Molecular identification of Ca2+ channels in human sperm

The acrosome reaction is a Ca2+-dependent exocytotic process that is a prerequisite step for fertilization. External calcium entry through voltage-activated Ca2+ channels is known to be essential in inducing the acrosome reaction of mammalian spermatozoa. Due to their complex geometry, however, electrophysiological identification of sperm Ca2+ channels has been limited. Here we identified Ca2+ channel mRNAs expressed in motile human sperm using RT-PCR and their levels were compared using RNase protection assays. L-type, non-L-type, and T-type Ca2+ channel mRNAs were detected by RT-PCR using degenerate primers. Cloning and sequencing of the PCR products revealed alpha1B, alpha1C, alpha1E, alpha1G, and alpha1H sequences. RT-PCR using specific primers repeatedly detected alpha1B, alpha1C, alpha1E, alpha1G, and alpha1H mRNAs, and additionally alpha1I mRNA. But alpha1A and alpha1D messages were not detected. Relative expression levels of the detected Ca2+ channel subtypes were compared by RNase protection assays. The abundance of detected mRNA messages was in the following order: alpha1H greater than or equal to alpha1G greater than or equal to alpha1E greater than or equal to alpha1B > alpha1C > alpha1I. These findings indicated that human motile sperm express multiple voltage-activated Ca2+ channel RNAs among which T-type and non-L-type channel messages are likely to be predominantly expressed. Based on their relative expression levels, we propose that not only T-type but also non-L-type calcium channels may be major gates for the external calcium influx, required for the acrosome reaction.