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First published online 11 July 2006
doi: 10.1242/jcs.03055

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Identification of SRC as a key PKA-stimulated tyrosine kinase involved in the capacitation-associated hyperactivation of murine spermatozoa

The ARC Centre of Excellence in Biotechnology and Development, Reproductive Science Group, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia

View larger version (77K): [in a new window]   Fig. 1. Identification of SRC and its binding partner PKAc in murine spermatozoa. (A) Samples were taken from either the caput or caudal regions of the epididymis, lysed and run in a 10% SDS-PAGE. Western blot analysis was performed using the anti-SRC monoclonal antibody. A positive control of SRC (A431 cell lysates) was included to ensure the antibody cross-reacted with a protein of the appropriate size. Visualization was performed with standard ECL chemiluminescence. (B,C) Back-flushed murine spermatozoa were sonicated and Percoll-purified to obtain populations consisting of pure (>95%) (B) sperm heads or (C) sperm tails. (D) Approximately 2 µg of these fractions were then lysed and subjected to 10% SDS PAGE followed by western-blot analysis with anti-SRC antibody. (E) To demonstrate an association between SRC and PKAc the above fractions were incubated with beads coated with anti-SRC antibodies and the precipitated proteins probed with an antibody against PKAc. (F) The importance of sperm capacitation in this association between SRC and PKAc was also confirmed in experiments involving the inmmunoprecitation of SRC-containing complexes from capacitated (incubated with 1 mM PTX and 1 mM dbcAMP for 45 minutes) and non-capacitated cells (freshly isolated from the cauda epididymis without incubation) followed by probing of these immunoprecipitates with anti-PKAc antibodies. The western blot shows non-capacitated spermatozoa (lane 1) capacitated spermatozoa (lane 2) and a control incubation (lane 3), in which beads were incubated with sperm lysates in the absence of antibody. Arrows indicate the location of the IgG heavy chains (top) and IgG light chains (bottom).

View larger version (45K): [in a new window]   Fig. 2. Identification of the phosphorylated forms of SRC. (A-D) Spermatozoa were pre-incubated for 5 minutes with either 10 µM H89 (B,D, non-capacitated cells) or the vehicle (A,C; capacitated cells) before the addition of pharmacological agents to drive sperm capacitation (1 mM dbcAMP and 1 mM PTX). After a further 40-minute incubation, the cells were assessed for hyperactivated motility. Populations containing at least 95% hyperactivated motility (A,C) or less than 5% hyperactivated motility (B,D) were lysed and subjected to 2D PAGE as described in Materials and Methods. The proteins were then transferred to nitrocellulose membranes and probed with anti-pY416 antibody. Arrows in A and B indicate the position of SRC. The membranes were then stripped and re-probed with anti-SRC as a positive loading control (C,D). Arrows in C and D indicate the position of SRC and its isoform phosphorylated at Y416. The encircled spots were also present when the membrane was probed with secondary antibody alone, indicating that these signals were the result of non-specific interactions.

View larger version (63K): [in a new window]   Fig. 3. Localization of pY416 in non-capacitating and capacitating spermatozoon. (A-F) Spermatozoa were pre-incubated for 5 minutes with either 10 µM H89 (C,D) or the vehicle (A,B,E,F) before addition of reagents (1 mM dbcAMP and 1 mM PTX) to drive sperm capacitation. After a further 40-minute incubation, the cells were assessed for hyperactivated motility as described in Materials and Methods. Mouse spermatozoa from the cauda epididymides were fixed, washed and subjected to immunocytochemistry using anti-pY416 antibody in sperm cell populations having less than 5% total hyperactivation (C,D) or at least 95% hyperactivation (E,F). The secondary antibody only controls are shown in panels A and B. The induction of a hyperactivated state is clearly associated with phosphorylated Tyr416 (Y416-P) on the sperm tail (E,F). In non-capacitated cells, in which PKA had been blocked with H89, cAMP failed to elicit this response. Corresponding phase-contrast (lower panels) and FITC images (upper panels) are displayed.

View larger version (47K): [in a new window]   Fig. 4. Tyrosine phosphorylation and sperm hyperactivation. (A,B) Treatment of murine spermatozoa with inhibitors of SRC such as (A) lavendustin A and (B) SU6656 inhibited phosphotyrosine expression. For this analysis, spermatozoa were pre-incubated with the inhibitors for 5 minutes before the addition of 1 mM dbcAMP and 1 mM PTX. After a 40-minute incubation the spermatozoa were analyzed for hyperactivated motility to confirm induction of capacitation in the vehicle-only controls. Approximately 2 µg of lystate was run in a 10% SDS-PAGE and western-blot analysis was performed with anti-phosphotyrosine antibodies as described. Arrowheads indicate the position of protein bands that were reduced in the presence of inhibitor; Hx indicates the location of hexokinase, a constitutively phosphorylated protein that served as a loading control (Nixon et al., 2006). When caudal epididymal murine spermatozoa were treated with PTX (1 mM) and dbcAMP (1 mM) tyrosine phosphorylation was observed along the length of the sperm tail in >95% of cells examined. (C,D) Phase-contrast and phosphotyrosine images of mouse spermatozoa, respectively. (E) Suppression of tyrosine phosphorylation had a dramatic effect on hyperactivated movement. Spermatozoa were pre-incubated for 5 minutes with the inhibitors indicated before the addition of 1 mM dbcAMP and 1 mM PTX to capacitate the cells. Following a 40-minute incubation, a 20 µl aliquot was taken and the spermatozoa were assessed for hyperactivated motility (presented as a percentage of the motile sperm population) as described in Materials and Methods.

View larger version (77K): [in a new window]   Fig. 5. Localization of CSK in spermatozoa. (A-C) Murine spermatozoa from the cauda epididymis were fixed, washed and subjected to immunocytochemistry with anti-CSK antibody as described in Materials and Methods. The no-primary antibody controls (A,B) and the anti-CSK images (C,D) were visualized by fluorescence (lower panels) or phase-contrast (upper panels) microscopy.

View larger version (68K): [in a new window]   Fig. 6. Phosphorylation of CSK in capacitating murine spermatozoa. (A-D) Spermatozoa were pre-incubated with either 10 µM H89 (A,C) or the vehicle (B,D) for 5 minutes before the addition of 1 mM dbcAMP and 1 mM PTX to drive capacitation. After a further 40-minute incubation, cells were assessed for hyperactivated motility to confirm the attainment of a capacitated state in the vehicle controls. Capacitated and non-capacitated mouse spermatozoa were lysed and subjected to 2D PAGE as described in Materials and Methods. Proteins were then transferred to nitrocellulose membranes and, in the first instance, probed with anti-CSK antibody (C,D). Membranes were stripped and re-probed with the anti-phosphoserine antibody (A,B). Circles and arrows indicate the same position on all four images and indicate the serine phosphorylation of CSK in capacitated spermatozoa (B).

View larger version (25K): [in a new window]   Fig. 7. A biochemical model to explain the tyrosine phosphorylation events leading to sperm hyperactivation. During capacitation, a variety of factors including Ca2+, HCO3 or H2O2 stimulate sAC. This is turn leads to production of cAMP and downstream activation of PKA. PKA is central to the induction of hyperactivation in a two step process: (1) it must phosphorylate SRC and activate this kinase. (2) PKA must also phosphorylate CSK, thereby inhibiting this enzyme and promoting further PKA activity. The activation of SRC by PKA leads to autophosphorylation and consequent tyrosine phosphorylation of a number of sperm targets, including enolase, HSP90 and tubulin.

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