Molecular identification of Ca(2+)channels in human sperm

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> or =alpha1G> or =alpha1E> or =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.

LOW RESPONSIVENESS OF CULTURED HUMAN INTESTINAL EPITHELIAL CELLS TO LPS STIMULATION AND ITS POSSIBLE MECHANISM / 解放军医学杂志

To investigate the mechanism of low responsiveness of cultured human intestinal epithelial cells (IECs) to LPS stimulation, IL-8 secretion level was detected by ELISA in supernatants harvested after the IECs were treated with LPS for 18h, and TLR4, TLR2, CD14 and MD2 mRNA were detected by reverse transcriptase-polymerase chain reaction(RT-PCR) and RNase protection assay(RPA). It was found that IL-8 was not detectable after IECs were treated with LPS and the expression levels of TLR4, TLR2, CD14 and MD2 mRNA of IECs were low and after exposed to LPS, the expressions of TLR4, CD14 and MD2 mRNA of IECs became further lower, whereas the expression of TLR2 mRNA didn't change obviously. The result suggests that low expressions of LPS signal transduction-associated receptors on IECs may be responsible for low response of IECs to LPS, so it confirms further that TLR4, CD14 and MD2 play important roles in transmembrane LPS signal transduction.