Src family kinases-mediated negative regulation of sperm acrosome reaction in chickens (Gallus gallus domesticus)

Abstract

The acrosome reaction (AR) is a strictly-regulated, synchronous exocytosis that is required for sperm to penetrate ova. This all-or-nothing process occurs only once in the sperm lifecycle through a sequence of signaling pathways. Spontaneous, premature AR therefore compromises fertilization potential. Although protein kinase A (PKA) pathways play a central role in AR across species, the signaling network used for AR induction is poorly understood in birds. Mechanistic studies of mammalian sperm AR demonstrate that PKA activity is downstreamly regulated by Src family kinases (SFKs). Using SFK inhibitors, our study shows that in chicken sperm, SFKs play a role in the regulation of PKA activity and spontaneous AR without affecting motility. Furthermore, we examined the nature of SFK phosphorylation using PKA and protein tyrosine phosphatase inhibitors, which demonstrated that unlike in mammals, SFK phosphorylation in birds does not occur downstream of PKA and is primarily regulated by calcium-dependent tyrosine phosphatase activity. Functional characterization of SFKs in chicken sperm showed that SFK activation modulates the membrane potential and plays a role in inhibiting spontaneous AR. Employing biochemical isolation, we also found that membrane rafts are involved in the regulation of SFK phosphorylation. This study demonstrates a unique mechanism for regulating AR induction inherent to avian sperm that ensure fertilization potential despite prolonged storage.

Fig 1. Localization of c-Src and p-SFK-Y416 in chicken sperm.

c-Src was immunodetected at a predicted molecular weight (A). Sperm acrosomes (arrows) were stained by FITC-PNA (B and C). c-Src was localized to the sperm head except for the acrosomal region (B). Similarly, p-SFK-Y416 was localized to the sperm head (C). Acrosomal localization was seen in some sperm. No signals were observed in control sperm (B and C). Images are representative of three replicate trials.

Fig 2. Regulation of PKA substrate protein phosphorylation by SFKs in chicken sperm.

Sperm were incubated in TES-NaCl + 2mM Ca²⁺ for 1 hour with 0–100 μM SFK inhibitors (SKI606 and SU6656) and then subjected to immunoblotting to obtain a phosphorylation profile of SFK-Y416 and PKA substrate proteins. No change was observed in p-SFK-Y416 (A) and p-PKA substrate protein (B) in the incubation period. Downregulation of p-SFK-Y416 in response to SFK inhibition by SKI606 (C) and SU6656 (D). SKI606 and SU6656 reduced p-SFK-Y416 (C and D) but increased p-PKA substrate proteins (E, F, G, and H). All immunoblotting analyses were repeated at least four times. Data are presented as mean ± SEM. ^adP < 0.05.

Fig 3. Mechanism of SFK phosphorylation and negative regulation of PKA in chicken sperm.

Sperm were incubated with 0–50 μM H-89 (PKA inhibitor), 25 μM KH7 (sAC inhibitor), 50 μM MDL (tmAC inhibitor), 0–250 μM PTP inhibitor, or EGTA-AM in the presence or absence of SKI606 and SU6656 and then subjected to immunodetection for p-SFK-Y416 or p-PKAs. Treatment with H89, KH7, and MDL did not change p-SFK-Y416 despite the reduction of p-PKAs (A and B). EGTA-AM treatment increased p-SFK-Y416 regardless of the presence of SKI606 and SU6656 (C and D), concomitant with abrogated increases in p-PKAs by SFK inhibition (E and F). Treatment with PTP inhibitor concurrently increased p-SFK-Y416 and decreased p-PKAs (G and H). All immunoblotting analyses are representative of four replicate trials. Data are presented as mean ± SEM. ^abP < 0.05.

Fig 4. The roles of SFK in the regulation of chicken sperm AR.

Sperm were incubated in TES-NaCl + 2mM Ca²⁺ with 0–100 μM SKI606 and SU6656 for 1 h, and subjected to evaluation of spontaneous and physiological AR (IPVL− and IPVL+). Both SFK inhibitors stimulated both AR types in a dose-dependent manner (A and B). Fluo 3-AM loaded sperm were treated with SFK inhibitors and subjected to [Ca²⁺]i measurements. [Ca²⁺]i increased in response to SKI606 (C) and SU6656 (D) treatments. Sperm were incubated with 0–100 μM SFK inhibitors and then treated with DiSBAC₂(3) for membrane polarization assay. Both SKI606 and SU6656 treatment induced hyperpolarization potential (E and F) representive from a dose-dependent reduction of fluroscent intensity. Sperm were loaded with 0, 10, or 100 μM EGTA-AM, incubated for 45 min with or without 1 μM SFK inhibitors, and examined with spontaneous AR. EGTA-AM treatment abrogated both SKI606- and SU6656-stimulated spontaneous AR (G and H). Data are presented as mean ± SEM. ^agP < 0.05.

Fig 5. The relationship between membrane hyperpolarization and spontaneous AR.

Sperm were incubated with 0 or 1 μM valinomycin, a K⁺ ionophore, for 60 min in TES-NaCl + 2mM Ca²⁺, and subjected to membrane polarization assay or evaluation of spontaneous and IPVL-induced AR. Valinomycin treatment induced hyperpolarizing potential (A) and elevation of spontaneous AR (B). Sperm were incubated for 5 and 60 min with 0 or 1 μM valinomycin. p-PKAs reduced in response to valinomycin treatment despite of no change in p-SFKs (C). All immunoblotting analyses are performed in triplicates. Data are presented as mean ± SEM. ^acP < 0.05.

Fig 6. Membrane rafts-mediated regulation of SFK phosphorylation.

Sperm membranes were separated into insoluble and soluble membranes (Raft and non-rafts) to TX-100 treatment. Immunoblots for p-SFK-Y416 and c-Src revealed their association with membrane rafts (A and B). Sperm were incubated with 0 or 1 mM 2-OHCD, and then immunoblotted for c-Src and p-SFK-Y416. Increases in p-SFK-Y416 were observed in response to 1 mM 2-OHCD treatment (D) despite no differences in c-Src (C). All immunoblotting analyses are representative of four replicate trials. Data are presented as mean ± SEM. ^abP < 0.05.

Fig 7. Schematic model of SFK-mediated regulation of sperm AR via PKA pathway.

The plasma membrane of sperm contains multiple membrane rafts where SFK are spatially and functionally associated. SFK phosphorylation/dephosphorylation are regulated by an equilibrium between C-terminal Src kinase (CSK) and Ca²⁺-dependent PTP. SFK phosphorylation/dephosphorylation is involved in the regulation of PKA activity and spontaneous AR through modulation of membrane potential.