Membrane Regulation of Protein Kinase Signaling Pathways in Chicken Sperm Function

概要

Both transcriptionally and translationally inactive sperm need preassembled pathways into specific cellular compartments to function. Although initiation of the acrosome reaction (AR) involves several signaling pathways including protein kinase A (PKA) activation, how these are regulated remains poorly understood in avian sperm. Membrane rafts (MR) are specific membrane regions enriched in sterols and functional proteins and play important roles in diverse cellular processes, including signal transduction. Our recent studies on chicken sperm demonstrated that MR exist and play roles in multistage fertilization. These, combined with the functional importance of membrane rafts in mammalian sperm AR, prompted us to investigate the roles of membrane rafts in signaling pathways leading to AR in chicken sperm.

As described in chapter 2, using 2-hydroxypropyl-β- cyclodextrin (2-OHCD), we found that the disruption of MR inhibits PKA activity and AR without affecting protein tyrosine phosphorylation; however, these inhibitions were abolished in the presence of a cyclic 3,5- adenosine monophosphate (cAMP) analog. In addition, biochemical experiments showed a decrease in cAMP content in 2-OHCD-treated sperm, suggesting the involvement of soluble adenylyl cyclase (sAC) and transmembrane adenylyl cyclase (tmAC). Pharmacological experiments, combined with transcriptome analysis, showed that sAC and tmAC are present and involved in AR induction in chicken sperm. Furthermore, stimulation of both isoforms reversed the inhibition of PKA activity and AR in 2-OHCD-treated sperm suggested that AC/cAMP/PKA pathway involvement of sperm AR in chickens.

Chapter 3 describes, the involvement of src family tyrosine kinases (SFK) in the regulation of sperm AR in chickens. Our previous study reported in avian sperm that the MR regulate AR induction via activation of cAMP/PKA pathway. However, the molecular mechanism connecting PKA activation to AR induction is not understood yet. It has been shown in mammalian sperm that phosphorylation of SFK is a downstream of PKA activation, consequently leading to elevation of acrosomal responsiveness. These encouraged us to characterize the functional roles of SFK in chicken sperm AR. Immunoblots for c-src and phosphorylated form of SFK (p-SFK-Y416) showed their expression at the predicted molecular weight (60kDa). Localization experiments found the presence of c-src and p-SFK-Y416 in the sperm head with multiple focal enrichments. Using two specific SFK inhibitors (SKI606 and SU6656), we found that SFK inhibition stimulated [Ca2+]i level, PKA activity and AR induction although no difference was seen in motility. Further, SFK inhibition hyperpolarized the sperm membrane. To better understand the relationship between PKA and SFK, we examined change in p-SFK under supplementation of PKA inhibitor, sAC inhibitor and tmAC inhibitor, demonstrating that SFK phosphorylation is not downstream of PKA. To seek regulation mechanism for SFK phosphorylation, changes in p-SFK in response to MR disruption was examined using 2OHCD, and showed the involvement of MR in the regulation of SFK phosphorylation. Taken together, our results demonstrated that SFK is involved in AR induction by regulating PKA pathway in avian sperm.

Chapter 4 describes, the functional and molecular changes occurs in sperm in response to the inner perivitelline layer (IPVL) treatment in-vitro. Although IPVL treatment did not affect PKA substrate protein phosphorylation, [Ca2+]i level was significantly increased in response to IPVL treatment, suggesting a calcium dependent signaling pathway(s) might be leading to AR at sperm-IPVL interaction in birds. Further, we found that progressive velocity (VSL), path velocity (VAP) and amplitude of lateral head displacement (ALH) motility parameters were significantly improved while there were no significant differences observed in motile sperm percentage, curvilinear velocity (VCL), linearity (LIN), straightness (STR) and beat cross frequency (BCF) in response to IPVL treatment. In conclusion, our results demonstrated for the first time that sperm-IPVL interaction makes significant Ca2+ influx which might lead to induce sperm AR and motility parameters in birds.

In conclusion, our results demonstrated for the first time that the MR and its associated kinases play an important role in chicken sperm AR induction by regulating membrane polarization, [Ca2+]i kinetics and PKA pathway. Further, IPVL might be involved in an initiation of Ca2+ dependent signaling pathway(s) to induce sperm AR in birds. Together, these findings will provide a mechanistic insight into membrane regulation of sperm function in birds and accumulated results will future contribute to the development of reproductive technologies for animal production, species conservation and population control programs in avian species.