
the exposed X-ray film were scanned and the intensities of control and
CaM-treated groups quantified. The asterisk (*) indicates a significant
difference (P<0.05) between the control and experimental
groups.
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Fig. 5. Proposed regulatory pathways to explain the effect of CaM antagonists on
sperm capacitation, sperm motility and protein tyrosine phosphorylation. All
antagonists used inhibited/prevented capacitation, as evident by the poor
response of spermatozoa to undergo agonist-induced AR
(Table 1). Three antagonists
(W7, OA and CZ) inhibited sperm motility before protein tyrosine
phosphorylation (A) or vice versa (B), and capacitation. The second set of
antagonists (i.e. compound 48/80, W13 and CBD) adversely affected capacitation
without altering sperm motility or protein tyrosine phosphorylation. Inclusion
of purified CaM in the capacitation medium significantly enhanced tyrosine
phosphorylation of the 95 kDa and 82 kDa proteins (A).
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© The Company of Biologists Ltd 2003
