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1.  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
2.  Sperm-Associated Antigen–17 Gene Is Essential for Motile Cilia Function and Neonatal Survival 
Primary ciliary dyskinesia (PCD), resulting from defects in cilia assembly or motility, is caused by mutations in a number of genes encoding axonemal proteins. PCD phenotypes are variable, and include recurrent respiratory tract infections, bronchiectasis, hydrocephaly, situs inversus, and male infertility. We generated knockout mice for the sperm-associated antigen–17 (Spag17) gene, which encodes a central pair (CP) protein present in the axonemes of cells with “9 + 2” motile cilia or flagella. The targeting of Spag17 resulted in a severe phenotype characterized by immotile nasal and tracheal cilia, reduced clearance of nasal mucus, profound respiratory distress associated with lung fluid accumulation and disruption of the alveolar epithelium, cerebral ventricular expansion consistent with emerging hydrocephalus, failure to suckle, and neonatal demise within 12 hours of birth. Ultrastructural analysis revealed the loss of one CP microtubule in approximately one quarter of tracheal cilia axonemes, an absence of a C1 microtubule projection, and other less frequent CP structural abnormalities. SPAG6 and SPAG16 (CP proteins that interact with SPAG17) were increased in tracheal tissue from SPAG17-deficient mice. We conclude that Spag17 plays a critical role in the function and structure of motile cilia, and that neonatal lethality is likely explained by impaired airway mucociliary clearance.
doi:10.1165/rcmb.2012-0362OC
PMCID: PMC3727877  PMID: 23418344
primary ciliary dyskinesia; axoneme; central pair; Spag17; cilia
3.  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
4.  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
5.  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
6.  Otitis Media in Sperm-Associated Antigen 6 (Spag6)-Deficient Mice 
PLoS ONE  2014;9(11):e112879.
Mammalian SPAG6 protein is localized to the axoneme central apparatus, and it is required for normal flagella and cilia motility. Recent studies demonstrated that the protein also regulates ciliogenesis and cilia polarity in the epithelial cells of brain ventricles and trachea. Motile cilia are also present in the epithelial cells of the middle ear and Eustachian tubes, where the ciliary system participates in the movement of serous fluid and mucus in the middle ear. Cilia defects are associated with otitis media (OM), presumably due to an inability to efficiently transport fluid, mucus and particles including microorganisms. We investigated the potential role of SPAG6 in the middle ear and Eustachian tubes by studying mice with a targeted mutation in the Spag6 gene. SPAG6 is expressed in the ciliated cells of middle ear epithelial cells. The orientation of the ciliary basal feet was random in the middle ear epithelial cells of Spag6-deficient mice, and there was an associated disrupted localization of the planar cell polarity (PCP) protein, FZD6. These features are associated with disordered cilia orientation, confirmed by scanning electron microscopy, which leads to uncoordinated cilia beating. The Spag6 mutant mice were also prone to develop OM. However, there were no significant differences in bacterial populations, epithelial goblet cell density, mucin expression and Eustachian tube angle between the mutant and wild-type mice, suggesting that OM was due to accumulation of fluid and mucus secondary to the ciliary dysfunction. Our studies demonstrate a role for Spag6 in the pathogenesis of OM in mice, possibly through its role in the regulation of cilia/basal body polarity through the PCP-dependent mechanisms in the middle ear and Eustachian tubes.
doi:10.1371/journal.pone.0112879
PMCID: PMC4231073  PMID: 25393619
7.  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
8.  Sperm-Associated Antigen 6 (SPAG6) Deficiency and Defects in Ciliogenesis and Cilia Function: Polarity, Density, and Beat 
PLoS ONE  2014;9(10):e107271.
SPAG6, an axoneme central apparatus protein, is essential for function of ependymal cell cilia and sperm flagella. A significant number of Spag6-deficient mice die with hydrocephalus, and surviving males are sterile because of sperm motility defects. In further exploring the ciliary dysfunction in Spag6-null mice, we discovered that cilia beat frequency was significantly reduced in tracheal epithelial cells, and that the beat was not synchronized. There was also a significant reduction in cilia density in both brain ependymal and trachea epithelial cells, and cilia arrays were disorganized. The orientation of basal feet, which determines the direction of axoneme orientation, was apparently random in Spag6-deficient mice, and there were reduced numbers of basal feet, consistent with reduced cilia density. The polarized epithelial cell morphology and distribution of intracellular mucin, α-tubulin, and the planar cell polarity protein, Vangl2, were lost in Spag6-deficient tracheal epithelial cells. Polarized epithelial cell morphology and polarized distribution of α-tubulin in tracheal epithelial cells was observed in one-week old wild-type mice, but not in the Spag6-deficient mice of the same age. Thus, the cilia and polarity defects appear prior to 7 days post-partum. These findings suggest that SPAG6 not only regulates cilia/flagellar motility, but that in its absence, ciliogenesis, axoneme orientation, and tracheal epithelial cell polarity are altered.
