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Expression and identification of CatSper3 channel in mouse brain and testis
Sperm specific non selective cation (CatSper) channels belong to the CatSper family of genes and are expressed only in sperm and testis. In general, gene expression profiles in the brains of humans and mice share the highest similarity with those in testis. Therefore, to identify whether CatSper genes are expressed in the mouse brain, we performed reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. RT-PCR detected all four CatSper mRNAs in both the testis and the brain, with CatSper3 being the most highly expressed. Consistent with RT-PCR data, Western blot analysis showed that CatSper3 was expressed in the brain. We cloned CatSper3 variant 2 from eight-week mouse brain. We named the gene as CatSper3 variant 1 (V1) because the mouse CatSper3 is orthologous to human CatSper3, and another mouse CatSper3 variant (variant 2, V2) with truncated second transmembrane helix was identified. The open reading frame of mCatSper3 V1 consists of 1185 nucleotides and encodes a putative 395-amino acid protein. At the amino acid level, CatSper3 isolated from brain is 100 and 64.8% identical to that isolated from mouse testis and human CatSper3, respectively. Based on comparison between the mCatSper3 V1 ORF and mCatSper3 V2 using TopPred software, the alignment of amino acid sequences shows that the differences exist mainly in segment 2. The CatSper3 transcript consists of eight exons and seven introns, and alternative splice is present within the third exon. In HT22 cell, a mouse hippocampal neuronal cell line, H2O2-induced changes in CatSper3 expression were studied. H2O2 dramatically increased CatSper3 expression in HT22 cells in a dose-and time-dependent manner. The H2O2-induced increase in CatSper3 expression was offset by the addition of N-acetylcysteine (NAC), which is an antioxidant. Taken together, these data strongly indicate that CatSper3 is expressed in mouse brain as variant 1 and suggest that CatSper3 could be a potential target for the modulation of ROS.
