A Heterozygous Mutation Disrupting the SPAG16 Gene Results in Biochemical Instability of Central Apparatus Components of the Human Sperm Axoneme

University of Arkansas at Little Rock, Little Rock, Arkansas, United States
Biology of Reproduction (Impact Factor: 3.32). 12/2007; 77(5):864-71. DOI: 10.1095/biolreprod.107.063206
Source: PubMed


The SPAG16 gene encodes two major transcripts, one for the 71-kDa SPAG16L, which is the orthologue of the Chlamydomonas rheinhardtii central apparatus protein PF20, and a smaller transcript, which codes for the 35-kDa SPAG16S nuclear protein that represents the C-terminus (exons 11-16) of SPAG16L. We have previously reported that a targeted mutation in exon 11 of the Spag16 gene impairs spermatogenesis and prevents transmission of the mutant allele in chimeric mice. In the present report, we describe a heterozygous mutation in exon 13 of the SPAG16 gene, which causes a frame shift and premature stop codon, affording the opportunity to compare mutations with similar impacts on SPAG16L and SPAG16S for male reproductive function in mice and men. We studied two male heterozygotes for the SPAG16 mutation, both of which were fertile. Freezing-boiling of isolated sperm from both affected males resulted in the loss of the SPAG16L protein, SPAG6, another central apparatus protein that interacts with SPAG16L, and the 28-kDa fragment of SPAG17, which associates with SPAG6. These proteins were also lost after freezing-boiling cycles of sperm extracts from mice that were heterozygous for an inactivating mutation (exons 2 and 3) in Spag16. Our findings suggest that a heterozygous mutation that affects both SPAG16L and SPAG16S does not cause male infertility in man, but is associated with reduced stability of the interacting proteins of the central apparatus in response to a thermal challenge, a phenotype shared by the sperm of mice heterozygous for a mutation that affects SPAG16L.

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    • "Although they are simply annotated as ‘sperm antigens’ because they were initially discovered as immunoreactive proteins in experiments of target proteins for contraception in humans [103], they are known to be structural components of the sperm cells. For example, sperm antigen 16 (SPAG16), expressed in the diploid meiotic stage, locates in the central apparatus of the sperm tail and is essential for flagellar motility [104]. Another constituent of the sperm tail is the transcript identified as outer dense fiber protein 3-like, also known as SHIPPO1, is only expressed in haploid cells, and is a testis-specific protein located in the flagella of elongated spermatids and along the entire length of the mature sperm [105]. "
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    ABSTRACT: Despite the great advances in sequencing technologies, genomic and transcriptomic information for marine non-model species with ecological, evolutionary, and economical interest is still scarce. In this work we aimed to identify genes expressed during spermatogenesis in the functional hermaphrodite scallop Nodipecten subnodosus (Mollusca: Bivalvia: Pectinidae), with the purpose of obtaining a panel of genes that would allow for the study of differentially transcribed genes between diploid and triploid scallops in the context of meiotic arrest and reproductive sterility. Because our aim was to isolate genes involved in meiosis and other testis maturation-related processes, we generated suppressive subtractive hybridization libraries of testis vs. inactive gonad. We obtained 352 and 177 ESTs by clone sequencing, and using pyrosequencing (454-Roche) we maximized the identified ESTs to 34,276 reads. A total of 1,153 genes from the testis library had a blastx hit and GO annotation, including genes specific for meiosis, spermatogenesis, sex-differentiation, and transposable elements. Some of the identified meiosis genes function in chromosome pairing (scp2, scp3), recombination and DNA repair (dmc1, rad51, ccnb1ip1/hei10), and meiotic checkpoints (rad1, hormad1, dtl/cdt2). Gene expression analyses in different gametogenic stages in both sexual regions of the gonad of meiosis genes confirmed that the expression was specific or increased towards the maturing testis. Spermatogenesis genes included known testis-specific ones (kelch-10, shippo1, adad1), with some of these known to be associated to sterility. Sex differentiation genes included one of the most conserved genes at the bottom of the sex-determination cascade (dmrt1). Transcript from transposable elements, reverse transcriptase, and transposases in this library evidenced that transposition is an active process during spermatogenesis in N. subnodosus. In relation to the inactive library, we identified 833 transcripts with functional annotation related to activation of the transcription and translation machinery, as well as to germline control and maintenance.
    Full-text · Article · Sep 2013 · PLoS ONE
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    • "We have also reported previously that sperm from human subjects heterozygous for a frame shift mutation in SPAG16 exhibited instability of important central apparatus components SPAG16L, SPAG17, and SPAG6 [18]. While both subjects in this report were fertile, observed biochemical instability of the sperm axoneme suggests that even heterozygous disruption of SPAG16 has phenotypic consequences that may reduce fecundity. "
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    ABSTRACT: Background SPAG16 is a critical structural component of motile cilia and flagella. In the eukaryotic unicellular algae Chlamydomonas, loss of gene function causes flagellar paralysis and prevents assembly of the “9 + 2” axoneme central pair. In mice, we have previously shown that loss of Spag16 gene function causes male infertility and severe sperm motility defects. We have also reported that a heterozygous mutation of the human SPAG16 gene reduces stability of the sperm axonemal central apparatus. Methods In the present study, we analyzed DNA samples from 60 infertile male volunteers of Western European (Italian) origin, to search for novel SPAG16 gene mutations, and to determine whether increased prevalence of SPAG16 single nucleotide polymorphisms (SNPs) was associated with infertility phenotypes. Semen parameters were evaluated by light microscopy and sperm morphology was comprehensively analyzed by transmission electron microscopy (TEM). Results For gene analysis, sequences were generated covering exons encoding the conserved WD40 repeat region of the SPAG16 protein and the flanking splice junctions. No novel mutations were found, and the four SNPs in the assessed gene region were present at expected frequencies. The minor alleles were not associated with any assessed sperm parameter in the sample population. Conclusions Analysis of the SPAG16 regions encoding the conserved WD repeats revealed no evidence for association of mutations or genetic variation with sperm motility and ultrastructural sperm characteristics in a cohort of Italian infertile males.
    Full-text · Article · Sep 2012 · BMC Urology
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    ABSTRACT: Spermiogenesis constitutes the steps involved in the metamorphosis of spermatids into spermatozoa. It involves modification of several organelles in addition to the formation of several structures including the flagellum and cytoplasmic droplet. The flagellum is composed of a neck region and middle, principal, and end pieces. The axoneme composed of nine outer microtubular doublets circularly arranged to form a cylinder around a central pair of microtubules is present throughout the flagellum. The middle and principal pieces each contain specific components such as the mitochondrial sheath and fibrous sheath, respectively, while outer dense fibers are common to both. A plethora of proteins are constituents of each of these structures, with each playing key roles in functions related to the fertility of spermatozoa. At the end of spermiogenesis, a portion of spermatid cytoplasm remains associated with the released spermatozoa, referred to as the cytoplasmic droplet. The latter has as its main feature Golgi saccules, which appear to modify the plasma membrane of spermatozoa as they move down the epididymal duct and hence may be partly involved in male gamete maturation. The end product of spermatogenesis is highly streamlined and motile spermatozoa having a condensed nucleus equipped with an acrosome. Spermatozoa move through the female reproductive tract and eventually penetrate the zona pellucida and bind to the egg plasma membrane. Many proteins have been implicated in the process of fertilization as well as a plethora of proteins involved in the development of spermatids and sperm, and these are high lighted in this review.
    No preview · Article · Jan 2009 · Microscopy Research and Technique
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