Pentoxifylline-stimulated capacitation and acrosome reaction in hamster spermatozoa: involvement of intracellular signalling molecules

Abstract

We investigated the role of cAMP/cGMP, protein kinases and intracellular calcium ( [Ca²⁺]_i) in pentoxifylline-stimulated hamster sperm capacitation and the acrosome reaction (AR) in vitro. Treatment with pentoxifylline (0.45 mM) initially increased sperm cAMP values 2.8-fold, compared with untreated controls (396 ± 9.2 versus 141 ± 6.0 fmoles/10⁶ spermatozoa; mean ± SEM, n = 6) after 15 min, although by 3 h, cAMP values were similar (503-531 fmoles/10⁶ spermatozoa). cGMP values (~27 fmoles/10⁶ spermatozoa) were the same in treated and control spermatozoa. Both sperm capacitation and the AR, determined from the absence of an acrosomal cap, were stimulated by pentoxifylline; these were almost completely inhibited by a Cl^-/ HCO₃^- antiporter inhibitor (4,4-diisothiocyanato-stilbene-2,2 disulphonic acid; 1 mM) defined from the degree of sperm motility and by a protein kinase A inhibitor (H89; 10 μM). A protein kinase C inhibitor (staurosporine, 1 nM) did not affect pentoxifylline-stimulated capacitation but inhibited the AR by 50%. A protein tyrosine kinase inhibitor (tyrphostin A-47, 0.1 mM) had no effect on either pentoxifylline-stimulated capacitation or AR. A phospholipase A₂ inhibitor (aristolochic acid, 0.4 mM) markedly inhibited the pentoxifylline-stimulated AR but not capacitation. When intracellular sperm calcium [Ca^2±]_i was measured using fura-2-AM, there was an early rise (271 nM at 0.5 h) in pentoxifylline-treated spermatozoa; this appeared to be due to intracellular mobilization rather than to uptake. In the absence of extracellular Ca²⁺, sperm motility was maintained in the presence of pentoxifylline, but capacitation did not occur; spermatozoa exhibited a low level of hyperactivated motility and had a poor rate of AR (20.5 ± 2.3%). These results suggest that: (i) the pentoxifylline-stimulated early onset of sperm capacitation may be mediated by an early rise in cAMP and [Ca^2±]_i and involves protein kinase A activity; and (ii) pentoxifylline-stimulated AR may require phospholipase A₂ and protein kinase C activity.