doi:10.1371/journal.pone.0107271
PMCID: PMC4204823  PMID: 25333478
9.  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
10.  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
11.  SEPT12/SPAG4/LAMINB1 Complexes Are Required for Maintaining the Integrity of the Nuclear Envelope in Postmeiotic Male Germ Cells 
PLoS ONE  2015;10(3):e0120722.
Male infertility affects approximately 50% of all infertile couples. The male-related causes of intracytoplasmic sperm injection failure include the absence of sperm, immotile or immature sperm, and sperm with structural defects such as those caused by premature chromosomal condensation and DNA damage. Our previous studies based on a knockout mice model indicated that SEPT12 proteins are critical for the terminal morphological formation of sperm. SEPT12 mutations in men result in teratozospermia and oligozospermia. In addition, the spermatozoa exhibit morphological defects of the head and tail, premature chromosomal condensation, and nuclear damage. However, the molecular functions of SEPT12 during spermatogenesis remain unclear. To determine the molecular functions of SEPT12, we applied a yeast 2-hybrid system to identify SEPT12 interactors. Seven proteins that interact with SEPT12 were identified: SEPT family proteins (SEPT4 and SEPT6), nuclear or nuclear membrane proteins (protamine 2, sperm-associated antigen 4, and NDC1 transmembrane nucleoproine), and sperm-related structural proteins (pericentriolar material 1 and obscurin-like 1). Sperm-associated antigen 4 (SPAG4; also known as SUN4) belongs to the SUN family of proteins and acts as a linker protein between nucleoskeleton and cytoskeleton proteins and localizes in the nuclear membrane. We determined that SEPT12 interacts with SPAG4 in a male germ cell line through coimmunoprecipitation. During human spermiogenesis, SEPT12 is colocalized with SPAG4 near the nuclear periphery in round spermatids and in the centrosome region in elongating spermatids. Furthermore, we observed that SEPT12/SPAG4/LAMINB1 formed complexes and were coexpressed in the nuclear periphery of round spermatids. In addition, mutated SEPT12, which was screened from an infertile man, affected the integration of these nuclear envelope complexes through coimmunoprecipitation. This was the first study that suggested that SEPT proteins link to the SUN/LAMIN complexes during the formation of nuclear envelopes and are involved in the development of postmeiotic germ cells.
doi:10.1371/journal.pone.0120722
PMCID: PMC4361620  PMID: 25775403
12.  A MEIG1/PACRG complex in the manchette is essential for building the sperm flagella 
Development (Cambridge, England)  2015;142(5):921-930.
A key event in the process of spermiogenesis is the formation of the flagella, which enables sperm to reach eggs for fertilization. Yeast two-hybrid studies revealed that meiosis-expressed gene 1 (MEIG1) and Parkin co-regulated gene (PACRG) interact, and that sperm-associated antigen 16, which encodes an axoneme central apparatus protein, is also a binding partner of MEIG1. In spermatocytes of wild-type mice, MEIG1 is expressed in the whole germ cell bodies, but the protein migrates to the manchette, a unique structure at the base of elongating spermatid that directs formation of the flagella. In the elongating spermatids of wild-type mice, PACRG colocalizes with α-tubulin, a marker for the manchette, whereas this localization was not changed in the few remaining elongating spermatids of Meig1-deficient mice. In addition, MEIG1 no longer localizes to the manchette in the remaining elongating spermatids of Pacrg-deficient mice, indicating that PACRG recruits MEIG1 to the manchette. PACRG is not stable in mammalian cells, but can be stabilized by MEIG1 or by inhibition of proteasome function. SPAG16L is present in the spermatocyte cytoplasm of wild-type mice, and in the manchette of elongating spermatids, but in the Meig1 or Pacrg-deficient mice, SPAG16L no longer localizes to the manchette. By contrast, MEIG1 and PACRG are still present in the manchette of Spag16L-deficient mice, indicating that SPAG16L is a downstream partner of these two proteins. Together, our studies demonstrate that MEIG1/PACRG forms a complex in the manchette and that this complex is necessary to transport cargos, such as SPAG16L, to build the sperm flagella.
