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1.  Male Infertility, Impaired Sperm Motility, and Hydrocephalus in Mice Deficient in Sperm-Associated Antigen 6 
Molecular and Cellular Biology  2002;22(17):6298-6305.
Gene targeting was used to create mice lacking sperm-associated antigen 6 (Spag6), the murine orthologue of Chlamydomonas PF16, an axonemal protein containing eight armadillo repeats predicted to be important for flagellar motility and stability of the axoneme central apparatus. Within 8 weeks of birth, approximately 50% of Spag6-deficient animals died with hydrocephalus. Spag6-deficient males surviving to maturity were infertile. Their sperm had marked motility defects and was morphologically abnormal with frequent loss of the sperm head and disorganization of flagellar structures, including loss of the central pair of microtubules and disorganization of the outer dense fibers and fibrous sheath. We conclude that Spag6 is essential for sperm flagellar motility and that it is important for the maintenance of the structural integrity of mature sperm. The occurrence of hydrocephalus in the mutant mice also implicates Spag6 in the motility of ependymal cilia.
doi:10.1128/MCB.22.17.6298-6305.2002
PMCID: PMC134010  PMID: 12167721
2.  Spag16, an Axonemal Central Apparatus Gene, Encodes a Male Germ Cell Nuclear Speckle Protein that Regulates SPAG16 mRNA Expression 
PLoS ONE  2011;6(5):e20625.
Spag16 is the murine orthologue of Chlamydomonas reinhardtii PF20, a protein known to be essential to the structure and function of the “9+2” axoneme. In Chlamydomonas, the PF20 gene encodes a single protein present in the central pair of the axoneme. Loss of PF20 prevents central pair assembly/integrity and results in flagellar paralysis. Here we demonstrate that the murine Spag16 gene encodes two proteins: 71 kDa SPAG16L, which is found in all murine cells with motile cilia or flagella, and 35 kDa SPAG16S, representing the C terminus of SPAG16L, which is expressed only in male germ cells, and is predominantly found in specific regions within the nucleus that also contain SC35, a known marker of nuclear speckles enriched in pre-mRNA splicing factors. SPAG16S expression precedes expression of SPAG16L. Mice homozygous for a knockout of SPAG16L alone are infertile, but show no abnormalities in spermatogenesis. Mice chimeric for a mutation deleting the transcripts for both SPAG16L and SPAG16S have a profound defect in spermatogenesis. We show here that transduction of SPAG16S into cultured dispersed mouse male germ cells and BEAS-2B human bronchial epithelial cells increases SPAG16L expression, but has no effect on the expression of several other axoneme components. We also demonstrate that the Spag16L promoter shows increased activity in the presence of SPAG16S. The distinct nuclear localization of SPAG16S and its ability to modulate Spag16L mRNA expression suggest that SPAG16S plays an important role in the gene expression machinery of male germ cells. This is a unique example of a highly conserved axonemal protein gene that encodes two protein products with different functions.
doi:10.1371/journal.pone.0020625
PMCID: PMC3105110  PMID: 21655194
3.  Functional Deficiencies and a Reduced Response to Calcium in the Flagellum of Mouse Sperm Lacking SPAG16L1 
Biology of Reproduction  2009;82(4):736-744.
The Spag16L gene codes for a protein that is localized to the central apparatus which is essential for normal sperm motility and male fertility. Sperm from mice homozygous for a targeted deletion of the Spag16L gene were examined to assess their flagellar motor functions compared with age- and strain-matched control sperm. Sperm were also demembranated with Triton X-100 and examined for their ability to respond to free calcium, as well as for their ability to undergo microtubule sliding driven by dynein action. In addition, the passive flagella, inhibited by sodium metavanadate to disable the dyneins, were examined for mechanical abnormalities. Live Spag16L-null sperm exhibited much less bending of the flagellum during the beat. The amount of microtubule sliding in the R-bend direction of the beat was selectively restricted, which suggests that there is limited activation of the dyneins on one side of the axoneme in the live cells. This is corroborated by the results on detergent-extracted sperm models. The flagellar response to calcium is greatly reduced. The calcium response requires the activation of the dyneins on outer doublets 1, 2, 3, and 4. These are the same dyneins required for R-bend formation. In axonemes prepared to disintegrate by microtubule sliding, we observed little or no extrusion of doublets 1 and 2, consistent with a reduced activity of their dyneins. This deficit in motor function, and an increased rigidity of the midpiece region which we detected in the passive flagella, together can explain the observed motility characteristics of the Spag16L-null sperm.
Mouse sperm lacking SPAG16L have altered motility and a reduced response to calcium.
doi:10.1095/biolreprod.109.080143
PMCID: PMC2842488  PMID: 20042536
axoneme; calcium; central pair; ciliopathies; dynein; hyperactivation
4.  Transcriptional Regulation of an Axonemal Central Apparatus Gene, Sperm-associated Antigen 6, by a SRY-related High Mobility Group Transcription Factor, S-SOX5* 
The Journal of Biological Chemistry  2010;285(40):30496-30505.
