The interaction of cAMP with modeled bull sperm

Abstract

Demembranated and membrane disrupted bull sperm models exhibit an increase in motility when exposed to cAMP. Tritium-labeled cAMP was used to locate the initial site of action of cAMP in the modeled sperm preparations. cAMP did not bind selectively to the modeled cells, and the presence or absence of plasma membrane fragments on the models did not significantly alter this result. When suspension medium taken from modeled sperm preparations was subjected to gel filtration on Sephadex G25-150 columns, cAMP bound to a high molecular weight component that eluted with the void volume. The responsible binding factor is a soluble component that is released when the plasma membranes of the sperm are disrupted during the modeling procedure. To test the importance of the cAMP binding factor, modeled sperm were centrifuged, the supernatant solution was decanted, and the cells were resuspended in fresh medium. After this treatment the cells could be restored to motility with Mg-ATP but no longer exhibited a response to cAMP. Furthermore, addition of cAMP binding factor isolated by gel filtration partially restored the response of these sperm to cAMP. Investigation of the properties of the cAMP-binding factor have confirmed that it is specific for cAMP, with a much lower affinity for AMP and cGMP. In the presence of a large excess of unlabeled cAMP the labeled complex has a half-life of approximately 1 hour. Our results indicate that the action of cAMP on the motility of modeled sperm is mediated by its attachment to a high molecular weight, soluble component of the cell cytoplasm.

Regulation of Mammalian Sperm Motility

“…The binding of cAMP to its regulatory subunit in bull sperm is competitively inhibited by cGMP, indicating both interact at the same binding site [96]. cAMP and cGMP do not appear to have identical effects on the Ca2' -mediated curvature response in rat sperm flagella [92], which is consistent with the Ca2' response of Paramecium cilia [15].…”

“…A soluble 56 kilodalton phosphoprotein called axokinin seems to play a key role in mediating the cAMP response [147,1481. Consistently, removal of the cytosol from sperm models renders the sperm incapable of responding to cAMP [96]. This suggests that some component of the cytosol, either axokinin or the 49 kilodalton Type I regulatory subunit of CAMP-dependent kinase (RI) that is removed by detergent extraction [76], is required for the cAMP response.…”

Stimulation of bovine sperm motility and respiration by the triazine dye cibacron blue F3GA

“…Similar patterns have been observed in bull (Cascieri et al, 1976), hamster (Morton et al, 1974), and guinea pig (Mukica et al, 1991) sperm in which quiescent epididymal sperm were stimulated by dilution. Furthermore, Lindemann et al (1983) found that theophylline treatment prior to demembranation and motility reactivation reduced the sperms' responsiveness to CAMP, suggesting that CAMP-dependent phosphorylation of motility-associated proteins was not necessary. However, the amount of free CAMPdependent protein kinase regulatory subunits was no different in control or theophylline-treated cells, indicating that the activation of CAMP-dependent protein kinases, like the pulse of cAMP produced by CB-V treatment, is quite shortlived.…”

“…Demembranation-reactivation experiments (Lindemann et al, 1983;Yeung, 1984) have firmly established the requirement for cAMP in mammalian sperm motility initiation and maintenance. The abundance of protein kinase A and a wide variety of phosphorylated axonemal proteins, as well as an apparent constitutive cAMP production, provide the basic elements for the control of motility through CAMP-dependent protein phosphorylation.…”

“…Although the mechanisms by which calcium affects motility are not clear, possible targets are the calcium-sensitive adenylyl cyclase (Gross et al, 1987), cyclic nucleotide phosphodiesterase (Wasco and Orr, 1984;Chaudry and Casillas, 19881, and protein phosphatase (Tash et al, 1988), all of which are also associated with the CAMPsensitive regulatory system. Demembranation-reactivation experiments (Lindemann et al, 1983;Yeung, 1984) have firmly established the requirement for cAMP in mammalian sperm motility initiation and maintenance. The abundance of protein kinase A and a wide variety of phosphorylated axonemal proteins, as well as an apparent constitutive cAMP production, provide the basic elements for the control of motility through CAMP-dependent protein phosphorylation.…”