Disruption of a Spermatogenic Cell-Specific Mouse Enolase 4 (Eno4) Gene Causes Sperm Structural Defects and Male Infertility
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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Available from: Sujeet Kumar
- "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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ABSTRACT: The hexokinase 1 variant in mammalian spermatozoa (HK1S) has a unique N-terminus and this isoform atypically localizes to the plasma membrane. However, the mechanism of this process currently remains ambiguous. In this report, we show that fatty acylation underlies the specific sorting of HK1S. Employing chimeric reporter constructs, we first established that compartmentalization of HK1S does not function exclusively in sperm cells and that this feature is swappable to somatic HEK293 cells. Although the N-terminus lacks the classical consensus signature for myristoylation and the sequence-based predictions fail to predict myristoylation of HK1S, complementary experimental approaches confirmed that HK1S is myristoylated. Using live-cell confocal microscopy, we show that the mutation of a single amino acid, the myristoyl recipient Gly(2), impedes the prominent feature of plasma membrane association and relocates the enzyme to the cytosol but not the nucleus. Additionally, substitutions of the putatively palmitoylated Cys(5) is also reflected in a similar loss of compartmentalization of the protein. Taken together, our findings conclusively demonstrate that the N-terminal 'MGQICQ' motif in the unique GCS domain of HK1S acquires hydrophobicity by dual lipidic modifications, N-myristoylation and palmitoylation, to serve the requirements for membranous associations and thus its compartmentalization.
Biology Open 11/2015; DOI:10.1242/bio.012831 · 2.42 Impact Factor
Available from: Alexandra Amaral
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ABSTRACT: Although mitochondria are best known for being the eukaryotic cell powerhouses, these organelles participate in various cellular functions besides ATP production, such as calcium homeostasis, generation of reactive oxygen species (ROS), the intrinsic apoptotic pathway and steroid hormone biosynthesis. The aim of this review was to discuss the putative roles of mitochondria in mammalian sperm function and how they may relate to sperm quality and fertilization ability, particularly in humans. Although paternal mitochondria are degraded inside the zygote, sperm mitochondrial functionality seems to be critical for fertilisation. Indeed, changes in mitochondrial integrity/functionality, namely defects in mitochondrial ultrastructure or in the mitochondrial genome, transcriptome or proteome, as well as low mitochondrial membrane potential or altered oxygen consumption have been correlated with loss of sperm function (particularly with decreased motility). Results from genetically engineered mouse models also confirmed this trend. On the other hand, increasing evidence suggests that mitochondrial-derived ATP is not crucial for sperm motility, and that glycolysis may be the main ATP supplier for this particular aspect of sperm function. However, there are contradictory data in the literature regarding sperm bioenergetics. The relevance of sperm mitochondria may thus be associated with their role in other physiological features, particularly with the production of ROS, which in controlled levels are needed for proper sperm function. Sperm mitochondria may also serve as intracellular Ca2+ stores, although their role in signalling is still unclear.
Reproduction 07/2013; 146(5). DOI:10.1530/REP-13-0178 · 3.17 Impact Factor
Available from: George L Gerton
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ABSTRACT: Triosephosphate isomerase 1 (TPI1) is a member of the glycolytic pathway, which is a critical source of energy for motility in mouse sperm. By immunoblotting, we detected two male, germ line-specific TPI1 bands (Mr 33,400 and 30,800) as well as the somatic - type band (Mr 27,700). Although all three bands were observed in spermatogenic cells, somatic - type TPI1 disappeared from sperm during epididymal maturation. In vitro dephosphorylation analysis suggested that the two male, germ line-specific TPI1 bands were not the result of phosphorylation of the 27,700 Mr TPI1 band. The Mr 33,400, 30,800, and 27,700 Mr TPI1 bands corresponded to the respective sizes of the proteins predicted to use the first, second, and third possible initiation codons of the Tpi1 cDNA. We performed immunofluorescence on epididymal sperm and determined that TPI1 specifically localized in the principal piece. The antibody staining was stronger in cauda epididymal sperm than in caput epididymal sperm, a finding consistent with the identification of TPI1 as a cauda epididymal sperm-enriched protein. Immunofluorescence with sodium dodecyl sulfate (SDS)-insoluble flagellar accessory structures showed a strong TPI1 signal only in the principal piece, indicating that TPI1 is a component of the fibrous sheath. Northern hybridization detected longer Tpi1 transcripts (1.56 kb) in mouse testis, whereas somatic tissues had shorter transcripts (1.32 kb). As there is only one triosephosphate isomerase gene in the mouse genome, we conclude that the three variants we see in sperm result from the use of alternative translation start codons in spermatogenic cells. Mol. Reprod. Dev. © 2013 Wiley Periodicals, Inc.
Molecular Reproduction and Development 10/2013; 80(10). DOI:10.1002/mrd.22217 · 2.53 Impact Factor
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