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First published online March 2, 2004
doi: 10.1242/10.1242/jcs.00931

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Increased phosphorylation of AKAP by inhibition of phosphatidylinositol 3-kinase enhances human sperm motility through tail recruitment of protein kinase A

¹ Andrology, Department of Physiopathology, University of Florence, Viale Pieraccini 6, I-50139 Florence, Italy
³ Endocrinology Units, Department of Physiopathology, University of Florence, Viale Pieraccini 6, I-50139 Florence, Italy
² Department of Internal Medicine, University of Florence, Viale Pieraccini 6, I-50139 Florence, Italy

View larger version (27K): [in a new window]   Fig. 1. Presence and activity of PI 3-kinase in human spermatozoa. Immunoprecipitation of (A) p85 regulatory subunit and (B) p110 catalytic subunit of PI 3-kinase from human sperm lysates and of MCF-7 or PC3 cells, respectively, as positive controls. Immunobeads obtained with anti-p85 PI 3-kinase or anti-p110 PI 3-kinase antibodies (IP) were separated by 8% SDS-PAGE and the transferred proteins were revealed by western blotting with the same antibodies used for immunoprecipitation (IB). Molecular mass markers are indicated on the right (A) and on the left (B) of the blots. (C) PI 3-kinase activity in sperm samples treated or not with 10 µM LY294002 was evaluated by in vitro assay after immunoprecipitation with anti-p85 antibody. The spots correspond to PI 3-kinase catalytic product [32P]phosphatidylinositol phosphate (PiP). Representative of four similar experiments. (D) Aliquot (1:4) of the p85 immunoprecipitates were subjected to western blot analysis with the same antibody, to ensure that protein G pulls down the same amount of the enzyme in both samples. Total lysates from PC3 cells and spermatozoa were run as positive controls.

View larger version (54K): [in a new window]   Fig. 2. Intracellular localization of PI 3-kinase in human spermatozoa. (A) Western blot analysis of tail and head protein extracts from swim-up-selected spermatozoa separated onto 8% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was first blotted with anti-p85 PI 3-kinase regulatory subunit antibody (upper panel). Anti-ERK2 antibody (lower panel) was used to check for equal lane loading after stripping and reprobing of the same membrane. MCF7 cell lysate was used as positive control. Molecular mass markers are indicated on the right of the blot. Representative of two similar experiments. (B) Immunofluorescence analysis of fixed and permeabilized human spermatozoa probed with the antibody against the anti-p85 regulatory subunit of PI 3-kinase (b). Negative control avoiding the primary antibody is shown in a, whereas the respective fields observed in light transmission microscopy are reported in c and d. Arrows indicate sperm heads. Representative of four similar experiments.

View larger version (23K): [in a new window]   Fig. 3. (A) Time-dependent effect of LY294002 on intracellular cAMP levels in swim-up-selected human spermatozoa. After different times of incubation with LY294002 (10 µM), sperm samples were washed and extracted in ethanol. Intracellular cAMP concentrations were evaluated by a RIA kit as described in Materials and Methods. Data represent means±s.e.m. from eight different experiments. *P<0.01, **P<0.001 versus control. Student's t-test for paired data. (B) Effects of dbcAMP and LY294002 on sperm motility. Swim-up-selected sperm samples were incubated for 1 hour in the presence of dbcAMP (1 mM) or LY294002 (10 µM) alone or in combination and forward and rapid motility was evaluated by CASA. Data represent means±s.e.m. from five different experiments. The percentage of sperm forward motility for swim-up-selected spermatozoa is indicated on the left ordinate, whereas the right ordinate indicates the same parameter for unselected spermatozoa. *P<0.001 versus respective controls, Student's t-test for paired data.

View larger version (43K): [in a new window]   Fig. 4. Dose- and time-dependent effects of PI 3-kinase inhibition on tyrosine phosphorylation of sperm p110 kDa protein. Western blot analysis of sperm lysates from swim-up-selected spermatozoa stimulated for 1 hour with increasing concentrations of LY294002 (LY, A), or treated with 10 µM LY294002 for the different times indicated (B) were separated by 8% SDS-PAGE. Equal amounts (30 µg) of sperm proteins were subjected to 8% SDS-PAGE. Tyrosine phosphorylated proteins were revealed with PY20-HRP antibody. C, control at 10 minutes. Molecular mass markers are indicated on the right of the blots. Arrowheads indicate the modulated band at about 110 kDa. Representative of three similar experiments.

