In this study we have provided evidence that sperm lacking the A1 receptors are less responsive to capacitating effectors and that there is a significant reduction in the number of pups produced by A1R–/– male mice, although reproductive parameters, that is, number, motility, and viability, of caudal epididymal spermatozoa from A1R–/– mice do not show significant differences compared with A1R+/+ mice. Although there are data in the literature reporting the involvment of A2A receptors in sperm motility, cAMP production, and protein phosphorylation (Fraser and Duncan, 1993; Shen et al, 1993; Fenichel et al, 1996), we have previously shown that the stimulation of A1 receptor has a capacitative effect on human spermatozoa (Minelli et al, 2000; Allegrucci et al, 2001). Therefore, signaling initiated by A1 receptors in response to either synthetic or endogenous agonists plays a role in promoting the attainment of capacitative status of spermatozoa. The capacitation percentage, that is, the difference between ZP-induced AR and spontaneous AR, of A1R+/– murine sperm is only slightly and not significantly reduced compared with that of A1R+/+ sperm, whereas significant differences in the percentage of capacitation are found in A1R−/− sperm. The attainment of the maximum percentage of capacitation is also greatly delayed in mice spermatozoa lacking the A1 receptors. A1R+/+ murine spermatozoa, treated with a high dose of caffeine, need 3 times longer to reach the maximum percentage of capacitation than untreated A1R+/+, a similar length of time to that observed in A1R−/− mouse sperm. Therefore, the total blocking of A1 receptors reproduces the situation found in knockout mice. This could be relevant for understanding the possible effects of caffeine on fertility. It has been found that 15 μM caffeine, an amount seen in body fluids after human consumption of 3 to 6 cups of coffee per day, is likely to bind to and inhibit half of the adenosine receptors (Fredholm et al, 1999). The present data, showing that this dose of caffeine had the same very low effect as that observed in A1R+/– mice, is entirely compatible with the previous results. By contrast, a very high dose of caffeine, 100 μM, did cause a significant reduction in sperm capacitation. However, concentrations of caffeine of this magnitude are unlikely to be reached by caffeine consumers. Indeed, strong side effects would preclude ingestion of this amount. Hence, our data suggest that regular caffeine consumption is unlikely to significantly affect spermatozoa function. This is reassuring for coffee drinkers. Interestingly, the lower concentration of caffeine had a clearly inhibitory effect on spermatozoa from A1R+/– mice, which have a reduced number of A1 receptors. It may be that individuals with low adenosine receptor number could be more susceptible to the inhibitory effects of caffeine on male fertility.
In conclusion, we have shown that the lack of expression of A1 receptors is associated with delayed capacitation. Very high doses of caffeine, unlikely to be reached by coffee drinkers due to strong side effects, mimic the lack of expression of A1 receptors, whereas caffeine, at levels achieved by regular human consumption, appears to minimally affect sperm capacitation. Our in vivo results show that A1R–/– mice are less fertile, implying that A1 receptors, although not indispensable for the completion of capacitation, are involved in the efficiency of the process. The reduced number of offspring indicates that several signaling pathways, including those activated by adenosine A1 receptors, must be interactive and fully operative to accomplish the maximum degree of capacitation and hence fertility.