Summary: In the manchette, a structure at the base of the elongating spermatid, the proteins MEIG1 and PACRG act in a complex to control cargo transport and direct formation of the flagellum.
doi:10.1242/dev.119834
PMCID: PMC4352978  PMID: 25715396
Cargo transport; Manchette; Sperm flagella; Spermiogenesis
13.  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
14.  Down regulation of SPAG9 reduces growth and invasive potential of triple-negative breast cancer cells: possible implications in targeted therapy 
Background
Recently, we reported an association of a novel cancer testis (CT) antigen, sperm-associated antigen 9 (SPAG9) expression in breast cancer clinical samples, indicating its potential role in carcinogenesis. Around 15% breast cancers are designated as triple-negative for which treatment modalities are limited. Therefore, in the present study, we assessed the role of SPAG9 in triple-negative breast cancer cells.
Methods
SPAG9 mRNA and protein expression was investigated in various breast cancer cells of different hormone receptor status and different subtypes by employing reverse transcriptase-polymerase chain reaction (RT-PCR), real time PCR, Western blotting, indirect immunofluorescence (IIF) and fluorescence activated cell sorting (FACS). Employing plasmid-based small interfering RNA (siRNA) approach, knockdown of SPAG9 was carried out in triple-negative breast cancer cells, MDA-MB-231, to assess its role on various malignant properties in vitro and in vivo.
Results
SPAG9 mRNA and protein expression was detected in all breast cancer cells. Further, IIF results showed that SPAG9 was predominantly localized in the cytoplasm of breast cancer cells. FACS analysis revealed distinct SPAG9 surface localization in breast cancer cells. Gene silencing of SPAG9 resulted in significant reduction in cellular proliferation, colony forming ability, migration, invasion and cellular motility of MDA-MB-231 cells. Further, ablation of SPAG9 expression resulted in reduction in the tumor growth of human breast cancer xenograft in nude mice in vivo.
Conclusions
In summary, our data indicated that down regulation of SPAG9 reduces growth and invasive potential of triple-negative breast cancer cells, suggesting that SPAG9 may be a potential target for therapeutic use.
doi:10.1186/1756-9966-32-69
PMCID: PMC3848771  PMID: 24330581
Migration; Invasion; CT antigens; SPAG9; Gene silencing
15.  Structural heterogeneity of the axonemes of respiratory cilia and sperm flagella in normal men. 
Journal of Clinical Investigation  1985;75(3):825-831.
The ultrastructure of normal human cilia and flagella was examined and quantitatively assessed to determine the normal variations in the structure of the axoneme. Ciliated respiratory epithelial cells and spermatozoa from 10 normal, nonsmoking male volunteers who had normal semen parameters were fixed for electron microscopy. Tannic acid and MgSO4 were included during fixation to enhance, in particular, axonemal components. In 75 axonemal cross sections per sample, the number of outer doublet and central singlet microtubules, outer and inner dynein arms, and radial spokes were recorded. Statistical analysis of the results showed a marked reduction, from the expected value of nine, in the numbers of inner dynein arms (mean +/- SE, cilia, 5.31 +/- 0.13; sperm, 5.38 +/- 0.16) and radial spokes (cilia, 4.95 +/- 0.22; sperm, 5.80 +/- 0.19). The ideal axoneme with all its structural components was seen in only 0.13% of cilia and 0.80% of sperm tails. Significantly more doublet microtubules (P less than 0.05) and less central microtubules (P less than 0.01) and radial spokes (P less than 0.01) were seen in cilia than in sperm tail axonemes. Between subjects there was little variation in the mean number of a structure seen per axoneme. However, within each sample, the variation was considerably higher, particularly for the inner and outer dynein arms and radial spokes. The doublet microtubules had significantly greater standard deviations in the sperm tails compared with the cilia (P less than 0.01), and furthermore, a significantly greater number of sperm tails compared with cilia showed the incorrect number of doublet microtubules (P less than 0.02). In one semen sample, with normal semen analysis, 20% of the sperm tails showed incorrect numbers of doublet microtubules, ranging from 12 + 2 to 5 + 2 compared with only 1.3% in cilia from this subject. This study has demonstrated that the ideal axoneme is rarely seen even in normal samples, probably because of the technical difficulties in resolution and visualization, and stresses the need for thorough documentation of axonemal ultrastructure. This work provides a normal data base for comparison with patients who have chronic respiratory disease and suspected infertility.
