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A novel gene required for male fertility and functional CATSPER channel formation in spermatozoa.

Nature communications | Jan 11, 2011

Calcium signalling is critical for successful fertilization. In spermatozoa, capacitation, hyperactivation of motility and the acrosome reaction are all mediated by increases in intracellular Ca(2+). Cation channels of sperm proteins (CATSPERS1-4) form an alkalinization-activated Ca(2+)-selective channel required for the hyperactivated motility of spermatozoa and male fertility. Each of the CatSper1-4 genes encodes a subunit of a tetramer surrounding a Ca(2+)-selective pore, in analogy with other six-transmembrane ion channel α subunits. In addition to the pore-forming proteins, the sperm Ca(2+) channel contains auxiliary subunits, CATSPERβ and CATSPERγ. Here, we identify the Tmem146 gene product as a novel subunit, CATSPERδ, required for CATSPER channel function. We find that mice lacking the sperm tail-specific CATSPERδ are infertile and their spermatozoa lack both Ca(2+) current and hyperactivated motility. We show that CATSPERδ is an essential element of the CATSPER channel complex and propose that CATSPERδ is required for proper CATSPER channel assembly and/or transport.

Pubmed ID: 21224844 RIS Download

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Associated grants

  • Agency: Howard Hughes Medical Institute, Id: P30 HD018655
  • Agency: NICHD NIH HHS, Id: R01 HD045339
  • Agency: NICHD NIH HHS, Id: U01 HD045857
  • Agency: NICHD NIH HHS, Id:

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Recombineering Information

Recombineering (recombination-mediated genetic engineering) is a powerful method for fast and efficient construction of vectors for subsequent manipulation of the mouse genome or for use in cell culture experiments. It is also an efficient way of manipulating the bacterial genome directly. Recombineering is a method based on homologous recombination in E. Coli using recombination proteins provided from ? phage. Our bacterial strains contain a defective ? prophage inserted into the bacterial genome. The phage genes of interest, exo, bet, and gam, are transcribed from the ?PL promoter. This promoter is repressed by the temperature-sensitive repressor cI857 at 32C and derepressed (the repressor is inactive) at 42C. When bacteria containing this prophage are kept at 32C no recombination proteins are produced. However, after a brief (15 minutes) heat-shock at 42C a sufficient amount of recombination proteins are produced. exo is a 5''-3'' exonuclease that creates single-stranded overhangs on introduced linear DNA. bet protects these overhangs and assists in the subsequent recombination process. gam prevents degradation of linear DNA by inhibiting E. Coli RecBCD protein. Linear DNA (PCR product, oligo, etc.) with sufficient homology in the 5'' and 3'' ends to a target DNA molecule already present in the bacteria (plasmid, BAC, or the bacterial genome itself) can be introduced into heat-shocked and electrocompetent bacteria using electroporation. The introduced DNA will now be modified by exo and bet and undergo homologous recombination with the target molecule. The method is so efficient that co-electroporation of a supercoiled plasmid and a linear piece of DNA into heat-shocked, electrocompetent bacteria will work as well.

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