Ram spermatozoa produce inositol 1,4,5-trisphosphate but not inositol 1,3,4,5-tetrakisphosphate during the Ca2+/ionophore-induced acrosome reaction.

An investigation was made of the production of inositol tris- and tetrakisphosphates concomitant with ionophore-stimulated breakdown of PtdIns(4,5)P2 in ram spermatozoa. As spermatozoa displayed very low rates of incorporation of [3H]inositol into their phosphoinositides, the studies were carried out using 32P-labelled cells. Using a specially developed procedure, inositol tris- and tetrakisphosphates were isolated, free of labelled ATP and P(i); they were then separated from each other (and from other minor labelled compounds) and analysed, using ionophoresis and HPLC. Levels of 32P-labelled material with the chromatographic characteristics of Ins(1,4,5)P3 were very low in untreated cells, but rose sharply with ionophore treatment, in parallel with rapid PtdInsP2 breakdown. No 32P-labelled material with the characteristics of Ins(1,3,4,5)P4 or Ins(1,3,4)P3 was found, and there was no evidence for phosphorylation of Ins(1,4,5)P3 in sperm homogenates. The implications of our findings are discussed with respect to the physiological modulation of Ca2+ influx that is required to initiate the acrosome reaction at fertilization.