SOX5 is a transcription factor with homology to the high mobility group box region of the testis-determining factor, SRY. Both the mouse and human SOX5 genes encode a 48-kDa SOX5 protein (S-SOX5) that is only present in tissues containing cells with motile cilia/flagella. The mammalian sperm-associated antigen 6 gene (SPAG6) encodes an axoneme central apparatus protein. Because human and mouse SPAG6 gene promoters contain multiple potential binding sites for SOX5, SPAG6 gene regulation by S-SOX5 was investigated in BEAS-2B cells, a line derived from human bronchial cells. Like FOXJ1, a transcription factor known to be essential for motile ciliogenesis, S-SOX5 stimulated mouse and human SPAG6 promoter function in BEAS-2B cells, but the effect was abrogated when the SOX5 binding sites were mutated or deleted. S-SOX5 and FOXJ1 functioned cooperatively in stimulating SPAG6 promoter activity. The SPAG6 message was up-regulated when S-SOX5 was overexpressed in BEAS-2B cells, and silencing of S-SOX5 by RNA interference down-regulated SPAG6 transcripts. Chromatin immunoprecipitation and EMSA experiments demonstrated that S-SOX5 associates with the SPAG6 promoter directly. The present study demonstrates that SPAG6 is a S-SOX5 target gene, indicating a key role for S-SOX5 in the formation and function of motile cilia.
doi:10.1074/jbc.M110.121590
PMCID: PMC2945543  PMID: 20668334
DNA-Protein Interaction; Gene Regulation; Promoters; Site-directed Mutagenesis; Sperm; Central Apparatus; Motile Cilia; SOX5; SPAG6; Transcription Regulation
5.  Phosphorylation of sperm axoneme central apparatus protein SPAG16L by a testis specific kinase, TSSK2* 
Biology of reproduction  2008;79(1):75-83.
Mammalian SPAG16L, the orthologue of Chlamydomonas Pf20, is an axoneme central apparatus protein necessary for flagellar motility. The SPAG16L protein sequence contains multiple potential phosphorylation sites and the protein was confirmed to be phosphorylated in vivo. A yeast-two-hybrid screen identified the testis-specific kinase, TSSK2, to be a potential SPAG16L binding partner. SPAG16L and TSSK2 interactions were confirmed by co-immunoprecipitation of both proteins from testis extracts and cell lysates expressing these proteins, and their co-localization was also noted by confocal microscopy in CHO cells where they were co-expressed. TSSK2 associates with SPAG16L via its C terminal domain bearing WD repeats. The N-terminal domain containing a coiled coil motif does not associate with TSSK2. SPAG16L can be phosphorylated by TSSK2 in vitro. Finally, TSSK2 is absent or markedly reduced from the testes in most of the SPAG16L null mice. These data support the conclusion that SPAG16L is a TSSK2 substrate.
doi:10.1095/biolreprod.107.066308
PMCID: PMC2660405  PMID: 18367677
SPAG16L; kinase; phosphorylation; sperm motility
6.  Identification, cloning and functional characterization of novel sperm associated antigen 11 (SPAG11) isoforms in the rat 
Background
Sperm binding proteins and their C-terminal peptides of the Sperm Associated Antigen 11 (SPAG11) family were found to play an important role in epididymal innate immunity in addition to their role in sperm maturation. However, the expression of Spag11 transcripts in rodents is not well documented.
Methods
Computational analysis was employed to identify novel Spag11 isoforms in the rat. RT-PCR analyses were carried out on RNAs isolated from the male reproductive tract tissues of rat using gene specific primers for Spag11c and Spag11t. The identities of PCR products were confirmed by sequencing. Tissue distribution, developmental expression and androgen regulation of Spag11t and Spag11c were studied using RT-PCR. The antimicrobial activities of recombinant Spag11t and Spag11c were tested against E coli in a colony forming unit assay.
Results
In this study, we identified two novel Spag11 transcripts, namely, Spag11t and Spag11c derived from the long arm of chromosome 16 in the rat (Rattus norvegicus), using both in silico and molecular biology approaches. Spag11c is expressed in all three regions of the epididymis, in testis and in ovary but is absent from the seminal vesicle. Spag11t expression is confined to the caput and it is not expressed in the testis, seminal vesicle or ovary. Age dependent expression of Spag11t and Spag11c was observed in the epididymides of rats (10–60 day old). Their expression was found to be most abundant in the adult rat (60 day) suggesting roles in mature reproductive function. Further, both Spag11t and Spag11c expression was down regulated in castrated rat epididymides and the expression was maintained in the testosterone replaced castrated rats. SPAG11C is a potent antibacterial agent. SPAG11T also displayed bactericidal capacity although weaker than SPAG11C and SPAG11E.
