Signaling by cAMP-dependent protein kinase (PKA) plays an important role in the regulation of mammalian sperm motility. However, it has not been determined how PKA signaling leads to changes in motility, and specific proteins responsible for these changes have not yet been identified as PKA substrates. Anti-phospho-(Ser/Thr) PKA substrate antibodies detected a sperm protein with a relative molecular weight of 270,000 (p270), which was phosphorylated within 1 min after incubation in a medium supporting capacitation. Phosphorylation of p270 was induced by bicarbonate or a cAMP analog, but was blocked by the PKA inhibitor H-89, indicating that p270 is likely a PKA substrate in sperm. In addition, phosphorylation of p270 was inhibited by stearated peptide st-Ht31, suggesting that p270 is phosphorylated by PKA associated with an A-kinase anchoring protein (AKAP). AKAP4 is the major fibrous sheath protein of mammalian sperm and tethers regulatory subunits of PKA to localize phosphorylation events. Phosphorylation of p270 occurred in sperm lacking AKAP4, suggesting that AKAP4 is not involved directly in the phosphorylation event. Phosphorylated p270 was enriched in fractionated sperm tails and appeared to be present in multiple compartments including a detergent-resistant membrane fraction. PKA phosphorylation of p270 within 1 min of incubation under capacitation conditions suggests that this protein may have an important role in the initial signaling events that lead to the activation and subsequent hyperactivation of sperm motility.
A collaborative study was conducted to determine useful and sensitive rat sperm motion parameters in a CellSoft Series 4000 semen analyzer to detect the effects of compounds on sperm motion. The effects on the sperm motion parameters were investigated using alpha-chlorohydrin, boric acid, ethinylestradiol, ethyl methanesulfonate, nitrazepam, nitrobenzene, ornidazole, sulfasalazine or valproic acid which are well known to induce reproductive or testicular toxicities. All compounds used in this study decreased percentage of motile sperm (% motile). Curvilinear velocity (VCL), maximum and mean amplitude of lateral head displacement (ALH max and ALH mean) were decreased by treatment with all compounds except for valproic acid. Treatment with alpha-chlorohydrin, ornidazole or sulfasalazine under mid-dosage regimens decreased only these parameters. Beat cross frequency (BCF) was increased by treatment with sulfasalazine. There were some treatments which caused either decreased or increased changes irrespective of dosage regimen in linearity, average radius, percentage of circular-swimming sperm out of motile sperm (circular/motile) and percentage of circular-swimming sperm out of all sperm (circular/all). Based on these results, we concluded that % motile, VCL, ALH max and ALH mean are considered useful and sensitive parameters for evaluating the effects of compounds on sperm motion. A parameter of BCF can be useful to detect the effects of specific compounds on sperm motion. Linearity, average radius, circular/motile and circular/all are not considered useful or sensitive indicators to detect the effects of compounds on sperm motion.
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