View larger version (29K): [in a new window]   Fig. 5. Tyrosine phosphorylation of sperm p110 kDa protein mediates LY294002 stimulatory effects on motility. (A) Effects of 1 hour treatment with LY294002 (LY, 10 µM) in the presence or absence of the tyrosine kinase inhibitor erbstatin (E, 12.5 or 25 µg/ml) on both forward and rapid motility of swim-up-selected spermatozoa. Sperm forward motility and rapid motility was evaluated by CASA. Data represent means±s.e.m. of five different experiments. #P<0.01 and *P<0.001 versus respective control, °P<0.001 versus respective LY; Student's t-test for paired data. (B) Western blot analysis of sperm lysates from swim-up-selected spermatozoa stimulated for 1 hour with 10 µM LY294002 and erbstatin (E, 12.5 or 25 µg/ml) together or alone. Equal amount (30 µg) of sperm proteins were subjected to 8% SDS-PAGE. Tyrosine phosphorylated proteins were revealed in chemiluminescence with PY20-HRP antibody. Molecular mass markers are indicated on the right of the blots. Arrowhead indicates the p110 kDa protein. Representative of five different experiments.

View larger version (32K): [in a new window]   Fig. 6. LY294002-induced tyrosine phosphorylation of AKAP3. (A,B) Western blot analysis of tail protein extracts from swim-up-selected spermatozoa treated or not with 10 µM LY294002, separated on 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was first blotted with anti-AKAP3 antibody (A) and, after stripping, re-probed with anti-phosphotyrosine antibody, PY20-HRP (B). (C,D) Western blot analysis of total protein extracts from swim-up-selected spermatozoa treated or not with 10 µM LY294002. Twenty µg of sperm lysates were run in duplicate onto 8% SDS-PAGE and transferred to nitrocellulose membrane. The membrane was cut longitudinally in two parts that were probed with anti-FSP95 (C) and anti-AKAP3 (D) respectively. Molecular mass markers are indicated on the right of the blots. Representative of three similar experiments.

View larger version (44K): [in a new window]   Fig. 11. Effect of inhibition of tyrosine phosphorylation of proteins by erbstatin on functional binding of RIIß to AKAP3 in human spermatozoa. Sperm proteins from spermatozoa treated with LY294002 (LY, 10 µM) or erbstatin (E, 25 µg/ml) alone or together for 1 hour, were separated by 8% SDS-PAGE and transferred to a nitrocellulose membrane. (A) RIIß overlay assay: the membrane was incubated with purified RIIß subunit followed by probing with anti-RIIß antibody revealed by chemiluminescence. After stripping, the same membrane was subsequently probed with anti-AKAP3 (B, IB: AKAP3) and anti-FSP95 (C, IB: FSP95) antibodies. Arrowheads in A-C indicate the AKAP3, which is tyrosine phosphorylated following LY294002 incubation and which binds RIIß regulatory subunits. Anti-ERK-2 antibody was used to check for equal protein loading (D, IB:ERK-2). Molecular mass markers are indicated on the right of the blots. Representative of three similar experiments.

View larger version (15K): [in a new window]   Fig. 7. Western blot analysis of immunoprecipitated AKAP3 from spermatozoa treated or not with 10 µM LY294002. After 1-hour treatment, sperm samples were extracted in SDS buffer (see Materials and Methods). Cells lysates were immunoprecipitated (IP) using an anti-AKAP3 antibody, electrophoresed on 8% SDS, and immunoblotted with anti-phosphotyrosine antibody (IB: PY20-HRP, upper panels), stripped and re-probed with anti-AKAP3 antibody (IB: AKAP3, A, lower panel) or anti-FSP95 antibody (IB: FSP95, B, lower panel). Molecular mass markers are indicated on the right of the blots. Representative of three similar experiments.

