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doi: 10.1242/jcs.00842

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Analysis of the mechanism by which calcium negatively regulates the tyrosine phosphorylation cascade associated with sperm capacitation

The ARC Centre of Excellence in Biotechnology and Development, Reproductive Science Group, School of Environmental and Life Science, and Hunter Medical Research Institute, University of Newcastle, NSW, Australia

View larger version (66K): [in a new window]   Fig. 1. Impact of calcium on tyrosine phosphorylation in human spermatozoa. Spermatozoa (10x106) were incubated in either BWW medium or in calcium-depleted medium (BWW -Ca2+) as indicated. (A) At the times indicated, the cells were centrifuged, washed and lysed in 2% SDS. 1 µg of each lysate was loaded onto a 10% polyacrylamide gel. Tyrosine phosphorylated proteins were detected with western blot analysis using the anti-phosphotyrosine antibody, 4G10. The positions of the molecular mass markers (kDa) are shown on the left hand side. (B) The nitrocellulose membrane was stripped (2% SDS, 2 mM DTE, 50 mM Tris, pH 8.8) at 65°C with shaking. Following this, the membrane was blocked and re-probed using -tubulin antibody, as a loading control. The western blot is representative of three independent experiments.

View larger version (16K): [in a new window]   Fig. 2. Impact of extracellular calcium on intracellular pH. (A) Spermatozoa (10x106/ml) were incubated in either complete BWW (circles) or BWW -Ca2+ (squares). Approximately 40 minutes before the time shown, 1 ml of sperm was harvested, washed and incubated with 9 µM BCECF. Following 40 minutes incubation, the cells were washed to remove free dye. A ratiometric analysis of 440/530 nm was performed and plotted against known standards to determine the [pH]i. The results are presented as the average of three experiments performed in triplicate; *P<0.05. (B) Spermatozoa (10x106/ml) were incubated in either BWW -Ca2+ (lane 1), complete BWW (lane 2) or BWW with a pH of 8.2 (lane 3). After 2 hours, 1 ml of sperm from each treatment was harvested, washed and incubated with 9 µM BCECF. Following 40 minutes incubation, the cells were washed to remove free dye. A ratiometric analysis of 440/530 nm was performed and plotted against known standards to determine the [pH]i. The results are presented as the average of three experiments performed in triplicate; *P<0.05. (C) Spermatozoa (10x106/ml) were incubated in either BWW -Ca2+ (lane 1), complete BWW (lane 2) or BWW with a pH of 8.2 (lane 3). Following a 3-hour incubation the cells were lysed (2% SDS) and subject to anti-phosphotyrosine western blot analysis as described in Materials and Methods. (D) The nitrocellulose membrane was stripped at 65°C with shaking. Following this, the membrane was blocked and re-probed using -tubulin antibody. The western blot is representative of three independent experiments.

View larger version (23K): [in a new window]   Fig. 3. Impact of tyrosine phosphorylation on intracellular pH. (A) Spermatozoa were incubated for 3 hours in either complete BWW (lane 1) BWW -Ca2+ (lane 2), or BWW -Ca2+ containing 1 (lane 3) 10 (lane 4) 20 (lane 5) or 50 (lane 6) µM H89. The cells were centrifuged, washed and lysed in 2% SDS. 1 µg of each lysate was loaded onto a 10% polyacrylamide gel. Tyrosine phosphorylated proteins were identified by western blot analysis using the antiphosphotyrosine antibody 4G10. The positions of the molecular mass markers (kDa) are shown on the left. (B) The nitrocellulose membrane was stripped at 65°C with shaking. Following this, the membrane was blocked and re-probed using -tubulin antibody. The western blot is representative of three similar experiments. (C) Spermatozoa were incubated for 3 hours in BWW (lane 1) or BWW -Ca2+ (lane 2), or BWW -Ca2+ containing 10 (lane 3), 20 (lane 4) or 50 (lane 5) µM H89. The cells were then harvested, and the [pH]i was determined as described. The graph is the average of three independent experiments; *P<0.05.

View larger version (15K): [in a new window]   Fig. 4. Relationship between intracellular and extracellular calcium. Spermatozoa were incubated in either complete BWW or BWW -Ca2+ as indicated. Approximately 40 minutes before the time shown, 10x106 cells were harvested and loaded with 2 µM FURA 2. Following a 20-minute incubation, the cells were centrifuged, washed and the [Ca2+]i was determined as described in Materials and Methods. This graph represents the data from three experiments performed in duplicate.

View larger version (13K): [in a new window]   Fig. 5. Impact of extracellular calcium on intracellular ATP. Spermatozoa (10x106/ml) were incubated in either complete BWW (circles) or BWW -Ca2+ (squares). At the times indicated, 50 µl of the cell suspension was taken for measurement of ATP, as described in Materials and methods. The graph represents the data from three experiments performed in quadruplicate; *P<0.05.

View larger version (39K): [in a new window]   Fig. 6. Relationship between extracellular calcium and tyrosine phosphorylation. (A) Spermatozoa were incubated in BWW -Ca2+ (lane 1), or BWW -Ca2+ supplemented with 50 (lane 2), 100 (lane 3), 300 (lane 4) or 600 (lane 5), µM CaCl2. After 120 minutes incubation, the cells were centrifuged, washed and lysed in 2% SDS. 1 µg of each lysate was loaded onto a 10% polyacrylamide gel. Tyrosine phosphorylated proteins were detected by western blot analysis using the anti-phosphotyrosine antibody 4G10. The positions of the molecular mass markers (kDa) are shown on the left hand side. (B) The nitrocellulose membrane was stripped at 65°C with shaking. Following this, the membrane was blocked and re-probed using -tubulin antibody. The western blot is representative of three similar experiments.

