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Regular Article |
a Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, Suita City, Osaka 565-0871, Japan
b Department of Anatomy, University of Turku, FIN-20520, Turku, Finland
c Department of Molecular Genetics, Research Institute For Microbial Diseases, Osaka University, Suita City, Osaka 565-0871, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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sperm, sperm maturation, sperm motility and transport, spermatid, spermatogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The specific isolation of ODF and FS protein fractions from sperm tail and the preparation of antisera has made possible the general biochemical characterization of their components [6–12]. The majority of these proteins, ranging from Mr 14 to 97 x 10-3, are highly phosphorylated at serine residues [13, 14] and show resistance to solubilization in ionic detergents (e.g., SDS) due to the presence of disulfide bonds [15–17]. Although some antibodies are able to recognize proteins in both the FS and ODF fractions, suggesting at least the presence of similar antigen determinants [18, 19], the molecular identity of these antigenic materials is not clear.
During the second half of spermiogenesis, substructural components of the mammalian spermatozoa, including the FS, ODF, perforatorium, and connecting piece, are assembled in developing spermatids [15]. Because few of these proteins derive from stored mRNA transcribed early in the spermatocyte nucleus, haploid-specific transcripts are thus expected to code many structural proteins involved in the morphogenesis of spermatids into spermatozoa [20].
Here we present the cloning and characterization of shippo 1, a new gene of the mouse testis exclusively transcribed in haploid germ cells. shippo 1 cDNA encodes a highly hydrophobic putative protein, SHIPPO 1, which exists in the cytoplasm of spermatids and along the entire length of the sperm tail of mature spermatozoa. Further localization of SHIPPO 1 to the ODF and FS was ascertained by fractionation of the sperm proteins. We have also isolated the human orthologue, h-shippo 1. The homology between the mouse and human sequences suggests that SHIPPO 1 proteins have an important functional role in the sperm tail.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Total RNA was extracted from C57BL/6 mouse testis by the guanidine thiocyanate/CsTFA method, and poly(A)+ RNA was purified [21]. Preparation of the cDNA library was described elsewhere [22]. The cDNA library we used had a complexity of 4.0 x 106 colony forming units (cfus).
A haploid germ cell-specific cDNA library was generated by subtracting mRNA of 17-day-old testis from an adult (35-day-old) mouse testis cDNA library [22]. Plasmid DNAs of randomly selected clones from the subtracted library were screened by Northern blot analysis using testicular RNA from both 17- and 35-day-old mice.
One of these clones, designated ß390, which is expressed in the adult but not in the infantile mouse testis, was used as a probe to screen an adult mouse testis cDNA library in order to isolate its complete sequence (shippo 1). Escherichia coli MC1061 cells carrying the adult testis cDNA library of pAP3neo were diluted to seed a final concentration of 1 x 10 5 cfus on a nitrocellulose filter placed on an LB plate (Nacalai, Kyoto, Japan). Cells were grown at 37°C, and colonies were transferred to 2 nylon replica filters. Transferred colonies were lysed by sequentially soaking the filters in the following solutions at room temperature: 0.5 N NaOH/1.5 M NaCl (15 min); 0.5 M Tris HCl pH 7.4/1.5 M NaCl (15 min), and 2x SSC (15 min). Filters were then baked at 80°C for 2 h and washed to remove bacterial debris. A partial 1-kilobase (kb) EcoRI-NotI cDNA fragment (ß390) was radiolabeled with [
-32P]dCTP using a BcaBest random primer kit (Takara, Siga, Japan). Filters were hybridized in a solution containing 4x SSC, 10x Denhardt solution, 0.1% SDS, and 100 µg/ml of denatured, sonicated salmon sperm DNA for 18 h at 65°C. Four positive clones were isolated and sequenced with a Li-COR 4000 sequencer (Li-COR, Lincoln, NE) and a computer-mediated sequence search was performed using the GenBank, European Molecular Biology Laboratory, DNA Data Bank of Japan (DDBJ), and Swiss-protein databases. The human orthologue cDNA was isolated from a human testicular cDNA library constructed with plasmid vector pAP3neo [23] using the [-32P]dCTP-labeled full-length cDNA of mouse shippo 1 as a probe.
