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First published online November 2, 2007
Journal of Experimental Biology 210, 4053-4064 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.007013

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Protein kinase C is an important signaling mediator associated with motility of intact sea urchin spermatozoa

Daniel White^*, Eve de Lamirande and Claude Gagnon

Urology Research Laboratory, Royal Victoria Hospital, McGill University Health Center and Faculty of Medicine, McGill University, Montréal, H3A 1A1, Canada

View larger version (22K): [in this window] [in a new window]   Fig. 1. PKC inhibitors dose-dependently inhibit the motility of intact sea urchin spermatozoa. Lytechinus pictus spermatozoa were pre-incubated in IM medium with one of the following inhibitors – chelerythrine, calphostin C or Gö6976 – and then diluted in ASW containing the same amount of inhibitor. The percentage of motile cells (A) and the spermatozoa velocity (B) were evaluated over a 15 min period as described in the Materials and methods. Values are means ± s.e.m. from three different experiments.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Increase in phospho-PKC substrate following motility initiation of intact sea urchin spermatozoa. The phosphorylation level, as detected by immunoblotting with an anti-phospho-PKC substrate antibody, of several proteins was markedly increased when immotile (i) L. pictus spermatozoa were diluted into ASW to initiate their motility (m) (). The increase in phosphorylation of four of these proteins (arrowheads at 200, 100, 65 and 28 kDa) was observed in all the experiments. The specificity of the antibody for these proteins is demonstrated by the disappearance of bands when the antibody was pre-incubated with its antigenic phospho-peptide (2261) but not after pre-incubation with a mixture of phospho-amino acids (phos-mix) or a different phospho-peptide (9621). A blot representative of two other blots is presented.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Time course of the phospho-PKC substrate levels following motility initiation of intact sea urchin spermatozoa. The phosphorylation level of the proteins detected with an anti-phospho-PKC substrate antibody increased rapidly (30 s) after transfer of immotile (i) L. pictus spermatozoa into ASW to initiate their motility (m). Protein phosphorylations further increased to a maximum at around 6 min and stayed almost at this level for up to an hour before making a significant drop after 5 h. The arrowheads indicate the position of the 200, 100, 65 and 28 kDa bands. A blot representative of two other blots is presented.

View larger version (37K): [in this window] [in a new window]   Fig. 4. Effect of various PKC inhibitors on the phospho-PKC substrate levels following motility initiation of intact sea urchin spermatozoa. The increase in phospho-PKC substrates, as detected with an anti-phospho-PKC substrate antibody, observed during motility (m) initiation was prevented when chelerythrine was present in IM and ASW at 1.5 µmol l–1 or more, with calphostin C at 10 µmol l–1 and with Gö6976 at 5 µmol l–1 (only bands at 65 and 28 kDa were affected in the latter case). The arrowheads indicate the position of the 200, 100, 65 and 28 kDa bands. i, immotile; m, motile. A blot representative of two other blots is presented.

View larger version (56K): [in this window] [in a new window]   Fig. 5. Cellular localization of phospho-PKC substrates. Sea urchin spermatozoa were mechanically separated into heads and flagella and then detergent fractionated with Triton X-100. The soluble phospho-PKC substrate proteins of spermatozoa, as detected with an anti-phospho-PKC substrate antibody, were predominantly found in the sperm heads while those recovered in the Triton X-100-insoluble sperm fraction were mainly associated with the flagella. The arrowheads indicate the position of the 200, 100, 65 and 28 kDa bands. i, immotile; m, motile; sol, Triton X-100 soluble; insol, Triton X-100 insoluble. A blot representative of two other blots is presented.

View larger version (14K): [in this window] [in a new window]   Fig. 6. PKC phosphorylation change following the initiation of motility in intact sea urchin spermatozoa. The presence of PKC and its catalytic cleavage product (arrowheads) was evidenced by immunoblotting with an anti-phospho-PKC antibody (pan). The protein phosphorylation profile obtained with the anti-phospho-PKC substrate antibody (subs) is presented for comparison. i, immotile; m, motile. A blot representative of two other blots is presented.

View larger version (51K): [in this window] [in a new window]   Fig. 7. Immunolocalization of PKC and phospho-PKC substrates on sea urchin spermatozoa. The presence of PKC and its phosphorylated substrates were evidenced by immunofluorescence with an anti-phospho-PKC antibody (p-PKC pan) and an anti-phospho-PKC substrate (p-PKC subst.) antibody in both immotile and motile spermatozoa (central columns). Labeling of flagellar ß-tubulin with a specific monoclonal antibody (see Materials and methods) is also shown for comparison purposes. In controls without any primary antibody (2nd Ab only), a weak spot of fluorescence was present at the base of sperm heads. The outer columns show similar images to those in the middle columns but viewed by phase-contrast. Scale bars, 10 µm.