Conclusion
The abundant expression of Spag11t and Spag11c in the male reproductive tract suggests an important role in male reproductive tract immunity. Their expression is developmentally regulated and androgen dependent. Characterization of novel SPAG11 isoforms will contribute to our understanding of the role of epididymal proteins in sperm maturation and innate immunity.
doi:10.1186/1477-7827-4-23
PMCID: PMC1524968  PMID: 16643671
7.  Sperm Associated Antigen 9 Plays an Important Role in Bladder Transitional Cell Carcinoma 
PLoS ONE  2013;8(12):e81348.
Background
Majority of bladder cancer deaths are caused due to transitional cell carcinoma (TCC) which is the most prevalent and chemoresistant malignancy of urinary bladder. Therefore, we analyzed the role of Sperm associated antigen 9 (SPAG9) in bladder TCC.
Methodology and Findings
We examined SPAG9 expression and humoral response in 125 bladder TCC patients. Four bladder cancer cell lines were assessed for SPAG9 expression. In addition, we investigated the effect of SPAG9 ablation on cellular proliferation, cell cycle, migration and invasion in UM-UC-3 bladder cancer cells by employing gene silencing approach. Our SPAG9 gene and protein expression analysis revealed SPAG9 expression in 81% of bladder TCC tissue specimens. High SPAG9 expression (>60% SPAG9 positive cells) was found to be significantly associated with superficial non-muscle invasive stage (P = 0.042) and low grade tumors (P = 0.002) suggesting SPAG9 putative role in early spread and tumorigenesis. Humoral response against SPAG9 was observed in 95% of patients found positive for SPAG9 expression. All four bladder cancer cell lines revealed SPAG9 expression. In addition, SPAG9 gene silencing in UM-UC-3 cells resulted in induction of G0–G1 arrest characterized by up-regulation of p16 and p21 and consequent down-regulation of cyclin E, cyclin D and cyclin B, CDK4 and CDK1. Further, SPAG9 gene silencing also resulted in reduction in cellular growth, and migration and invasion ability of cancer cells in vitro.
Conclusions
Collectively, our data in clinical specimens indicated that SPAG9 is potential biomarker and therapeutic target for bladder TCC.
doi:10.1371/journal.pone.0081348
PMCID: PMC3857194  PMID: 24349057
8.  Targeted Disruption of the Testicular SPAG5/Deepest Protein Does Not Affect Spermatogenesis or Fertility 
Molecular and Cellular Biology  2002;22(7):1993-1997.
In an effort to define the molecular basis for morphogenesis of major sperm tail structures, including outer dense fibers, we recently cloned the Spag5 gene by virtue of its strong and specific leucine-zipper-mediated interaction with Odf1, the 27-kDa major outer dense fiber protein. Spag5 is expressed during meiosis and in round spermatids and is similar, if not identical, to Deepest, a putative spindle pole protein. Here we report the disruption of the Spag5 gene by homologous recombination. Spag5-null mice lack Spag5 mRNA and protein. However, male mice are viable and fertile. Analysis of the process of spermatogenesis and sperm produced in Spag5-null mice did not reveal a major phenotype as a consequence of the knockout event. This result suggests that if Spag5 plays a role in spermatogenesis it is likely compensated for by unknown proteins.
doi:10.1128/MCB.22.7.1993-1997.2002
PMCID: PMC133686  PMID: 11884588
9.  Mice Deficient in the Axonemal Protein Tektin-t Exhibit Male Infertility and Immotile-Cilium Syndrome Due to Impaired Inner Arm Dynein Function 
Molecular and Cellular Biology  2004;24(18):7958-7964.
The haploid germ cell-specific Tektin-t protein is a member of the Tektin family of proteins that form filaments in flagellar, ciliary, and axonemal microtubules. To investigate the physiological role of Tektin-t, we generated mice with a mutation in the tektin-t gene. The homozygous mutant males were infertile, while the females were fully fertile. Sperm morphology and function were abnormal, with frequent bending of the sperm flagella and marked defects in motility. In vitro fertilization assays showed that the defective spermatozoa were able to fertilize eggs. Electron microscopic examination showed that the dynein inner arm structure was disrupted in the sperm flagella of tektin-t-deficient mice. Furthermore, homozygous mutant mice had functionally defective tracheal cilia, as evidenced by altered dynein arm morphology. These results indicate that Tektin-t participates in dynein inner arm formation or attachment and that the loss of Tektin-t results in impaired motility of both flagella and cilia. Therefore, the tektin-t gene is one of the causal genes for immotile-cilium syndrome/primary ciliary dyskinesia.
doi:10.1128/MCB.24.18.7958-7964.2004
PMCID: PMC515054  PMID: 15340058
10.  The mitotic spindle protein SPAG5/Astrin connects to the Usher protein network postmitotically 
Cilia  2012;1:2.