Northern Blot Analyses
Total RNA was extracted from various organs of C57BL /6 mice using RNAzol B (Tel-Test Inc., Friendswood, TX). The isolation of germ cells and other somatic cells of the testis was performed as previously described [24]. After quantification based on optical density, 20 µg of mRNA containing 2.2 M formaldehyde was separated on a 1.0% agarose gel in 0.66 M formaldehyde, transferred to nitrocellulose filters, and probed with the full-length shippo 1 cDNA. Hybridization using the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as a probe was performed to check RNA integrity. Filters were prehybridized for 4 h, and then hybridized overnight in 5x SSC, 5x Denhardt solution, 0.1% SDS, and 100 µg/ml of sheared salmon sperm DNA at 42°C with the labeled probe. Washes were performed at 60°C in 0.2x SSC and 0.1% SDS. Radioactive signals were detected using an Image Analyzer (Fuji Film, Tokyo, Japan).
Reverse Transcription-Polymerase Chain Reaction Analyses
First, a cDNA chain was generated using 5 µg of total RNA from mouse testes at different ages as a template (the Thermoscript reverse transcription-polymerase chain reaction [RT-PCR] system was provided by Gibco BRL, Rockville, MD). Messenger RNA-cDNA hybrids were then digested with RNaseH and used as templates for PCR amplification of a 240-base pair (bp) internal fragment of shippo 1 cDNA with specific primers (5'-CTCTACAGCAGCCCTGGACCCAAG-3' and 5'-CACCACAGGCAGCATGTATGCAGC-3'). PCR conditions were as follows: 25 cycles of denaturation at 96°C for 45 sec, annealing at 64°C for 45 sec, and extension at 72°C for 1 min. Amplification of the mouse nicotinamide adenine dinucleotide-dependent glycerol-3-phosphate dehydrogenase (G3PDH) gene was used as the control. Primers for the mouse G3PDH (5'-GGTCCAGGGGTTTCTTACTC-3' and 5'-AGGTCGGTGTGAACGGATTT-3') amplified a 1000-bp fragment under the following conditions: 94°C for 1 min, 54°C for 1 min, and 72°C for 1 min for 25 cycles. PCR products were separated in a 0.8% agarose gel, stained with ethidium bromide (0.5 mg/ml), and visualized under UV light.
Antibody
Digestion of the full-length shippo 1 cDNA with NcoI yielded a 720-bp fragment that covers 
of the coding region, from amino acid residue 1 to 202 of the shippo 1 open reading frame (ORF). This fragment was then subcloned in-frame into pET30a vector (Novagen, Madison, WI). The protein tagged with hexa-histidines at the N-terminus was expressed in E. coli BL21 treated with isopropyl-ß-D-thiogalactopyranoside. Purified SHIPPO 1 recombinant protein was obtained using His Bind Resin (Novagen) according to the manufacturer's protocol, and used as antigen to raise polyclonal antiserum in rabbits by injection with Gerbu Adjuvant (Gerbu Biotechnik, Heidelberg, Germany).
Western Blot Analyses
Protein samples of various organs of C57BL/6 mice were lysed in RIPA buffer (10 mM Tris-HCl pH 7.5, 0.15 M NaCl, 1% NP40, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, and 1 mM PMSF), centrifuged, and quantified. Mouse sperm from the epididymis and vas deferens were collected in PBS with 1 mM PMSF, filtered, and washed 3 times in PBS before being homogenized in sperm extraction buffer (SEB) containing 62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, and 5% ß-mercaptoethanol [25]. About 50 µg of protein per lane was separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) filters (Millipore, Bedford, MA). The filters were blocked overnight at 4°C with 5% skim milk in TBS-T (2 mM Tris HCl pH 7.5, 150 mM NaCl, 50 mM KCl, and 0.05% Tween 20), incubated with antibodies, diluted (1:500) in TBS-T, and then treated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) antibody (Amersham Pharmacia Biotech, Tokyo, Japan). After being extensively washed, filters were developed using a POD staining kit (Wako, Osaka, Japan).