View larger version (25K): [in a new window]   Fig. 7. Relationship between ATP and tyrosine phosphorylation. (A) Human spermatozoa were incubated for 120 minutes in either complete BWW (open bars) or BWW -Ca2+ (closed bars). The four treatments consisted of the vehicle control BWW (lane 1), BWW-glucose (lane 2) BWW +2-deoxyglucose (lane 3) and BWW +1 µM rotenone (lane 4). 50 µl of cell suspension was taken for ATP measurements, as described in Materials and Methods. The graph represents the data from three experiments performed in quadruplicate; *P<0.05, **P<0.01. (B) Cells were incubated in either complete BWW (lanes 1-4) or BWW -Ca2+ (lanes 5-8) as shown. Following a 120-minute incubation, cells incubated in complete BWW (lanes 1, 5), BWW -glucose (lanes 2, 6), BWW +2-deoxyglucose (lanes 3, 7) or BWW +1 µM rotenone (lanes 4, 8) were harvested, washed and lysed in 2% SDS. 1 µg of each lysate was loaded onto a 10% polyacrylamide gel. Tyrosine phosphorylated proteins were detected by western blot analysis using the anti-phosphotyrosine antibody 4G10. The positions of the molecular mass markers (kDa) are shown on the left. (C) The nitrocellulose membrane was stripped at 65°C with shaking. Following this, the membrane was blocked and re-probed using -tubulin antibody. The western blot is representative of three independent experiments.

View larger version (25K): [in a new window]   Fig. 8. Extracellular calcium ATP and tyrosine phosphorylation in mouse spermatozoa. (A) Mouse spermatozoa obtained from the cauda epididymides were incubated in either BWW (circles) or BWW -Ca2+ (squares). At the times indicated, 50 µl of each cell suspension was taken for ATP measurements as described in Materials and methods. The graph represents data from three experiments performed in duplicate; *P<0.05. (B) Caudal mouse preparations were harvested and incubated in either complete BWW (lane 1) or BWW -Ca2+ (lane 2). Following a 90-minute incubation, the cells were lysed and 1 µg of each lysate was loaded onto a 10% polyacrylamide gel. Tyrosine phosphorylated proteins were detected by western blot analysis using the anti-phosphotyrosine antibody, 4G10. The positions of the molecular mass markers (kDa) are shown on the left hand side. This western blot is representative of four independent experiments.

View larger version (27K): [in a new window]   Fig. 9. Sperm function correlates with tyrosine phosphorylation. (A) Cells were placed into either complete BWW (lane 1) or BWW -Ca2+ (lane 2). Approximately 2.5 hours later, an aliquot of cells in BWW -Ca2+ was placed into complete BWW (lane 3) and challenged with 2.5 µM A23187 for a further 30 minutes. The level of acrosome reaction was then counted as described in Materials and Methods. The graph represents the mean and standard deviation of three independent experiments; *P<0.05. (B) Phosphotyrosine expression in the treatments illustrated in A. (C) The nitrocellulose membrane was stripped at 65°C with shaking. Following this, the membrane was blocked and re-probed using -tubulin antibody. The western blot is representative of three independent experiments.

View larger version (33K): [in a new window]   Fig. 10. Sperm function and calcium-dependent ATP binding proteins. (A) Approximately 10x106 human spermatozoa were harvested, washed and lysed in either 5% CHAPS, with 0.6 mM CaCl2 (lane 1) or without CaCl2 (lane 2). Approximately 2 µCi -32P-labeled 8-azidoadenosine-5'-triphosphate [8-N3ATP; 20 Ci/mmol, 2 mCi/ml] was added. Following 10 minutes incubation, the cells lysates were UV irradiated (2 minutes at 1200 MW/m2). An equal amount of loading dye was then added to 1 µg of protein, which was subject to 10% SDS-PAGE. Autoradiography was performed using X-ray film according to the manufacturer's instructions. (B) Cells were incubated in BWW -Ca2+, (1) or BWW medium (vehicle control, 2), or BWW medium containing 1 (3), 5 (4) or 10 (5) µM thapsigargin. After 3 hours, 50 µl of cell suspension was taken and ATP measured as described in Materials and Methods. Inset shows relative increase in [Ca2+]i following the addition of thapsigargin (Tps, 10 µM). The graph represents data from three experiments performed in duplicate; **P<0.01. (C) Cells were incubated in BWW medium (vehicle control, lane 1,), or BWW medium containing 1 (lane 3), 5 (lane 4) or 10 (lane 5) µM thapsigargin. As a positive control, cells were incubated in BWW -Ca2+ (lane 2). After 3-hours incubation, cells were harvested, washed and lysed in 2% SDS. 1 µg of each lysate was loaded onto a 10% polyacrylamide gel. Tyrosine phosphorylated proteins were detected by western blot analysis using the anti-phosphotyrosine antibody, 4G10. The positions of the molecular mass markers (kDa) are shown on the left. (D) The nitrocellulose membrane was stripped at 65°C with shaking. Following this, the membrane was blocked and re-probed using -tubulin antibody. The western blot is representative of three independent experiments.

View larger version (35K): [in a new window]   Fig. 11. Proposed mechanism of sperm capacitation.