Abstract
The relative importance of plasma membrane Ca2+-ATPase (PMCA) 1 and PMCA4 was assessed in mice carrying null mutations in their genes (Atp2b1 and Atp2b4). Loss of both copies of the gene encoding PMCA1 caused embryolethality, whereas heterozygous mutants had no overt disease phenotype. Despite widespread and abundant expression of PMCA4, PMCA4 null (Pmca4-/-) mutants exhibited no embryolethality and appeared outwardly normal. Loss of PMCA4 impaired phasic contractions and caused apoptosis in portal vein smooth muscle in vitro; however, this phenotype was dependent on the mouse strain being employed. Pmca4-/- mice on a Black Swiss background did not exhibit the phenotype unless they also carried a null mutation in one copy of the Pmca1 gene. Pmca4-/- male mice were infertile but had normal spermatogenesis and mating behavior. Pmca4-/- sperm that had not undergone capacitation exhibited normal motility but could not achieve hyperactivated motility needed to traverse the female genital tract. Ultrastructure of the motility apparatus in Pmca4-/- sperm tails was normal, but an increased incidence of mitochondrial condensation indicated Ca2+ overload. Immunoblotting and immunohistochemistry showed that PMCA4 is the most abundant isoform in testis and sperm and that it is localized to the principle piece of the sperm tail, which is also the location of the major Ca2+ channel (CatSper) required for sperm motility. These results are consistent with an essential housekeeping or developmental function for PMCA1, but not PMCA4, and show that PMCA4 expression in the principle piece of the sperm tail is essential for hyperactivated motility and male fertility.
Highlights
Ʈ To whom correspondence should be addressed: Dept. of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0524
If there had been any cell types in which PMCA4 was essential for cell viability, it would have indicated that any Ca2ϩ extrusion mediated by PMCA1 or other isoforms in those cells was not sufficient to maintain viability and that PMCA4 served an essential housekeeping role
In contrast to PMCA4 null mutants, mice lacking PMCA1, which is expressed throughout the embryo [22], did not survive to birth, thereby showing that PMCA1 is needed for embryonic development
Summary
Preparation of Mutant Mice and Genotype Analysis—Mouse Pmca and Pmca genomic sequences from a 129/SvJ library were used to prepare targeting constructs. Genotyping of mice was performed by blot hybridization of tail (or spleen) DNA or by PCR using a combination of three primers that simultaneously amplify wild-type and mutant alleles. For Pmca genotyping, forward (5Ј-GTGAGACACCTGGACGCTTGTGAGACC-3Ј) and reverse (5Ј-TCCCCGTTACCAGGTAGGACAGGA-3Ј) primers, corresponding to sequences from the 5Ј and 3Ј ends of exon 10, respectively, amplified a 183-bp product (codons 457–517) from the wild-type allele. A reverse primer complimentary to sequences in the phosphoglycerate kinase promoter (5Ј-GCATGCTCCAGACTGCCTTG-3Ј) that drives the neoR gene, and the forward primer from the 5Ј end of exon 10 amplified a 126-bp product. Sperm were isolated from epididymides of 8- to 12-week-old Pmca4Ϫ/Ϫ and Pmca4ϩ/ϩ mice, and their motility prior to hyperactivation was assessed as described previously [19]. After mounting with VectaMount (Vector Laboratories) and staining with 4,6-diamidino-2-phenylindole (DAPI) (1:10), which binds to DNA and yields a blue fluorescence, images were obtained by phase contrast microscopy
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