Mouse Sperm Protein Fractionation
The sperm collected from 10 mice were fractionated according to an adapted protocol described previously [16]. Briefly, sperm washed from epididymis and vas deferens were submitted to 3 sequential extractions at 4°C in 500 µl of a solution containing 1% Triton X-100 and 2 mM dithiothreitol (DTT) in 50 mM sodium borate buffer at pH 9.0 for 40 min each. After each extraction, samples were centrifuged at 400 x g using a Tomy MRX-150 centrifuge (Tomy, Tokyo, Japan), and the supernatants were collected (membrane soluble fractions: M1, M2, and M3). The pellet was washed 3 times with 50 mM sodium borate buffer and suspended in 500 µl of a 0.6 M potassium thiocyanate (KSCN), 2 mM DTT, and 50 mM Tris-HCl pH 8.0 solution for 2 h at 4°C. After centrifugation at 800 x g, the supernatant was collected (central axoneme fraction—Ax) and the pellet extracted overnight at 4°C in 500 µl of a solution containing 4 M urea, 50 mM Tris-HCl pH 8.0, and 2 mM DTT. A final centrifugation at 14 200 x g was performed to separate the urea-extracted fraction (urea fraction—Ur). Finally, the resulting nonextracted pellet was washed in borate buffer 3 times, suspended in SEB, and sonicated on ice for 10 min (fibrous sheath, head fraction—FS/H). Protein concentrations of all fractions (M1, M2, M3, Ax, Ur, and FS/H) were estimated by the Bradford Protein Assay (Nacalai Tesque Inc., Kyoto, Japan). Fractions M3, Ax, and Ur were precipitated with 10% trichloroacetic acid. About 20 µg of protein of each fraction was separated by SDS-PAGE in a 12% polyacrylamide gel. Western blotting was performed as described above. Control antibodies for the membrane fraction (CD46) [26], FS/H fraction (anti-AKAP82) [27], and ODF fraction (anti-ODF-1) [28] at a final dilution of 500x were used for verifying the extracted proteins.
Immunohistochemistry
Testis and epididymis Testes fixed with Bouin solution were processed, and 4-µm sections were used for immunohistochemical analyses. The sections were sequentially blocked with the following solutions at room temperature for 30 min each: 5% skim milk, 5% whole goat serum in PBS, avidin blocking solution, and biotin blocking solution (Vector Laboratories, Burlingame, CA). After being washed with PBS, the sections on slides were incubated overnight at 4°C with anti-SHIPPO 1 antiserum (diluted 500x) or preimmune serum as a control (diluted 500x) in PBS. Anti-rabbit IgG of goat serum conjugated with biotin was used as secondary antibody. Sections were then treated with avidin/biotin (Vector Laboratories) and developed with diaminobenzidine. After being counterstained with hematoxylin, sections were examined under a light microscope.
Sperm Mouse sperm from the vas deferens and caudal epididymis suspended in PBS were filtered through nylon mesh and centrifuged at 400 x g. The pellet was washed in PBS, and a few drops were placed on glass slides and dried at 55°C for 10 min. The slides were blocked with 5% skim milk for 30 min followed by a 5% goat whole serum in PBS for 30 min at room temperature. Drying and blocking conditions were kept the same for all immunohistochemical proceedings. Blocked samples were incubated with anti-SHIPPO 1 rabbit serum or preimmune serum, both diluted (500x) in PBS overnight at 4°C. After a wash, sections were treated with diluted (50x) anti-rabbit IgG goat serum conjugated with rhodamine or fluorescein isothiocyanate (FITC) for 1 h at room temperature. Slides were then washed and examined under a fluorescent microscope. Triton X-100 and urea-treated sperm obtained from the fractionation of sperm proteins were also investigated immunohistochemically using the same protocol.
In order to visualize individual fibers of the ODF, mature sperm were treated with a solution containing 10 mM Tris-HCl, 30 mM ß-mercaptoethanol, 0.2 mM PMSF, and 0.05% cetyltrimethylammonium bromide (CTAB), a cationic detergent that under reducing conditions extracts all tail structures except for the ODFs, which are released from the tight native form [13], for 1 h at room temperature. Colocalization of SHIPPO 1 with ODF-1 and ODF-2 proteins was accomplished by incubating intact and CTAB-treated sperm with anti-SHIPPO 1 antibody that had been conjugated to FITC with a Fluorotag FITC conjugation kit (Sigma Chemical Company, St. Louis, MO) overnight at 4°C. After being thoroughly washed in PBS, samples were incubated with anti-ODF-1 monoclonal antibody [28] or anti-ODF-2 rabbit polyclonal antibody for 2 h at room temperature, washed in PBS, and treated with diluted (50x) anti-mouse (ODF-1) or rabbit (ODF-2) IgG antibody conjugated with rhodamine for 1 h at room temperature.