Background
Mutations in the gene for Usher syndrome 2A (USH2A) are causative for non-syndromic retinitis pigmentosa and Usher syndrome, a condition that is the most common cause of combined deaf-blindness. To gain insight into the molecular pathology underlying USH2A-associated retinal degeneration, we aimed to identify interacting proteins of USH2A isoform B (USH2AisoB) in the retina.
Results
We identified the centrosomal and microtubule-associated protein sperm-associated antigen (SPAG)5 in the retina. SPAG5 was also found to interact with another previously described USH2AisoB interaction partner: the centrosomal ninein-like protein NINLisoB. Using In situ hybridization, we found that Spag5 was widely expressed during murine embryonic development, with prominent signals in the eye, cochlea, brain, kidney and liver. SPAG5 expression in adult human tissues was detected by quantitative PCR, which identified expression in the retina, brain, intestine, kidney and testis. In the retina, Spag5, Ush2aisoB and NinlisoB were present at several subcellular structures of photoreceptor cells, and colocalized at the basal bodies.
Conclusions
Based on these results and on the suggested roles for USH proteins in vesicle transport and providing structural support to both the inner ear and the retina, we hypothesize that SPAG5, USH2AisoB and NINLisoB may function together in microtubule-based cytoplasmic trafficking of proteins that are essential for cilium formation, maintenance and/or function.
doi:10.1186/2046-2530-1-2
PMCID: PMC3541543  PMID: 23351521
11.  Loss of SPEF2 Function in Mice Results in Spermatogenesis Defects and Primary Ciliary Dyskinesia1  
Biology of Reproduction  2011;85(4):690-701.
Primary ciliary dyskinesia (PCD) results from defects in motile cilia function. Mice homozygous for the mutation big giant head (bgh) have several abnormalities commonly associated with PCD, including hydrocephalus, male infertility, and sinusitis. In the present study, we use a variety of histopathological and cell biological techniques to characterize the bgh phenotype, and we identify the bgh mutation using a positional cloning approach. Histopathological, immunofluorescence, and electron microscopic analyses demonstrate that the male infertility results from shortened flagella and disorganized axonemal and accessory structures in elongating spermatids and mature sperm. In addition, there is a reduced number of elongating spermatids during spermatogenesis and mature sperm in the epididymis. Histological analyses show that the hydrocephalus is characterized by severe dilatation of the lateral ventricles and that bgh sinuses have an accumulation of mucus infiltrated by neutrophils. In contrast to the sperm phenotype, electron microscopy demonstrates that mutant respiratory epithelial cilia are ultrastructurally normal, but video microscopic analysis shows that their beat frequency is lower than that of wild-type cilia. Through a positional cloning approach, we identified two sequence variants in the gene encoding sperm flagellar protein 2 (SPEF2), which has been postulated to play an important role in spermatogenesis and flagellar assembly. A causative nonsense mutation was validated by Western blot analysis, strongly suggesting that the bgh phenotype results from the loss of SPEF2 function. Taken together, the data in this study demonstrate that SPEF2 is required for cilia function and identify a new genetic cause of PCD in mice.
Sperm flagellar protein 2 is required for spermatogenesis and regulation of ciliary motility in mice.
doi:10.1095/biolreprod.111.091132
PMCID: PMC3184289  PMID: 21715716
cilia; flagella; genetics; hydrocephalus; male infertility; primary ciliary dyskinesia; sinusitis; SPEF2; spermatogenesis
12.  CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs 
Nature genetics  2010;43(1):72-78.
Primary ciliary dyskinesia (PCD) is an inherited disorder characterized by recurrent infections of the upper and lower respiratory tract, reduced fertility in males and situs inversus in about 50% of affected individuals (Kartagener syndrome). It is caused by motility defects in the respiratory cilia that are responsible for airway clearance, the flagella that propel sperm cells and the nodal monocilia that determine left-right asymmetry1. Recessive mutations that cause PCD have been identified in genes encoding components of the outer dynein arms, radial spokes and cytoplasmic pre-assembly factors of axonemal dyneins, but these mutations account for only about 50% of cases of PCD. We exploited the unique properties of dog populations to positionally clone a new PCD gene, CCDC39. We found that loss-of-function mutations in the human ortholog underlie a substantial fraction of PCD cases with axonemal disorganization and abnormal ciliary beating. Functional analyses indicated that CCDC39 localizes to ciliary axonemes and is essential for assembly of inner dynein arms and the dynein regulatory complex.
doi:10.1038/ng.726
PMCID: PMC3509786  PMID: 21131972
13.  Novel Partners of SPAG11B Isoform D in the Human Male Reproductive Tract1 
Biology of Reproduction  2009;81(4):647-656.
