Abstract
The capacitation of mammalian spermatozoa involves the activation of a cAMP-mediated signal transduction pathway that drives tyrosine phosphorylation via mechanisms that are unique to this cell type. Controversy surrounds the impact of extracellular calcium on this process, with positive and negative effects being recorded in independent publications. We clearly demonstrate that the presence of calcium in the external medium decreases tyrosine phosphorylation in both human and mouse spermatozoa. Under these conditions, a rise in intracellular pH was recorded, however, this event was not responsible for the observed changes in phosphotyrosine expression. Rather, the impact of calcium on tyrosine phosphorylation in these cells was associated with an unexpected change in the intracellular availability of ATP. Thus, the ATP content of both human and mouse spermatozoa fell significantly when these cells were incubated in the presence of external calcium. Furthermore, the removal of glucose, or addition of 2-deoxyglucose, decreased ATP levels within human spermatozoon populations and induced a corresponding decline in phosphotyrosine expression. In contrast, the mitochondrial inhibitor rotenone had no effect on either ATP levels or tyrosine phosphorylation. Addition of the affinity-labeling probe 8-N3 ATP confirmed our prediction that spermatozoa have many calcium-dependent ATPases. Moreover, addition of the ATPase inhibitor thapsigargin, increased intracellular calcium levels, decreased ATP and suppressed tyrosine phosphorylation. Based on these findings, the present study indicates that extracellular calcium suppresses tyrosine phosphorylation by decreasing the availability of intracellular ATP, and not by activating tyrosine phosphatases or inhibiting tyrosine kinases as has been previously suggested.
Highlights
During the process of mammalian fertilization, spermatozoa undergo a priming process, known as capacitation, before they are capable of fertilizing the oocyte
The capacitation of mammalian spermatozoa involves the activation of a cAMP-mediated signal transduction pathway that drives tyrosine phosphorylation via mechanisms that are unique to this cell type
We clearly demonstrate that the presence of calcium in the external medium decreases tyrosine phosphorylation in both human and mouse spermatozoa
Summary
During the process of mammalian fertilization, spermatozoa undergo a priming process, known as capacitation, before they are capable of fertilizing the oocyte. Functional maturation of the spermatozoa is achieved as these cells ascend the female reproductive tract; a process that can take anywhere from 6 to 30 hours depending on the species (Chang, 1951). In the context of assisted reproduction, the capacitation of spermatozoa is performed in vitro In this procedure, freshly ejaculated or epididymal sperm are washed and incubated in a simple defined medium that mimics oviductal fluid. Freshly ejaculated or epididymal sperm are washed and incubated in a simple defined medium that mimics oviductal fluid The latter normally contains electrolytes, metabolic energy sources and a macromolecule such as serum albumin to allow for cholesterol efflux (Yanagimachi, 1969; Yanagimachi and Usui, 1974; Aitken et al, 1983; Yanagimachi and Bhattacharyya, 1988; Yanagimachi, 1994a). Biochemical changes occurring during capacitation have been reported and include a decrease in membrane cholesterol content (Aitken et al, 1983; Cross, 1998), an increase in intracellular pH (Schackmann et al, 1981; Carr and Acott, 1989; Parrish et al, 1989) changes in intracellular Ca2+ concentration (Baldi et al, 1991) and the production of reactive oxygen species (Aitken and Clarkson, 1987; Aitken and Buckingham, 1992; Aitken et al, 1992a; Aitken et al, 1992b; Aitken and Fisher, 1994; Aitken et al, 1995; De Lamirande and Gagnon, 1995; Aitken et al, 1997; Aitken et al, 1995; De Lamirande et al, 1998)
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