Squashed preparations of living cells Segments of seminiferous tubules of 0.5–1 mm in length from stages I–XII were identified by transillumination, microdissected under a stereomicroscope, and transferred to glass slides with 15 µl of PBS [29]. A coverslip (18 x 18 mm) was carefully placed over the tubule segment, avoiding the formation of air bubbles. The excess fluid was removed by blotting, which allowed cells of the seminiferous epithelium to float out. The slides were monitored under phase contrast optics (40x) to adjust the slightly flattened monolayer. The edge of the coverslip was then carefully sealed with paraffin oil to achieve a complete immobilization of cells. Germ cell types were identified based on morphological criteria [30] under phase-contrast optics with a 100x oil immersion objective. After ascertaining the respective stage of spermatogenesis, dissected tubules were rapidly frozen in liquid nitrogen for a few seconds, fixed with 70% ethanol on ice for 10 min, and used for immunocytochemistry as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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From a subtracted cDNA library of mouse testis [22], we isolated a new haploid germ cell-specific cDNA clone of 406 bp, designated ß390. Because this clone did not have a full-length ORF as judged from the size of the band obtained on Northern blotting (1.2 kb) and from sequence analysis, we rescreened an adult mouse testicular cDNA library of pAP3neo using ß390 as a probe. All 4 independently isolated clones were of similar size (1.2 kb) and had the same putative ORF (DDBJ accession number AB067773; Fig. 1A). A stop codon was present 42 bp upstream from the first Met, and the entire ORF consisted of 762 nucleotides (corresponding to nt 112 to 781 of the mouse sequence) encoding a predicted 254 amino acids. A polyadenylation signal was also found 80 bp downstream from the stop codon. We named this new gene shippo 1, and its predicted protein, SHIPPO 1. Using shippo 1 as a probe, we screened a human testis cDNA library and isolated 5 independent clones carrying approximately 1.5 kb of human cDNA sequence (DDBJ accession number AB067774; Fig. 1A). The human cDNA (h-shippo 1) showed a longer 3' untranslated region, but was otherwise similar to the mouse sequence (i.e., a 762 bp ORF encoding a 254 amino acid putative protein [hSHIPPO 1] and a polyadenylation signal at nt 1372 as in the mouse). The predicted amino acid sequence of the human cDNA is 93% identical to the mouse sequence (Fig. 1A). A computer-assisted search using the predicted amino acid sequence of isolated mouse cDNA revealed homology with 2 new D. melanogaster sequences; 32% identity with CG8086 (GenBank accession number AE003620), and 31% identity with CG10252 (GenBank accession number AE003744), whose functions and localization in the fly have not yet been addressed. Although no previously described domains were found in isolated mouse and human cDNAs, a series of Pro-Gly-Pro repeats were present in both the human and mouse proteins as well as in CG8086 and CG10252 (Fig. 1B).
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A basic local alignment search in GenBank using the human orthologue sequence localized the gene in the short arm of human chromosome 11p15.5 (GenBank accession number NT009407.3) spread over 200 kb in a region in synteny with mouse chromosome 7 (between LOC51238 and LOC51272 contigs), where such genes as Fadd (Fas-associated protein with death domain), Tnnt3 (troponin T3), Igf2 (insulin growth factor 2), and the tumor suppressor gene, H-19, are also located.
Messenger RNA Expression of the Mouse Shippo 1 in Various Organs and in Germ Cells
A transcript of about 1.2 kb was present specifically in the mouse testis (Fig. 2A) and was restricted to the germ cell fraction, but absent in somatic cell fractions such as Sertoli and Leydig cells (Fig. 2B). To investigate the developmental changes of gene expression in the mouse testis, RT-PCR was performed using total RNA of the testis at the ages of 2–5, 8, 17, 23, 29, and 35 days. Transcription was found to start in the third week postpartum (23 days) after haploid germ cells developed, and to increase with age (Fig. 2C).
