First published online July 13, 2004
doi: 10.1242/10.1242/jcs.01214
Journal of Cell Science 117, 3645-3657 (2004)
Published by The Company of Biologists 2004
Tyrosine phosphorylation activates surface chaperones facilitating sperm-zona recognition
Kelly L. Asquith1,
Rosa M. Baleato1,
Eileen A. McLaughlin1,2,
Brett Nixon1 and
R. John Aitken1,2,*
1 Reproductive Science Group, School of Environmental and Life Sciences, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia
2 ARC Centre of Excellence in Biotechnology and Development, School of Environmental and Life Sciences, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia
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Fig. 1. Tyrosine phosphorylation of mouse spermatozoa. Cauda epididymal sperm were capacitated in BWW for 1 hour followed by 30 minutes with zona pellucidae. Sperm from the free-swimming population and recovered from the zona were fixed, permeabilised and labelled with anti-phosphotyrosine. (A) Cells displayed either partial flagellum (principal piece only) or complete flagellum labelling (midpiece and principal piece). Corresponding phase-contrast images are included. Scale bar, 10 µm. (B) Comparison of percentages of spermatozoa displaying partial (white bar) and complete (black bars) flagellum labelling in free-swimming and zona pellucida (ZP)-bound populations. The experiment was repeated four times and a minimum of 100 cells were scored for each experiment. **, P<0.005.
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Fig. 2. Tyrosine phosphorylation and zona pellucida binding. Cauda epididymal spermatozoa were capacitated for 90 minutes in complete BWW (1), BWW–HCO3– (2), BWW–Ca2+ + pentoxifylline (ptx) + dibutyryl cyclic-AMP (cAMP) (3) or BWW–Ca2++Sr2++ptx+cAMP (4). (A) Representative western blot of sperm proteins probed with anti-phosphotyrosine. The experiment was repeated three times. (B) Percentage of spermatozoa displaying partial (white bars) and complete (black bars) flagellum labelling following immunofluorescence with anti-phosphotyrosine. The experiment was repeated three times, with a minimum of 200 cells scored for each one. Treatment 2 led to a significant (P<0.05) reduction in the percentage of cells exhibiting phosphotyrosine expression over the entire tail when compared to control treated cells (1). By contrast, treatment 3 and 4 resulted in significant (P<0.005) increases in the proportion of spermatozoa exhibiting partial and complete labelling. *, P<0.05; **, P<0.005. (C) Correlation between tyrosine phosphorylation and zona pellucida binding. Cauda spermatozoa were prepared as described followed by a 30-minute capacitation with salt-stored oocytes. The mean number of sperm bound to each zona is expressed as a percentage of the control (treatment 1) for four repeats. Cells capacitated in solution 2 express low levels of flagellar tyrosine phosphorylation exhibited a significant (P<0.05) decrease in zona binding compared with the controls. Conversely, the enhanced tyrosine phosphorylation status of spermatozoa capacitated in solution 4 was associated with a significant (P<0.005) elevation in the zona binding capacity (Fig. 2C). *, P<0.05; **, P<0.005.
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Fig. 3. Localisation of phosphotyrosine residues on live spermatozoa. Cauda epididymal spermatozoa were capacitated for 90 minutes in either complete BWW (1), BWW–HCO3– (2) or BWW–Ca2++Sr2++ptx+cAMP (4). Tyrosine phosphorylation was assessed by immunofluorescence or by a 30-minute capacitation with anti-phosphotyrosine-coated magnetic beads. Controls were included where beads were preabsorbed with 20 mM O-phospho-L-tyrosine (pY). (A) Representative images of live spermatozoa displaying phosphotyrosine residues on the surface of the head visualised by immunofluorescence and immunobead assay. Scale bar, 10 µm. (B) Percentage of viable spermatozoa expressing phosphotyrosine residues on the head as assessed by immunobead assay. The experiment was repeated three times, with a minimum of 200 viable cells scored for each experiment. Surface tyrosine phosphorylation was observed on the head of approximately 9% of spermatozoa capacitated in complete media. This was significantly (P<0.05) decreased in uncapacitated cells (solution 2). By contrast, capacitation in solution 4 induced significantly (P<0.01) increased surface labelling compared with uncapacitated cells. Pre-incubation of phosphotyrosine beads with O-phospho-L-tyrosine significantly (P<0.01) reduced the labelling of cells in solution 4 to approximately 2.5%. *, P<0.05; **, P<0.01.