Images
PMCID: PMC423610  PMID: 3156880
16.  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
17.  MNS1 Is Essential for Spermiogenesis and Motile Ciliary Functions in Mice 
PLoS Genetics  2012;8(3):e1002516.
During spermiogenesis, haploid round spermatids undergo dramatic cell differentiation and morphogenesis to give rise to mature spermatozoa for fertilization, including nuclear elongation, chromatin remodeling, acrosome formation, and development of flagella. The molecular mechanisms underlining these fundamental processes remain poorly understood. Here, we report that MNS1, a coiled-coil protein of unknown function, is essential for spermiogenesis. We find that MNS1 is expressed in the germ cells in the testes and localizes to sperm flagella in a detergent-resistant manner, indicating that it is an integral component of flagella. MNS1–deficient males are sterile, as they exhibit a sharp reduction in sperm production and the remnant sperm are immotile with abnormal short tails. In MNS1–deficient sperm flagella, the characteristic arrangement of “9+2” microtubules and outer dense fibers are completely disrupted. In addition, MNS1–deficient mice display situs inversus and hydrocephalus. MNS1–deficient tracheal motile cilia lack some outer dynein arms in the axoneme. Moreover, MNS1 monomers interact with each other and are able to form polymers in cultured somatic cells. These results demonstrate that MNS1 is essential for spermiogenesis, the assembly of sperm flagella, and motile ciliary functions.
Author Summary
Cilia are microtubule-based structures present in virtually all cells in vertebrates. Cilia have diverse functions in development, growth, signaling, and fertilization. Primary ciliary dyskinesia (PCD) affects one in 16,000 individuals. PCD is characterized by bronchiectasis and chronic sinusitis, and is often associated with situs inversus and male infertility. The genetic cause of PCD is heterogeneous. Some cases of PCD in humans and animals are caused by single genic mutations such as mutations in genes encoding microtubule-based dynein arm components. We have characterized a protein called MNS1 and found that it plays an essential role in ciliary functions in mice. MNS1 is a novel and integral component of sperm flagella. Mice lacking MNS1 exhibit male sterility as evidenced by abnormal assembly of sperm flagella. MNS1–deficient mice also display defects in left–right asymmetry patterning of internal organs and hydrocephalus. Therefore, mutations in MNS1 may contribute to male infertility and PCD in humans.
doi:10.1371/journal.pgen.1002516
PMCID: PMC3291534  PMID: 22396656
18.  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
19.  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
20.  Spag17 Deficiency Results in Skeletal Malformations and Bone Abnormalities 
PLoS ONE  2015;10(5):e0125936.
Height is the result of many growth and development processes. Most of the genes associated with height are known to play a role in skeletal development. Single-nucleotide polymorphisms in the SPAG17 gene have been associated with human height. However, it is not clear how this gene influences linear growth. Here we show that a targeted mutation in Spag17 leads to skeletal malformations. Hind limb length in mutants was significantly shorter than in wild-type mice. Studies revealed differences in maturation of femur and tibia suggesting alterations in limb patterning. Morphometric studies showed increased bone formation evidenced by increased trabecular bone area and the ratio of bone area to total area, leading to reductions in the ratio of marrow area/total area in the femur. Micro-CTs and von Kossa staining demonstrated increased mineral in the femur. Moreover, osteocalcin and osterix were more highly expressed in mutant mice than in wild-type mice femurs. These data suggest that femur bone shortening may be due to premature ossification. On the other hand, tibias appear to be shorter due to a delay in cartilage and bone development. Morphometric studies showed reduction in growth plate and bone formation. These defects did not affect bone mineralization, although the volume of primary bone and levels of osteocalcin and osterix were higher. Other skeletal malformations were observed including fused sternebrae, reduced mineralization in the skull, medial and metacarpal phalanges. Primary cilia from chondrocytes, osteoblasts, and embryonic fibroblasts (MEFs) isolated from knockout mice were shorter and fewer cells had primary cilia in comparison to cells from wild-type mice. In addition, Spag17 knockdown in wild-type MEFs by Spag17 siRNA duplex reproduced the shorter primary cilia phenotype. Our findings disclosed unexpected functions for Spag17 in the regulation of skeletal growth and mineralization, perhaps because of its role in primary cilia of chondrocytes and osteoblasts.