Human sperm-associated antigen 11 (SPAG11) is closely related to beta-defensins in structure, expression, and function. Like the beta-defensins, SPAG11 proteins are predominantly expressed in the male reproductive tract, where their best-known major roles are in innate host defense and reproduction. Although several hypotheses have emerged to describe the evolution of beta-defensin and SPAG11 multifunctionality, few describe these multiple functions in terms of defensin interactions with specific proteins. To gain insight into the protein interaction potentials of SPAG11 and the signaling pathways that SPAG11 may influence, we used a yeast two-hybrid screening of a human testis-epididymis library. The results reveal human SPAG11B isoform D (SPAG11B/D) interactions with tryptase alpha/beta 1 (TPSAB1), tetraspanin 7 (TSPAN7), and attractin (ATRN). These interactions were confirmed by coimmunoprecipitation and glutathione S-transferase affinity matrix binding. SPAG11B/D and the three interacting proteins are expressed in the proximal epididymis, and all function in immunity and fertility pathways. We analyzed the functional consequences of SPAG11B/D interaction with TPSAB1 and showed that SPAG11B/D is both a substrate and a potent inhibitor of TPSAB1 activity. Furthermore, we show that (like SPAG11B/D) TSPAN7 and ATRN are associated with spermatozoa.
Human sperm associated antigen SPAG11B (isoform D) is both a substrate and a potent inhibitor of tryptase alpha/beta 1 activity, and interacts with tetraspanin 7 and attractin in the proximal epididymis and spermatazoa..
doi:10.1095/biolreprod.109.077545
PMCID: PMC2754882  PMID: 19535787
attractin; beta-defensin; epididymis; gene regulation; male reproductive tract; signal transduction; sperm; spermatozoa; tetraspanin; tryptase
14.  Conserved structural motifs in the central pair complex of eukaryotic flagella 
Cytoskeleton (Hoboken, N.J.)  2012;70(2):101-120.
Cilia and flagella are conserved hair-like appendages of eukaryotic cells that function as sensing and motility generating organelles. Motility is driven by thousands of axonemal dyneins that require precise regulation. One essential motility regulator is the central pair complex (CPC) and many CPC defects cause paralysis of cilia/flagella. Several human diseases, such as immotile cilia syndrome, show CPC abnormalities, but little is known about the detailed three-dimensional structure and function of the CPC. The CPC is located in the center of typical [9+2] cilia/flagella and is composed of two singlet microtubules, each with a set of associated projections that extend toward the surrounding nine doublet microtubules. Using cryo-electron tomography coupled with subtomogram averaging, we visualized and compared the three-dimensional structures of the CPC in both the green alga Chlamydomonas and the sea urchin Strongylocentrotus at the highest resolution published to date. Despite the evolutionary distance between these species, their CPCs exhibit remarkable structural conservation. We identified several new projections, including those that form the elusive sheath, and show that the bridge has a more complex architecture than previously thought. Organism-specific differences include the presence of microtubule inner proteins in Chlamydomonas, but not Strongylocentrotus, and different overall outlines of the highly connected projection network, which forms a round-shaped cylinder in algae, but is more oval in sea urchin. These differences could be adaptations to the mechanical requirements of the rotating CPC in Chlamydomonas, compared to the Strongylocentrotus CPC which has a fixed orientation.
doi:10.1002/cm.21094
PMCID: PMC3914236  PMID: 23281266
cryo-electron tomography; axoneme; flagella; Chlamydomonas; Strongylocentrotus
15.  Sperm-associated antigen 11A is expressed exclusively in the principal cells of the mouse caput epididymis in an androgen-dependent manner 
Background
Epididymal sperm maturation occurs via interactions between sperm and proteins secreted by the epididymal epithelium. Although this is an important process, the genes that encode the involved proteins remain largely uncharacterized. Previous studies have demonstrated that the genes involved in sperm maturation are regulated by androgen. Spag11a is an epididymal gene that is influenced by androgen. However, little is known about the putative role of this gene in the sperm maturation process. The objective of this study was to characterize Spag11a in the mouse epididymis.
Methods
In silico analyses were performed to predict signal peptides and functional domains. Spag11a expression was measured by quantitative real-time RT-PCR. Western blots and immunocytochemistry were performed to determine protein expression.
Results
SPAG11A is a member of the beta defensin protein family and constitutes a secretory protein. Spag11a was expressed exclusively in the epididymis. Moreover, it exhibited region-specific expression in the caput, which is typical for genes that are involved in creating a suitable microenvironment for sperm maturation. Mouse Spag11a was regulated by androgen. A significant decrease of Spag11a expression was observed at third day following a gonadectomy (P < 0.001). Interestingly, testosterone replacement therapy was able to maintain the expression almost at the normal level, indicating a dependency on androgen. Besides androgen, testicular factors influenced Spag11a expression in a different way. This was revealed by efferent duct ligation in which Spag11a was transiently up-regulated at the third day following the ligation before returning to the normal level at day 5. Spag11a regional expression was also observed at protein level detected by western immunoblotting which revealed a clear band in the caput but not in other regions. The prediction that SPAG11A is a secretory protein was confirmed by immunocytochemical analyses indicating cell-specific expression mainly in the caput principal cells and detection of the protein in epididymal luminal fluid and spermatozoa.
