Zona pellucida agonists activate phosphoinositide signaling pathways in sperm. (A) Examples of the early effects of ZP3 on sperm phosphoinositides are shown. ZP3 (5 μg/ml) stimulated labeling of PI(3,4,5)P3 but not of PI(3)P. Enhanced labeling was inhibited by 100 nM wortmannin. Relative migration of sperm phosphoinositides and other phospholipids were identified using purified standards. Abbreviations: PC, phosphatidylcholine; PA, phosphatidic acid; phosphatidylinositides were defined previously. The spot migrating beneath PI(3)P could not be identified but did not change during treatment with ZPse or ZP3. (B) Time courses of the effects of ZP3 or of ZPse (5 and 20 μg/ml, respectively) on sperm phosphoinositides are shown. Data (mean ± SD, 7-10 experiments) were obtained from chromatograms of response time courses, similar to those in panel A, and expressed as the ratio of labeling at the indicated time, P_n, to the initial activity prior to addition of agonist or control proteins, P_i. ZP_se and ZP3 had similar effects and results are pooled for presentation (ZP3 and ZP_se, ). PI3 kinase antagonists (100 nM wortmannin and 10 uM LY294002) had similar inhibitory effects on the ZP3/ZP_se-evoked responses and data are pooled for presentation (). Similarly, control proteins (10 ug/ml ZP1 or ZP2; 20 ug/ml BSA, a1-acid glycoprotein) failed to evoke a phosphoinositide response and these results are pooled for presentation ().

PI3 kinase and PTEN distribution in mouse sperm. (A) Surface domains of mouse sperm. (B, C) Immunolocalization of PI3 kinase catalytic and regulatory subunits and of PTEN. For each panel >100 sperm were examined. A typical labeling pattern is illustrated in (B) and results are summarized in (C). (B) Panel shows paired phase contrast and fluorescence images (a, b; scale bar, 10 μm) and an enlarged fluorescence image of the sperm head (c; scale bar, 5 μm). Arrows facilitate orientation. Immunoblots of PI3 kinase catalytic and regulatory subunits, of PTEN, and of a control (no primary antibody). (C) Summary of immunolocalization results. Class 1A (catalytic subunits: p110α, -δ; a regulatory subunit, p85α) and IB (catalytic subunit, p110γ) PI3 kinase isoforms are detected in sperm and specifically in the acrosomal crescent region, as is PTEN. A third class IA PI3 kinase catalytic subunit (p110β) was not detected. ++, strong labeling; +, weak labeling; blank, no labeling detected.

Role of Akt in the ZP3-evoked acrosome reaction. (A) Akt is present in the acrosomal crescent, posterior head, and flagellum (total Akt). Treatment with ZP_se or ZP3 (5 and 20 ug/ml, respectively; data pooled for presentation) resulted in enhanced T³⁰⁸ phosphorylation of Akt. Paired phase contrast and fluorescence images (a,b; scale bar, 10 μm), and magnified fluorescence images (c; scale bar, 5 μm) are shown. Arrows facilitate orientation. Data are quantified in panel B. (B) Effects of ZP_se and ZP3 total and active Akt. Pixel intensities were integrated across the sperm head (mean ± SD, triplicate experiments with >25 sperm observed/group in each experiment). ZP3/ZP_se treatment produced a 2-3-fold increase in pT³⁰⁸-Akt and this was reduced to basal levels by 100 nM wortmannin. During these treatments there were no significant changes in the total Akt levels. (C) Akt inhibitors block the ZP3/ZP_se-induced acrosome reaction. Dashed lines indicate acrosome reaction levels in buffer-treated controls and in ZP3/ZP_se-treated samples. Data represent the mean (± SD) of 3-4 separate experiments (>200 sperm observed/group in each experiment).

Effects of PI3 kinase inhibitors on the zona pellucida-induced acrosome reaction and capacitation. (A) Acrosome reactions are triggered by ZP_se (20 μg/ml) or by ZP3 (5 μg/ml) and these were inhibited by PI3 kinase antagonists (100 nM wortmannin, 10 uM LY294002). Dashed lines indicate acrosome reaction levels in sperm suspensions treated with buffer and with ZP_se. The maximum exocytotic response of the preparation is evoked by the Ca²⁺-conducting ionophore (10 μM ionomycin). Data represents mean (± SD) of 3-5 separate experiments with >200 sperm assessed in each experiment. (B) Distribution of tyrosine phosphorylated proteins in sperm. Sperm were incubated 75 min in a capacitating medium. Wortmannin or solvent were added for the final 15 min.

Effects of a D3-phosphoinositide regulation on acrosome reactions. (A) D3 phosphinositide kinase/phosphatase cycle, illustrating the targets of the PI3 kinase activator, 740 Y-P, and the lipid phosphatase inhibitor, pbV(pic). (B) 740 Y-P stimulated acrosome reactions in the absence of ZP3/ZP_se and exocytosis was inhibited by 100 nM wortmannin. 740 Y-P effects were quantitatively similar to and were not additive with those of ZP_se. Inset: 740 Y-P treatment also enhanced ³²P-labeling of PI(3,4,5)P3 as determined by thin layer chromatography. (C) pbV(pic) produced increased ³²P-labeling of PI(3,4,5)P3 (inset) and induced wortmannin-sensitive acrosome reactions. (D) bpV(pic) accelerates the time course of the ZP_se-induced acrosome reaction: , bpV(pic); , ZP3/ZP_se; , pbV(pic) + ZP3/ZP_se. (B-D) Data represents the mean (± SD) of 5-7 separate experiments with >300 sperm assessed/experiment. Dashed lines in B-C represent minimum acrosome reaction levels in buffer-treated sperm and maximal signals in ZP3/ZP_se-treated sperm, respectively. Dashed line in D represents the half-maximal acrosome reaction response. ZP3 and ZP_se doses were 5 ug/ml and 20 ug/ml, respectively.

PI3 kinase effectors, PDK1 and PKCζ, in mouse sperm. (A) PDK1 is present in the acrosomal crescent and in the flagellum. (B) PKCζ in restricted to the acrosomal crescent. (A, B) Panels show paired phase contrast and fluorescence images (scale bar, 10 μm), and a magnified fluorescence image (scale bar, 5 μm). Arrows facilitate orientation. Images are representative of 50-100 sperm observed. (C) Effects of PKC isoform-specific pseudosubstrate antagonists on the ZP_se- or ZP3-evoked acrosome reaction (20 and 5 ug/ml, respectively). Myristolated (myr-) inhibitors directed against ζ, α/β, and ε isoforms were tested, as well as a myr-scrambled (scr-) PKCζ sequence and a non-myristolated PKCζ reagent. All inhibitors were used at 50 uM. Data represents the mean (± SD) of 3-5 separate experiments, with >300 sperm assessed/experiment. Dashed lines represent the levels of acrosome reaction in sperm treated with buffer or with ZP_se or ZP3 in the absence of inhibitors and represent minimal and maximal responses, respectively. ZP_se and ZP3 data are pooled.

Model of phosphoinositide signaling during the ZP3-evoked acrosome reaction. PI(3,4,5)P3 levels are regulated by a constitutively active D3-phosphorylation/dephosphorylation cycle. ZP3 stimulation during sperm contact with the zona pellucida drives this cycle towards enhanced production of PI(3,4,5)P3. PI(3,4,5)P3 provides binding sites for PDK1 and facilitates it’s activation of the downstream targets, Akt and PKCζ. These downstream effectors are both required to drive sperm exocytosis.