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Western Blotting and Immunohistochemical Analyses of SHIPPO 1 Protein
Rabbit polyclonal antiserum raised against histidine-tagged recombinant SHIPPO 1 was used for Western blot analysis and to investigate the localization of SHIPPO 1 in the testis. A single band of about Mr 32 x 10-3 was exclusively detected in the testis (Fig. 3A). The positive signal was specific to the cytoplasmic fraction of germ cells and not detected in somatic cells such as Sertoli or Leydig cells (Fig. 3B). During germ cell development, SHIPPO 1 was first detected at the age of 4 wk, then increased in the adult testis, where it was eventually detected in sperm extracts (Fig. 3C). Immunohistochemical preparation of testicular cross-sections showed a positive signal in the tail of elongated spermatids sticking out toward the tubular lumen, and also in cytoplasmic droplets still attached to the spermatid tail membrane just before release from the seminiferous epithelium (Fig. 4, A–C). Mature spermatozoa that had moved to the epididymis also showed positive signals in the tail as well as in the discarded cytoplasmic droplets (Fig 4, D and E). Isolated mouse sperm from the vas deferens showed positive signal from the midpiece, extending through the principal and end pieces of the tail (Fig. 4, F and G). Due to its presence in the sperm tail, we named this new protein SHIPPO 1, from the Japanese word for "tail". No positive signal was detected when preimmune serum was used (data not shown). To investigate the appearance of SHIPPO 1 in individual germ cells, squashed preparations from tubules at specific stages of the spermatogenic cycle were used, and isolated cells were submitted to immunohistochemistry with anti-SHIPPO 1 antibody (Fig. 5). Although the absence of SHIPPO 1 was confirmed in premeiotic germ cells (spermatocytes) (Fig. 5, A and a), it was detected in the Golgi-acrosome region of early haploid cells (round spermatids at step 4), where the development of the tail had not started yet (Fig. 5, B and b). In late round spermatids, where the tail precursor is visible and the cells have acquired a polarized asymmetry, the signal was detected clearly in the Golgi region, which by then had drifted apart from the acrosome vesicle and also in some of the vesicles scattered in the cytoplasm (Fig. 5, C and c). It is interesting that a limited region of the membrane in these cells was also marked, especially the posterior area, where the new tail is erupting. As the elongating spermatid approaches the top layer of the tubular epithelium, the cytoplasm shrinks and the development of the major structures of the spermatozoa is almost complete. At this terminal stage of spermatid maturation, SHIPPO 1 protein was restricted to the tail (Fig. 5, D and d). In the mature sperm in the epididymis, positive signal was also confined to the tail region (Fig. 5, E and e).
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Western Blotting and Immunostaining Analysis of Subcellularly Fractionated Sperm
To further examine the localization of SHIPPO 1 in the sperm (membrane/cytoplasm, axoneme, ODF, or FS), the subcellular fractionation of sperm proteins was performed [16]. Soluble fractions and the final FS-H unsolubilized fraction were separated by SDS-PAGE, transferred to a filter, and subjected to Western Blotting with anti-SHIPPO 1 antibody. SHIPPO 1 was resistant to nonionic detergent (Triton X-100) extraction and potassium thiocyanate treatment, but could mostly be solubilized with urea. The majority of SHIPPO 1 was indeed recovered in the ODF (urea-extracted) fraction, although some protein remained in the nonextracted pellet (FS and head fraction; Fig. 6). Although AKAP82, an FS protein, was present in the FS fraction and not the ODF fraction, ODF1, the major ODF protein [28], was only extracted with urea (Fig. 6). SHIPPO 1 was extracted together with the core of the cytoskeletal elements that make up the ODF and FS. To confirm these observations, the presence of SHIPPO 1 in sperm treated with 0.05% CTAB (1 h at room temperature) and in samples from fractionation experiments after 1% Triton X-100 (1 h at room temperature) and 4 M urea (15 min at room temperature) treatments, was investigated by immunocytochemistry (Fig. 7). Nontreated sperm showed the basic staining pattern of SHIPPO 1, the entire length of the sperm tail being homogeneously stained (Fig. 7, A, a, B, and b). It has been shown that in sperm treated with 1% Triton X-100, membranes, organelles (including the mitochondrial sheath in the middle piece), and cytosolic proteins are solubilized, the head bends over due to the weakened support offered by the neck, and the ODF fibers can sometimes be seen sticking out from the weakened annulus region [16]. In Triton X-100-treated sperm, SHIPPO 1 signal was observed in the fibers projecting from the annulus (Fig. 7, C and c) as well as along the rest of the tail, indicating that SHIPPO 1 was associated with the ODF. As determined by fractionated Western blotting, treatment of sperm with 4 M urea efficiently extracted most of the ODF protein [16] together with SHIPPO 1 from the sperm tail. After only 15 min exposure to urea, SHIPPO 1 signal was scattered, and the fiberlike view of SHIPPO 1 in freed ODF was lost (Fig. 7, D and d).
