Abstract
The acrosome reaction is a Ca(2+)-dependent exocytotic process that is a prerequisite step for fertilization. External calcium entry through voltage-activated Ca(2+) channels is known to be essential in inducing the acrosome reaction of mammalian spermatozoa. Due to their complex geometry, however, electrophysiological identification of sperm Ca(2+) channels has been limited. Here we identified Ca(2+) 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 Ca(2+) 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 Ca(2+) channel subtypes were compared by RNase protection assays. The abundance of detected mRNA messages was in the following order: alpha1H alpha1G alpha1E alpha1B alpha1C alpha1I. These findings indicated that human motile sperm express multiple voltage-activated Ca(2+) 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.
Highlights
The acrosome reaction (AR) is an obligatory event happening prior to sperm penetration into eggs
Some pharmacological studies have suggested that L-type channels were involved in the AR, which was supported by direct detections of α1C mRNA messages from human and rodent sperm by Reverse transcription-polymerase chain reaction (RT-PCR) (Florman et al, 1992; Goodwin et al, 1997; Goodwin et al, 2000)
We made an attempt to identify voltage-activated Ca2+ channels (VACCs) mRNA subtypes in human spermatozoa and compare their relative expression levels using the combined tools of RT-PCR and RNase protection assays
Summary
The acrosome reaction (AR) is an obligatory event happening prior to sperm penetration into eggs. In the process of sperm contacting eggs, chemical signals such as ZP3 from zona pellucida of eggs were identified to induce increases of pH and membrane depolarization, subsequently coupled with calcium influx through activated VACCs in the sperm membrane (Babcock and Pfeiffer, 1987; Foresta et al, 1993) Due to their pivotal involvement in the AR, VACCs in mammalian sperm have been extensively explored using pharmacological, electrophysiological, or molecular-biological tools. In contrast to the published reports addressing possible roles of high voltage-activated (HVA) Ca2+ channels, direct electrophysiological recordings of Ca2+ currents in mouse spermatogenic cells displayed typical low voltage-activated (LVA) T-type Ca2+ currents, characterized by low voltage activation, slow deactivation, and fast activation and inactivation kinetics, suggesting that T-type channels might be major Ca2+ entry pathways in the spermatogenic cells (Livano et al, 1996; Santi et al, 1996; Arnoult et al, 1998; 1999) Due to their complex geometry, electrophysiological characterization of VACCs in mammalian mo-. We made an attempt to identify VACC mRNA subtypes in human spermatozoa and compare their relative expression levels using the combined tools of RT-PCR and RNase protection assays
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