Disruption of a Spermatogenic Cell-Specific Mouse Enolase 4 (Eno4) Gene Causes Sperm Structural Defects and Male Infertility

Biology of Reproduction (Impact Factor: 3.32). 02/2013; 88(4). DOI: 10.1095/biolreprod.112.107128
Source: PubMed


Sperm utilize glycolysis to generate ATP required for motility and several spermatogenic cell-specific glycolytic isozymes are associated with the fibrous sheath (FS) in the principle piece of the sperm flagellum. We used proteomics and molecular biology approaches to confirm earlier reports that a novel enolase is present in mouse sperm. We then found that a pan-enolase antibody, but not antibodies to ENO2 and ENO3, recognized a protein in the principal piece of the mouse sperm flagellum. Database analyses identified two previously uncharacterized enolase family-like candidate genes, 64306537H0Rik and Gm5506. Northern analysis indicated that 64306537H0Rik (renamed Eno4) was transcribed in testes of mice by Postnatal Day 12. To determine the role of ENO4, we generated mice using ES cells in which an Eno4 allele was disrupted by a gene trap containing a beta galactosidase (beta-gal) reporter (Eno4+/Gt). Expression of beta-gal occurred in the testis and male mice homozygous for the gene trap allele (Eno4Gt/Gt) were infertile. Epididymal sperm numbers were two-fold lower and sperm motility was reduced substantially in Eno4Gt/Gt mice compared to wild type (WT) mice. Sperm from Eno4Gt/Gt mice had a coiled flagellum and a disorganized FS. The Gm5506 gene encodes a protein identical to ENO1 and also is transcribed at a low level in testis. We conclude that ENO4 is required for normal assembly of the FS and provides most of the enolase activity in sperm, and that Eno1 and/or Gm5506 may encode a minor portion of the enolase activity in sperm.

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    • "The male germ cells (spermatozoa) have a conserved structural organization among mammals which is specialized both morphologically and biochemically to deliver the male genome to the egg. The sperm flagellum contains the machinery required for motility as demonstrated by the targeted gene disruption of enzymes; that confirmed ATP generated via glycolysis is essential for sperm motility and male fertility (Danshina et al., 2010; Miki et al., 2004; Nakamura et al., 2013; Narisawa et al., 2002; Odet et al., 2013). The tethering of multiple glycolytic enzymes to the FS of the mammalian sperm is highly resistant to extraction; however, HK1 as an exception is readily solubilized by detergents and is not identified in the isolated fibrous sheath preparation (Kim et al., 2007; Nakamura et al., 2010). "
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