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In sperm that were heated at 55°C but not fixed chemically, ODF-1 and ODF-2 were seen as scattered patches in contrast with the continuous SHIPPO 1 signal along the tail (Fig. 8, A and B). After 1 h of CTAB treatment, sperm released individual ODFs that were set free from the central core of the tail, spraying apart distally to the connecting piece, although some were still bound together to some degree in the middle piece (Fig. 8C). In these incompletely extracted sperm, the fibers were strongly labeled with anti-ODF-2 antibody, but the SHIPPO 1 signal was progressively lost in the principal piece and exhibited a dotlike distribution in the middle and connecting pieces (Fig. 8C). Most sperm heads were separated from the tail; resistant ODFs were completely frayed and joined together only at the connecting piece [13]. Whereas ODF-2 signal was clearly seen in individual fibers and the connecting piece, SHIPPO 1 signal weakened in the fibers (Fig. 8D) until it was expressed only in the connecting piece, colocalizing with ODF-2 (Fig. 8E). Taken together, these results demonstrated that SHIPPO 1 associated with ODFs and the connecting piece in a nonionic detergent-resistant form (Triton X-100). In the fibrous proteins known to constitute the ODF, SHIPPO-1 signal eventually disappeared following exposure to an ionic detergent (CTAB). The association of SHIPPO 1 with the connecting piece, however, appeared to be stronger than that to the fibers, because SHIPPO 1 in this region was not extracted even by CTAB treatment.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Here we have cloned and characterized a new sperm tail protein, SHIPPO 1, from a haploid-specific cDNA library of mouse testis and its human orthologue, hSHIPPO 1. The 11p15 band, to which the human gene is mapped, harbors many tumor suppressor genes, and loss of heterozygosity in this region is frequently associated with different types of human cancers (e.g., Wilms tumors) [35]. Furthermore, several paternally (IGF2, LIT1, PEG8, MTR1) and maternally (IPL, IMPT1, P57, KVLQT1, ASCL1, H19) imprinted genes are also clustered at 11p15.5, constituting a major imprinted chromosome domain in the human genome [36]. It is during gametogenesis that allele-specific epigenetic blueprints of imprinted genes are initially erased to be reintroduced before fertilization. Whether shippo 1, a gene solely expressed in male germ cells, is imprinted or not deserves further investigation.
The 93% identity in predicted amino acid sequence between mouse and human SHIPPO 1 suggests that these proteins are indispensable and under strong selection pressure. Fifty-two percent of residues of SHIPPO 1 are hydrophobic, especially proline, which alone accounts for 13% of the total number of residues. SHIPPO 1, together with the homologous D. melanogaster proteins CG8086 and CG10252, show 6 Pro-Gly-Pro repeats, which, interestingly, are evenly distributed along the length of these proteins. Taking into account that proline is a known -helix breaker residue, and that the predicted secondary structure of SHIPPO 1 is a highly coiled protein, we speculate that Pro-Gly-Pro regions play an important role in the final conformation of these molecules. Although no concrete information exists concerning the transcription pattern and protein localization of CG8086 and CG10252 in D. melanogaster, it may be reasonable to classify these 3 sequences into a new protein family.
SHIPPO 1 starts to be transcribed in early haploid spermatids and the mRNA is translated as early as step 4, when it accumulates in the Golgi and is subsequently driven to the cytoplasmic lobe of late round and early elongating spermatids (Fig. 5). Although not a common feature among cytoskeletal proteins of the sperm tail, ODF- and FS-associated proteins are localized to the Golgi of early spermatogenic cells before targeting the tail [25, 37]. Moreover, the murine hexokinase type 1 (HK1-sc) is present in the Golgi and later also targets the fibrous sheath, although lacking a classical Golgi-target motif like SHIPPO 1 [38].