Conclusions
Based on the characteristics of Spag11a, it is likely that this gene has a specific role in epididymal sperm maturation. Further studies using functional assays are necessary to confirm this finding.
doi:10.1186/1477-7827-11-59
PMCID: PMC3710511  PMID: 23815807
Spag11a; Epididymis; Androgen; Secretory protein; Principal cells
16.  Drosophila Bld10 Is a Centriolar Protein That Regulates Centriole, Basal Body, and Motile Cilium Assembly 
Molecular Biology of the Cell  2009;20(10):2605-2614.
Cilia and flagella play multiple essential roles in animal development and cell physiology. Defective cilium assembly or motility represents the etiological basis for a growing number of human diseases. Therefore, how cilia and flagella assemble and the processes that drive motility are essential for understanding these diseases. Here we show that Drosophila Bld10, the ortholog of Chlamydomonas reinhardtii Bld10p and human Cep135, is a ubiquitous centriolar protein that also localizes to the spermatid basal body. Mutants that lack Bld10 assemble centrioles and form functional centrosomes, but centrioles and spermatid basal bodies are short in length. bld10 mutant flies are viable but male sterile, producing immotile sperm whose axonemes are deficient in the central pair of microtubules. These results show that Drosophila Bld10 is required for centriole and axoneme assembly to confer cilium motility.
doi:10.1091/mbc.E08-11-1115
PMCID: PMC2682601  PMID: 19321663
17.  PF20 gene product contains WD repeats and localizes to the intermicrotubule bridges in Chlamydomonas flagella. 
Molecular Biology of the Cell  1997;8(3):455-467.
The central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components, we generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen contains an allele of a previously identified mutation, pf20. Mutant cells have paralyzed flagella, and the entire central apparatus is missing in isolated axonemes. We have cloned the wild-type PF20 gene and confirmed its identity by rescuing the pf20 mutant phenotype upon transformation. Rescued transformants were wild type in motility and in axonemal ultrastructure. A cDNA clone containing a single, long open reading frame was obtained and sequenced. Database searches using the predicted 606-amino acid sequence of PF20 indicate that the protein contains five contiguous WD repeats. These repeats are found in a number of proteins with diverse cellular functions including beta-transducin and dynein intermediate chains. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunogold labeling of wild-type axonemes indicates that the PF20 protein is localized along the length of the C2 microtubule on the intermicrotubule bridges connecting the two central microtubules. We suggest that the PF20 gene product is a new member of the family of WD repeat proteins and is required for central microtubule assembly and/or stability and flagellar motility.
Images
PMCID: PMC276097  PMID: 9188098
18.  Assessment of SPAG9 Transcript in Fine Needle Aspirates of Thyroid Nodules 
European Thyroid Journal  2012;1(2):118-121.
Objectives
Sperm-associated antigen 9 (SPAG9) has been suggested as a possible biomarker in several malignancies including thyroid cancer. We investigated the expression of SPAG9 mRNA in fine needle aspiration (FNA) material from papillary thyroid carcinoma (PTC) and benign thyroid nodules.
Study Design
SPAG9 expression was assessed in 36 FNA samples corresponding to 16 PTC and 20 benign nodules using the original method detecting the SPAG9 transcript containing intron 21 (NCBI X91879). The presence of the BRAF V600E point mutation was also analyzed by pyrosequencing.
Results
Six of 16 (38%) PTC samples were positive for X91879 SPAG9 transcript compared to 8 of 20 (40%) benign samples (p = 0.88). Out of 12 BRAF-positive PTC, 3 (25%) also expressed the SPAG9 transcript compared to 3 out of 4 BRAF-negative PTC (75%; p = 0.12).
Conclusions
The X91879 SPAG9 transcript originally described does not appear to be overexpressed in FNA material from PTC or to be clinically relevant in the diagnosis of thyroid nodules.
doi:10.1159/000338922
PMCID: PMC3821466  PMID: 24783006
Sperm-associated antigen 9; Fine needle aspiration; Thyroid; Diagnosis; Cancer
19.  A Sperm-Associated WD Repeat Protein Orthologous to Chlamydomonas PF20 Associates with Spag6, the Mammalian Orthologue of Chlamydomonas PF16 
Molecular and Cellular Biology  2002;22(22):7993-8004.