doi:10.1371/journal.pone.0125936
PMCID: PMC4446355  PMID: 26017218
21.  Regulation of flagellar motility by the conserved flagellar protein CG34110/Ccdc135/FAP50 
Molecular Biology of the Cell  2011;22(7):976-987.
This study reports the function of a conserved flagellar protein CG34110, which has highly conserved orthologues in species containing motile cilia. It appears to represent a novel regulatory protein located on the outer doublet microtubules of the axoneme across a broad range of species.
Eukaryotic cilia and flagella are vital sensory and motile organelles. The calcium channel PKD2 mediates sensory perception on cilia and flagella, and defects in this can contribute to ciliopathic diseases. Signaling from Pkd2-dependent Ca2+ rise in the cilium to downstream effectors may require intermediary proteins that are largely unknown. To identify these proteins, we carried out genetic screens for mutations affecting Drosophila melanogaster sperm storage, a process mediated by Drosophila Pkd2. Here we show that a new mutation lost boys (lobo) encodes a conserved flagellar protein CG34110, which corresponds to vertebrate Ccdc135 (E = 6e-78) highly expressed in ciliated respiratory epithelia and sperm, and to FAP50 (E = 1e-28) in the Chlamydomonas reinhardtii flagellar proteome. CG34110 localizes along the fly sperm flagellum. FAP50 is tightly associated with the outer doublet microtubules of the axoneme and appears not to be a component of the central pair, radial spokes, dynein arms, or structures defined by the mbo waveform mutants. Phenotypic analyses indicate that both Pkd2 and lobo specifically affect sperm movement into the female storage receptacle. We hypothesize that the CG34110/Ccdc135/FAP50 family of conserved flagellar proteins functions within the axoneme to mediate Pkd2-dependent processes in the sperm flagellum and other motile cilia.
doi:10.1091/mbc.E10-04-0331
PMCID: PMC3069022  PMID: 21289096
22.  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
23.  Bug22 influences cilium morphology and the post-translational modification of ciliary microtubules 
Biology Open  2014;3(2):138-151.
Summary
Cilia and flagella are organelles essential for motility and sensing of environmental stimuli. Depending on the cell type, cilia acquire a defined set of functions and, accordingly, are built with an appropriate length and molecular composition. Several ciliary proteins display a high degree of conservation throughout evolution and mutations in ciliary genes are associated with various diseases such as ciliopathies and infertility. Here, we describe the role of the highly conserved ciliary protein, Bug22, in Drosophila. Previous studies in unicellular organisms have shown that Bug22 is required for proper cilia function, but its exact role in ciliogenesis has not been investigated yet. Null Bug22 mutant flies display cilia-associated phenotypes and nervous system defects. Furthermore, sperm differentiation is blocked at the individualization stage, due to impaired migration of the individualization machinery. Tubulin post-translational modifications (PTMs) such as polyglycylation, polyglutamylation or acetylation, are determinants of microtubule (MT) functions and stability in centrioles, cilia and neurons. We found defects in the timely incorporation of polyglycylation in sperm axonemal MTs of Bug22 mutants. In addition, we found that depletion of human Bug22 in RPE1 cells resulted in the appearance of longer cilia and reduced axonemal polyglutamylation. Our work identifies Bug22 as a protein that plays a conserved role in the regulation of PTMs of the ciliary axoneme.
doi:10.1242/bio.20146577
PMCID: PMC3925317  PMID: 24414207
Basal bodies; Cilia; Sperm individualization; Spermatogenesis; Tubulin post translation modifications
24.  Androgenic regulation of beta-defensins in the mouse epididymis 
Background
The majority of beta-defensin family members are exclusively expressed in the epididymis, and some members have been shown to play essential roles in sperm maturation and fertility in rats, mice and humans. Therefore, beta-defensins are hypothesized to be potential targets for contraception and infertility diagnosis and treatment. Clarifying the regulatory mechanisms for the expression of these genes is necessary. Androgen/androgen receptor (AR) signaling plays an important regulatory role in epididymal structure and function. However, very little is known about the androgenic regulation on the production and secretion of the epididymal beta-defensins.
