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 ( [Ca 2⍣ ] i ) in pentoxifyllinestimulated 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/10 6 spermatozoa; mean ⍨ SEM, n ⍧ 6) after 15 min, although by 3 h, cAMP values were similar (503-531 fmoles/10 6 spermatozoa). cGMP values (~27 fmoles/10 6 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 -/ HCO 3 -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 A 2 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 Ca 2ϩ , 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 A 2 and protein kinase C activity.

Cryopreservation of sperm ofXenopus laevis andXenopus tropicalis

“…Chicken egg yolk was included in the cryoprotectant, as in other protocols (Holt, 2000a), because it significantly improved viability of X. laevis sperm after a freeze-thaw cycle. Agents that raise intracellular cyclic AMP are said to improve fertilisation of mammalian sperm, including bicarbonate ions (Visconti et al, 1999) and pentoxyfylline, a phosphodiesterase inhibitor of the xanthine family (Ain et al, 1999;Esteves et al, 1998;Gradil and Ball, 2000;Stanic et al, 2002). Sodium bicarbonate was mildly beneficial in maintaining motility after cryopreservation of X. laevis sperm.…”

Localizations of intracellular calcium and Ca2+-ATPase in hamster spermatogenic cells and spermatozoa

“…Sperm concentration was determined using a hemocytometer to adjust to 4 3 10 5 sperm/mL. Suspensions were incubated in 200 lL droplets (modified Tyrode's medium plus 4 mg/mL BSA) under paraffin oil at 378C in a moist atmosphere of 5% CO 2 for up to 4 h. These conditions are known to induce capacitation and the acrosome reaction (Ain et al, 1999).…”

Methylxanthines and Reproduction

“…Hence, the data suggest that regular caffeine consumption is unlikely to significantly affect spermatozoa function, thereby reassuring all coffee drinkers of the lack of negative effects of caffeine on male fertility. The scientific literature dealing with the AR contains references to the use of methylxanthines as PDE inhibitors (Kopf et al 1983a, b;Carr and Acott 1990;Ain et al 1999;Lachance et al 2007) that maintain intracellular cAMP levels and induce the AR.…”