cDNAs were cloned for the murine and human orthologues of Chlamydomonas PF20, a component of the alga axoneme central apparatus that is required for flagellar motility. The mammalian genes encode transcripts of 1.4 and 2.5 kb that are highly expressed in testis. The two transcripts appear to arise from alternative transcription start sites. The murine Pf20 gene was mapped to chromosome 1, syntenic with the location of the human gene on chromosome 2. An antibody generated against an N-terminal sequence of mouse Pf20 recognized a 71-kDa protein in sperm and testis extracts. Immunocytochemistry localized Pf20 to the tails of permeabilized sperm; electron microscope immunocytochemistry showed that Pf20 was located in the axoneme central apparatus. A murine Pf20-green fluorescent protein fusion protein expressed in Chinese hamster ovary cells accumulated in the cytoplasm. When coexpressed with Spag6, the mammalian orthologue of Chlamydomonas PF16, Pf20 was colocalized with Spag6 on polymerized microtubules. Yeast two-hybrid assays demonstrated interaction of the Pf20 WD repeats with Spag6. Pf20 was markedly reduced in sperm collected from mice lacking Spag6, which are infertile due to a motility defect. Our observations provide the first evidence for an association between mammalian orthologues of two Chlamydomonas proteins known to be critical for axoneme structure and function.
doi:10.1128/MCB.22.22.7993-8004.2002
PMCID: PMC134734  PMID: 12391165
20.  Identification of predicted human outer dynein arm genes: candidates for primary ciliary dyskinesia genes 
Journal of Medical Genetics  2005;43(1):62-73.
Background
Primary ciliary dyskinesia (PCD) is a severe inherited disorder characterised by chronic respiratory disease, male infertility, and, in ∼50% of affected individuals, a left‐right asymmetry defect called situs inversus. PCD is caused by defects in substructures of the ciliary and flagellar axoneme, most commonly loss of the outer dynein arms. Although PCD is believed to involve mutations in many genes, only three have been identified.
Methods
To facilitate discovery of new PCD genes, we have used database searching and analysis to systematically identify the human homologues of proteins associated with the Chlamydomonas reinhardtii outer dynein arm, the best characterised outer arm of any species.
Results
We find that 12 out of 14 known Chlamydomonas outer arm subunits have one or more likely orthologues in humans. The results predict a total of 24 human genes likely to encode outer dynein arm subunits and associated proteins possibly necessary for outer arm assembly, plus 12 additional closely related human genes likely to encode inner dynein arm subunits.
Conclusion
These genes, which have been located on the human chromosomes for easy comparison with known or suspected PCD loci, are excellent candidates for screening for disease‐causing mutations in PCD patients with outer and/or inner dynein arm defects.
doi:10.1136/jmg.2005.033001
PMCID: PMC2593024  PMID: 15937072
Chlamydomonas; cilia; dynein; flagella; immotile cilia syndrome; Kartagener's syndrome; primary ciliary dyskinesia
21.  PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella 
The Journal of Cell Biology  1996;132(3):359-370.
Several studies have indicated that the central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components we have generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen, D2, is an allele of a previously identified mutant, pf16. Mutant cells have paralyzed flagella, and the C1 microtubule of the central apparatus is missing in isolated axonemes. We have cloned the wild-type PF16 gene and confirmed its identity by rescuing pf16 mutants upon transformation. The rescued pf16 cells were wild-type in motility and in axonemal ultrastructure. A full-length cDNA clone for PF16 was obtained and sequenced. Database searches using the predicted 566 amino acid sequence of PF16 indicate that the protein contains eight contiguous armadillo repeats. A number of proteins with diverse cellular functions also contain armadillo repeats including pendulin, Rch1, importin, SRP-1, and armadillo. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunofluorescence labeling of wild-type flagella indicates that the PF16 protein is localized along the length of the flagella while immunogold labeling further localizes the PF16 protein to a single microtubule of the central pair. Based on the localization results and the presence of the armadillo repeats in this protein, we suggest that the PF16 gene product is involved in protein-protein interactions important for C1 central microtubule stability and flagellar motility.
PMCID: PMC2120723  PMID: 8636214
22.  Impacts of Oxidative Stress and Antioxidants on Semen Functions 
Oxidative stress (OS) has been considered a major contributory factor to the infertility. Oxidative stress is the result of imbalance between the reactive oxygen species (ROS) and antioxidants in the body which can lead to sperm damage, deformity, and eventually male infertility. Although high concentrations of the ROS cause sperm pathology (ATP depletion) leading to insufficient axonemal phosphorylation, lipid peroxidation, and loss of motility and viability but, many evidences demonstrate that low and controlled concentrations of these ROS play an important role in sperm physiological processes such as capacitation, acrosome reaction, and signaling processes to ensure fertilization. The supplementation of a cryopreservation extender with antioxidant has been shown to provide a cryoprotective effect on mammalian sperm quality. This paper reviews the impacts of oxidative stress and reactive oxygen species on spermatozoa functions, causes of ROS generation, and antioxidative strategies to reduce OS. In addition, we also highlight the emerging concept of utilizing OS as a tool of contraception.
doi:10.4061/2011/686137
PMCID: PMC2943128  PMID: 20871827
23.  Combined exome and whole-genome sequencing identifies mutations in ARMC4 as a cause of primary ciliary dyskinesia with defects in the outer dynein arm 
Journal of Medical Genetics  2013;51(1):61-67.