SHIPPO 1 occupies all of the tail of mature sperm, from the connecting piece proximal to the head, along the middle and principal pieces, down to the distal end piece (Fig. 5E). This widespread localization suggests that SHIPPO 1 is not a particular component of any one individual element (e.g., ODF, FS, or connecting piece), but rather associates with different structures that characterize the domains of the sperm tail. Consistent with this assumption, immunohistochemistry revealed that the major signal of SHIPPO 1 is associated with the fibers of the tail cytoskeleton, in particular ODF, and the connecting piece (Figs. 6–8). Thus, SHIPPO 1 protein was partially resistant to ionic (CTAB) detergent, and completely resistant to nonionic (Triton X-100) detergent, a common feature of cytoskeletal proteins of the sperm tail. The treatment of demembranated sperm with urea is a well-described procedure for specifically extracting components of the ODF [16]. Indeed, the bulk of SHIPPO 1 was extracted with urea under reducing conditions, indicating that the protein is related to ODF elements (Figs. 6 and 7D). However, treatment with 0.05% CTAB, which does not solubilize ODF fibers, even under reducing conditions [13], was able to extract most of the SHIPPO 1 seen to associate with ODFs in the middle and principal pieces, but not the connecting piece, where SHIPPO 1 still colocalized with ODF-2 (Fig. 8, C–E). The connecting piece itself represents a modified continuation of the ODF and would be expected to share structural proteins with the ODF [39]. Further, in contrast to the continuity of the signal for SHIPPO 1, antibodies against ODF-1 and ODF-2 that strongly detected individual ODFs in CTAB-treated sperm, showed a discontinuous distribution of signal along the tail of intact sperm (Fig. 8, A and B). In fact, our results revealed that in heated sperm but not in CTAB-treated sperm, the accessibility of ODF epitopes to their respective antibodies seems to be somehow limited. In contrast, SHIPPO 1 epitopes are equally accessible in intact (heated) and fully demembranated (Triton X-100 treated) sperm. Hence, although associated with the tail cytoskeleton, SHIPPO 1, or at least part of it, likely occupies an outer position in these fibers (Fig. 7C). Electron microscopy would clarify its exact position. Thus, the staining patterns in dried, Triton X-100-treated and CTAB-treated sperm indicate that, although not a classical constitutive protein of the ODFs, SHIPPO 1 nevertheless directly or indirectly associates with these fibers in the middle and principal pieces and is also present in an extraction-resistant form in the connecting piece region. The difference in susceptibility to extraction by CTAB in the middle/principal piece and in the connecting piece implies that SHIPPO 1 probably associates differently with the cytoskeletal structures in these 2 tail regions, or that it is rendered inaccessible to CTAB on occupying an internal domain in the connecting piece.
ODF-1 (ODF27, RT7), a major ODF component, is a Mr 27 x 10-3 protein characterized by Cys-Gly-Pro repeats at its C-terminal end [28, 40]. It is curious that it was initially identified as the rat orthologue of a D. melanogaster sperm protein (mst(3)gl-9), which also carries Cys-Gly-Pro repeats [41]. Indeed, although ODF and FS are exclusively present in mammals, the sperm of certain insects, including D. melanogaster, have a remarkably similar structure (accessory fibers) that is also assembled in a similar fashion to the ODF around the axoneme [42, 43]. In fact, at least 5 such male-specific transcripts (msts) of D. melanogaster, grouped in the Mst(3)CGP gene family, are clustered on chromosome 3 of the fly and encode proteins with Cys-Gly-Pro repeats [43]. Taking into account the presence of Pro-Gly-Pro repeats identified here, it would be worthwhile to investigate whether CG8086 and CG10252 are the D. melanogaster counterparts of the mammalian SHIPPO 1 in the fly's accessory fibers, as a step to better understanding the origin of these structures.
ODF-1 is able to homodimerize partially through an amphipathic leucine zipper domain in the N-terminus [44], which can also be used to bind to leucine zippers of Spag4 [33] (an axonemal microtubule-binding protein) and ODF-2 (another major ODF protein) [45]. It is surprising that ODF-2 (ODF 84), which was previously described as a sperm tail-specific protein [46], was recently shown to be a widespread scaffold component of the centrosome in somatic cells, where it is likely involved in microtubule nucleation during mitosis and maturation of daughter centrioles [47]. Moreover, ODF-2 has 2 leucine zippers, but only one is committed to ODF-1 binding. Considering that ODF-1 is restricted to the medulla of the ODF, whereas ODF-2 is present in both the cortex and medulla, a role as a linker protein has been proposed for ODF-2 in the fibers of the sperm tail [45]. In one of the present models, ODF-2 would bind ODF-1, and a third, still nonidentified protein through its second leucine zipper domain [48]. In fact, association via leucine zippers appears to be a general mechanism among cytoskeletal proteins of the flagellum [39]. Although SHIPPO 1 protein does not have such a domain, it nevertheless shows weak homology to an -collagen-related protein of Leishmania major (GenBank accession number AL352980). Collagen and related fibrous proteins are rich in proline residues that play a role in stabilizing strand conformation of the fibril after hydroxylation in the cytoplasm. In these proteins, Gly-Prohydr-Pro repeats form -chain structures that associate to generate the typical triple-strand helix conformation of collagenlike fibers. Elucidating whether SHIPPO 1 is able to self-associate or associates to other structural proteins would provide a clue as to a possible cytoskeletal role.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by a grant from the Ministry of Education, Science and Culture of the Government of Japan (Mombusho). C.E. de C. was the recipient of a Foreigner Graduate Student Scholarship from Mombusho. 