View larger version (28K): [in a new window]   Fig. 8. Effect of LY294002 on localization of PKA regulatory and catalytic subunits in sperm tails and heads. Western blot analysis of tail and head protein extracts from swim-up-selected spermatozoa treated or not with 10 µM LY294002, separated onto 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blotted with antibodies against RIIß (A) and PKA catalytic (B) subunits. Anti-ERK2 antibody (C) was used to check for equal lane loading after stripping and re-probing of the same membrane. Jurkat cells have been used as positive controls for PKA subunits. Molecular mass markers are indicated on the right of the blots. Representative of three similar experiments.

View larger version (28K): [in a new window]   Fig. 9. Effects of Ht31 on LY294002-stimulated increase in sperm motility, RIIß localization and interaction with AKAP3 in spermatozoa. (A) Swim-up-selected spermatozoa were incubated for 1 hour in the presence of LY294002 (10 µM) with or without the active inhibitor Ht31 (10 µM) or the inactive molecule P-Ht31 (10 µM) and sperm forward and rapid motility was evaluated by CASA. Data represent means±s.e.m. of seven different experiments. *P<0.01 and **P<0.001 versus respective controls, #P<0.001 versus Ht31, Student's t-test for paired data. (B) Western blot analysis of insoluble protein extracts from swim-up-selected spermatozoa treated or not with 10 µM LY294002 in the presence or absence of 10 µM Ht31. Insoluble protein fractions separated by 8% SDS-PAGE and transferred to a nitrocellulose membrane were revealed first with anti-RIIß antibody (upper panel) and, after stripping, with anti-ERK2 antibody to check for equal lane loading (lower panel). Human endothelial cells were used as positive control. Molecular mass markers are indicated to the right of the blot. Representative of three similar experiments. (C) Effects of Ht31 and LY294002 on RIIß-AKAP3 interaction as revealed by co-immunoprecipitation of RIIß with AKAP3. Swim-up-selected spermatozoa treated with Ht31 or LY294002 alone or in combination were extracted in immunoprecipitation buffer containing 0.1% SDS and immunoprecipitated with anti-AKAP3 antibody (see Materials and Methods). After 8% SDS-PAGE of the immunobeads, proteins transferred to nitrocellulose membranes were revealed with anti-FSP95 (upper panel) or anti-RIIß (lower panel) antibodies. Molecular mass markers are indicated on the left of the blot. Representative of two similar experiments.

View larger version (35K): [in a new window]   Fig. 10. RII overlay assay (A,B) and western blot analysis (C,D) of protein extracts from swim-up-selected spermatozoa treated or not with LY294002. After treatment with 10 µM LY294002, sperm samples were lysed and proteins were separated by 8% SDS-PAGE and transferred to nitrocellulose membranes. RII overlay assay: For RII overlay assay, membranes were first incubated with purified RIIa (A) or RIIß (B) PKA regulatory subunits followed by probing with anti-RII (A) or anti-RIIß (B) antibodies. After stripping, the same membrane was subsequently subjected to western blot analysis using PY20 antibody (C) and, finally, anti-ERK2 antibody (D) to check for equal protein loading (IB: western blot analysis). Arrowheads in A-C indicate the AKAP3, which is tyrosine phosphorylated following LY294002 incubation and which binds PKA RII regulatory subunits. Molecular mass markers are indicated on the right of the blots. Representative of three similar experiments.

View larger version (34K): [in a new window]   Fig. 12. Effect of PKA inhibition by H89 on sperm forward motility of swim-up-selected spermatozoa stimulated with LY294002 and dbcAMP. Swim-up-selected sperm samples were incubated for 1 hour in the presence of dbcAMP (1 mM), LY294002 (10 µM) and H89 (50 µM) alone or in combination, and forward and rapid motility were evaluated by CASA. Data represent means±s.e.m. of five different experiments. *P<0.01 versus respective stimulus with H89 and #P<0.01 versus respective controls (C) with or without H89; P<0.01 versus C without H89; Student's t-test for paired data.

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