Background
Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous ciliopathy disorder affecting cilia and sperm motility. A range of ultrastructural defects of the axoneme underlie the disease, which is characterised by chronic respiratory symptoms and obstructive lung disease, infertility and body axis laterality defects. We applied a next-generation sequencing approach to identify the gene responsible for this phenotype in two consanguineous families.
Methods and results
Data from whole-exome sequencing in a consanguineous Turkish family, and whole-genome sequencing in the obligate carrier parents of a consanguineous Pakistani family was combined to identify homozygous loss-of-function mutations in ARMC4, segregating in all five affected individuals from both families. Both families carried nonsense mutations within the highly conserved armadillo repeat region of ARMC4: c.2675C>A; pSer892* and c.1972G>T; p.Glu658*. A deficiency of ARMC4 protein was seen in patient's respiratory cilia accompanied by loss of the distal outer dynein arm motors responsible for generating ciliary beating, giving rise to cilia immotility. ARMC4 gene expression is upregulated during ciliogenesis, and we found a predicted interaction with the outer dynein arm protein DNAI2, mutations in which also cause PCD.
Conclusions
We report the first use of whole-genome sequencing to identify gene mutations causing PCD. Loss-of-function mutations in ARMC4 cause PCD with situs inversus and cilia immotility, associated with a loss of the distal outer (but not inner) dynein arms. This addition of ARMC4 to the list of genes associated with ciliary outer dynein arm defects expands our understanding of the complexities of PCD genetics.
doi:10.1136/jmedgenet-2013-101938
PMCID: PMC3888613  PMID: 24203976
Clinical Genetics; Developmental; Genetics; Molecular Genetics; Other Respiratory Medicine
24.  Ktu/PF13 is required for cytoplasmic pre-assembly of axonemal dyneins 
Nature  2008;456(7222):611-616.
Summary
Cilia/flagella are highly conserved organelles that play diverse roles in cell motility and sensing extracellular signals. Motility defects in cilia/flagella often result in primary ciliary dyskinesia (PCD). However, the mechanisms underlying cilia formation and function, and in particular the cytoplasmic assembly of dyneins that power ciliary motility, are only poorly understood. Here we report a novel gene, kintoun (ktu), involved in this cytoplasmic process. This gene was first identified in a medaka mutant, and found to be mutated in PCD patients from two affected families as well as in the pf13 mutant of Chlamydomonas. In the absence of Ktu/PF13, both outer and inner dynein arms are missing or defective in the axoneme, leading to a loss of motility. Biochemical and immunohistochemical studies show that Ktu/PF13 is one of the long-sought proteins involved in pre-assembly of dynein arm complexes in the cytoplasm before intraflagellar transport loads them for the ciliary compartment.
doi:10.1038/nature07471
PMCID: PMC3279746  PMID: 19052621
25.  RFX2 is a candidate downstream amplifier of A-MYB regulation in mouse spermatogenesis 
Background
Mammalian spermatogenesis involves formation of haploid cells from the male germline and then a complex morphological transformation to generate motile sperm. Focusing on meiotic prophase, some tissue-specific transcription factors are known (A-MYB) or suspected (RFX2) to play important roles in modulating gene expression in pachytene spermatocytes. The current work was initiated to identify both downstream and upstream regulatory connections for Rfx2.
Results
Searches of pachytene up-regulated genes identified high affinity RFX binding sites (X boxes) in promoter regions of several new genes: Adam5, Pdcl2, and Spag6. We confirmed a strong promoter-region X-box for Alf, a germ cell-specific variant of general transcription factor TFIIA. Using Alf as an example of a target gene, we showed that its promoter is stimulated by RFX2 in transfected cells and used ChIP analysis to show that the promoter is occupied by RFX2 in vivo. Turning to upstream regulation of the Rfx2 promoter, we identified a cluster of three binding sites (MBS) for the MYB family of transcription factors. Because testis is one of the few sites of A-myb expression, and because spermatogenesis arrests in pachytene in A-myb knockout mice, the MBS cluster implicates Rfx2 as an A-myb target. Electrophoretic gel-shift, ChIP, and co-transfection assays all support a role for these MYB sites in Rfx2 expression. Further, Rfx2 expression was virtually eliminated in A-myb knockout testes. Immunohistology on testis sections showed that A-MYB expression is up-regulated only after pachytene spermatocytes have clearly moved away from the tubule wall, which correlates with onset of RFX2 expression, whereas B-MYB expression, by contrast, is prevalent only in earlier spermatocytes and spermatogonia.
Conclusion
With an expanding list of likely target genes, RFX2 is potentially an important transcriptional regulator in pachytene spermatocytes. Rfx2 itself is a good candidate to be regulated by A-MYB, which is essential for meiotic progression. If Alf is a genuine RFX2 target, then A-myb, Rfx2, and Alf may form part of a transcriptional network that is vital for completion of meiosis and preparation for post-meiotic differentiation.
doi:10.1186/1471-213X-9-63
PMCID: PMC2797782  PMID: 20003220

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