Ca2+ stores and hyperactivation of human sperm. (360G-Wellcome-086470_Z_08_Z)

The nature, location and regulation of Ca2+-release channels in the store situated at the neck/midpiece of human sperm will be established by a combination of immunolocalisation, radioligand binding and experimental/pharmacological manipulation of cells in which this store has been labelled with magfluo-4. High speed, high-sensitivity fluorescent imaging will be used to monitor Ca2+ mobilisation, Ca2+ signal spread, store mobilisation and flagellar activity in loosely-tethered cells stimulated by ryanodine receptor agonists and inducers of hyperactivation, including 4-aminopyridine (an extremely effective agonist previously characterised in this laboratory). We will then extend the imaging work to investigate signal propagation between the sperm head/midpiece and flagellum using localised uncaging of Ca2+ and Ca2+-buffers. In particular we will investigate the response of the neck/midpiece Ca2+ store to activation of CatSpers (sperm-specific, plasma membrane Ca2+ channels) in the f lagellum and the effects on flagellar [Ca2+]i of store mobilisation, thus elucidating the interaction between these two primary regulators of hyperactivation. To investigate the clinical significance of our findings we will screen sperm donated by patients with motility-related sub-fertility for responsiveness to (i) progesterone, (ii) 4-aminopyridine and (iii) elevation of pHi (to activate CatSpers) and we will assess functional characteristics of the sperm neck/midpiece store