2 Correspondence: Yoshitake Nishimune, Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita City, Osaka 565-0871, Japan. FAX: 81 6 6879 8339; nishimun{at}biken.osaka-u.ac.jp
Accepted: October 26, 2001.
Received: August 2, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  K. Tokuhiro, M. Hirose, Y. Miyagawa, A. Tsujimura, S. Irie, A. Isotani, M. Okabe, Y. Toyama, C. Ito, K. Toshimori, et al. Meichroacidin Containing the Membrane Occupation and Recognition Nexus Motif Is Essential for Spermatozoa Morphogenesis J. Biol. Chem., July 4, 2008; 283(27): 19039 - 19048. [Abstract] [Full Text] [PDF]
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 A. Yano, K. Suzuki, and G. Yoshizaki Flow-Cytometric Isolation of Testicular Germ Cells from Rainbow Trout (Oncorhynchus mykiss) Carrying the Green Fluorescent Protein Gene Driven by Trout vasa Regulatory Regions Biol Reprod, January 1, 2008; 78(1): 151 - 158. [Abstract] [Full Text] [PDF]
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 Y. Liu, N. Pathak, A. Kramer-Zucker, and I. A. Drummond Notch signaling controls the differentiation of transporting epithelia and multiciliated cells in the zebrafish pronephros Development, March 15, 2007; 134(6): 1111 - 1122. [Abstract] [Full Text] [PDF]
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I. Nagamori, K. Yomogida, P. D. Adams, P. Sassone-Corsi, and H. Nojima Transcription Factors, cAMP-responsive Element Modulator (CREM) and Tisp40, Act in Concert in Postmeiotic Transcriptional Regulation J. Biol. Chem., June 2, 2006; 281(22): 15073 - 15081. [Abstract] [Full Text] [PDF]
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 Y. Nishimune and H. Tanaka Infertility Caused by Polymorphisms or Mutations in Spermatogenesis-Specific Genes J Androl, May 1, 2006; 27(3): 326 - 334. [Full Text] [PDF]
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 R. M. Zayas, A. Hernandez, B. Habermann, Y. Wang, J. M. Stary, and P. A. Newmark The planarian Schmidtea mediterranea as a model for epigenetic germ cell specification: Analysis of ESTs from the hermaphroditic strain PNAS, December 20, 2005; 102(51): 18491 - 18496. [Abstract] [Full Text] [PDF]
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K. Kitamura, N. Iguchi, Y. Kaneko, H. Tanaka, and Y. Nishimune Characterization of a Novel Postacrosomal Perinuclear Theca-Specific Protein, CYPT1 Biol Reprod, December 1, 2004; 71(6): 1927 - 1935. [Abstract] [Full Text] [PDF]
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K. Kitamura, H. Tanaka, and Y. Nishimune Haprin, a Novel Haploid Germ Cell-specific RING Finger Protein Involved in the Acrosome Reaction J. Biol. Chem., November 7, 2003; 278(45): 44417 - 44423. [Abstract] [Full Text] [PDF]
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A. Miranda-Vizuete, K. Tsang, Y. Yu, A. Jimenez, M. Pelto-Huikko, C. J. Flickinger, P. Sutovsky, and R. Oko Cloning and Developmental Analysis of Murid Spermatid-specific Thioredoxin-2 (SPTRX-2), a Novel Sperm Fibrous Sheath Protein and Autoantigen J. Biol. Chem., November 7, 2003; 278(45): 44874 - 44885. [Abstract] [Full Text] [PDF]
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Y. Nakamura, H. Tanaka, M. Koga, Y. Miyagawa, N. Iguchi, C. E. de Carvalho, K. Yomogida, M. Nozaki, H. Nojima, K. Matsumiya, et al. Molecular Cloning and Characterization of oppo 1: A Haploid Germ Cell-Specific Complementary DNA Encoding Sperm Tail Protein Biol Reprod, July 1, 2002; 67(1): 1 - 7. [Abstract] [Full Text] [PDF]
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