Methods
The expression of beta-defensins was detected by quantitative RT-PCR. The androgen dependence of beta-defensins was determined by bilateral orchiectomy and androgen supplementation. The androgen response elements (AREs) in the promoters of beta-defensins were identified using the MatInspector software. The binding of AR to AREs was assayed by ChIP-PCR/qPCR.
Results
We demonstrated that 23 mouse caput epididymal beta-defensins were differentially regulated by androgen/androgen receptor. Six genes, Defb18, 19, 20, 39, 41, and 42, showed full regulation by androgens. Ten genes, Defb15, 30, 34, 37, 40, 45, 51, 52, 22 and Spag11a, were partially regulated by androgens. Defb15, 18, 19, 20, 30, 34, 37, 39, 41, 42, 22 and Spag11a were associated with androgen receptor binding sites in their promoter or intronic regions, indicating direct regulation of AR. Six genes, Defb1, 12, 13, 29, 35, and spag11b/c, exhibited an androgen-independent expression pattern. One gene, Defb25, was highly dependent on testicular factors rather on androgens.
Conclusions
The present study provides novel insights into the mechanisms of androgen regulation on epididymal beta-defensins, enabling a better understanding of the function of beta-defensins in sperm maturation and fertility.
doi:10.1186/1477-7827-12-76
PMCID: PMC4127520  PMID: 25099571
Androgen; Androgen receptor; Epididymis; Beta-defensins
25.  RAB-Like 2 Has an Essential Role in Male Fertility, Sperm Intra-Flagellar Transport, and Tail Assembly 
PLoS Genetics  2012;8(10):e1002969.
A significant percentage of young men are infertile and, for the majority, the underlying cause remains unknown. Male infertility is, however, frequently associated with defective sperm motility, wherein the sperm tail is a modified flagella/cilia. Conversely, a greater understanding of essential mechanisms involved in tail formation may offer contraceptive opportunities, or more broadly, therapeutic strategies for global cilia defects. Here we have identified Rab-like 2 (RABL2) as an essential requirement for sperm tail assembly and function. RABL2 is a member of a poorly characterized clade of the RAS GTPase superfamily. RABL2 is highly enriched within developing male germ cells, where it localizes to the mid-piece of the sperm tail. Lesser amounts of Rabl2 mRNA were observed in other tissues containing motile cilia. Using a co-immunoprecipitation approach and RABL2 affinity columns followed by immunochemistry, we demonstrated that within developing haploid germ cells RABL2 interacts with intra-flagella transport (IFT) proteins and delivers a specific set of effector (cargo) proteins, including key members of the glycolytic pathway, to the sperm tail. RABL2 binding to effector proteins is regulated by GTP. Perturbed RABL2 function, as exemplified by the Mot mouse line that contains a mutation in a critical protein–protein interaction domain, results in male sterility characterized by reduced sperm output, and sperm with aberrant motility and short tails. Our data demonstrate a novel function for the RABL protein family, an essential role for RABL2 in male fertility and a previously uncharacterised mechanism for protein delivery to the flagellum.
Author Summary
A greater understanding of the mechanism of male fertility is essential in order to address the medical needs of the 1 in 20 men of reproductive age who are infertile. Conversely, there remains a critical need for additional contraceptive options, including those that target male gametes. Towards the aim of filling these knowledge gaps, we have used random mutagenesis to produce the Mot mouse line and to identify RABL2 as an essential regulator of male fertility. Mice carrying a mutant Rabl2 gene are sterile as a consequence of severely compromised sperm motility. Using biochemical approaches we have revealed that RABL2 binds to components of the intraflagellar transport machinery and have identified a number of RABL2 binding (effector) proteins. The presence of the Mot mutation in RABL2 leads to a significantly compromised ability to deliver binding proteins into the sperm tail. RABL2 is predominantly produced in male germ cells; however, lower levels are notably produced in organs that contain motile cilia (hair like structures involved in fluid/cell movement), thus raising the possibility that RABL2 may be involved in a broader set of human diseases collectively known as primary cilia dyskinesia.
doi:10.1371/journal.pgen.1002969
PMCID: PMC3464206  PMID: 23055941

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