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Fig. 4. Tyrosine phosphorylation of live spermatozoa bound to the zona pellucida (ZP). Cauda epididymal spermatozoa were capacitated for 1 hour in BWW–Ca2++Sr2++ptx+cAMP followed by a 30-minute incubation with salt-stored oocytes and FITC-conjugated anti-phosphotyrosine. Sperm-ZP complexes were washed and labelling of bound spermatozoa was scored. (A) Representative fluorescent (top) and phase-contrast (bottom) images of phosphotyrosine labelling of sperm bound to ZP. Analysis revealed that 100% of the spermatozoa bound to the surface of the ZP displayed a punctate pattern of fluorescence over the head, particularly localised in the region of plasma membrane overlying the acrosome. Scale bar, 6 µm. (B) Labeled spermatozoa adhered to the ZP were compared to the free-swimming population. Phosphotyrosine was detected on the head but not the flagellum of viable spermatozoa; a highly significant increase (P<0.001) in the proportion of phosphorylated spermatozoa compared with the free-swimming sperm population was detected. The experiment was repeated three times and a minimum of 100 cells were scored for each experiment. **, P<0.001.
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Fig. 5. Identification of tyrosine-phosphorylated proteins on the surface of spermatozoa. (A) Cauda epididymal spermatozoa were capacitated for 90 minutes in BWW–Ca2++Sr2++ptx+cAMP. Surface proteins were affinity-purified and resolved by SDS-PAGE. Tyrosine-phosphorylated surface proteins were immunoblotted with anti-phosphotyrosine. Three major bands (P1, P2 and P3) were identified and corresponding bands sequenced from a Coomassie-stained gel by tandem mass spectrometry. (B-D) Alignment of peptide sequences from (B) P1 with mouse hexokinase type 1 (hk1, accession number P19376), (C) P2 with mouse endoplasmin (erp99, accession number P08113) and (D) P3 with mouse heat shock protein 60 (hsp60, accession number NP_034607). Sequenced peptides are underlined. Potential tyrosine phosphorylation sites predicted by NetPhos 2.0 (Blom, 1999) appear in bold italics.
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Fig. 6. Confirmation of tyrosine phosphorylation and surface localisation of erp99 and hsp60 on mouse spermatozoa. (A) Cauda epididymal spermatozoa were either freshly isolated (lanes 1 and 3) or capacitated for 90 minutes in BWW–Ca2++Sr2++ptx+cAMP (lanes 2 and 4). CHAPS detergent lysates (5 µg protein/lane) (lanes 1 and 2) and purified surface-protein extracts from 100 µg lysate (lanes 3 and 4) were prepared, resolved by SDS-PAGE and immunoblotted with anti-hsp60 and anti-erp99 monoclonal antibodies. (B) Cauda epididymal spermatozoa were capacitated for 90 minutes in BWW. Proteins were solubilised, resolved by two-dimensional electrophoresis and immunoblotted with anti-phosphotyrosine (a), anti-erp99 (b) and anti-hsp60 (c) antibodies.
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Fig. 7. Immunolocalisation of chaperones on fixed capacitated mouse spermatozoa. Representative images of mouse sperm labelled with anti-hsp60 and anti-erp99 monoclonal antibodies followed by FITC-conjugated secondary antibodies (left). Corresponding phase contrast images (right) are included. Scale bar, 10 µm.
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Fig. 8. Proposed model for the involvement of tyrosine-phosphorylated molecular chaperones in the acquisition of mammalian sperm fertilizing ability. We hypothesise that a zona pellucida (ZP) receptor complex present in the plasma membrane of spermatozoa might encompass a ZP-binding molecule and also the chaperone proteins heat shock protein 60 (hsp60) and endoplasmin (erp99). In freshly ejaculated spermatozoa, these molecules might be on the cytoplasmic side of the membrane. During capacitation, cholesterol efflux from the membrane promotes increased fluidity, which facilitates changes in protein distribution. Tyrosine phosphorylation of hsp60 and erp99 on the inner surface of the membrane might activate this receptor complex and allow conformational changes such that the chaperone proteins and ZP binding molecule are exposed to the cell surface. This would account for the appearance of phosphotyrosine residues observed on the sperm head following capacitation, and the association of this event with the attainment to recognise and bind the ZP.
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© The Company of Biologists Ltd 2004
