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J. Biol. Chem., Vol. 278, Issue 45, 44874-44885, November 7, 2003
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From the aCenter for Biotechnology, Department of Biosciences at Novum, Karolinska Institutet, S-14157 Huddinge, Sweden, the cDepartment of Anatomy and Cell Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada, the eDepartment of Developmental Biology, Tampere University Medical School, and the Department of Pathology, Tampere University Hospital, Fin-33101 Tampere, Finland, the gDepartment of Cell Biology, University of Virginia, Charlottesville, Virginia 22908, and the iDepartments of Animal Science and Obstetrics and Gynecology, University of Missouri, Columbia, Missouri 65211-5300
Received for publication, May 24, 2003 , and in revised form, July 9, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The primary function of the spermatozoon is to contribute the paternal half of chromosomes to the offspring. In higher vertebrates, the complexity of the fertilization process is increased by the advent of internal fertilization and by the functional adaptations of the oocyte vestments. To circumvent these hurdles, the spermatozoon has acquired a highly specialized morphology comprising cytoskeletal components, which appear to have no structural counterpart in somatic cells. The most evident among these cytoskeletal structures are the perinuclear theca of the sperm head and the outer dense fibers (ODFs)1 and fibrous sheath (FS) of the sperm tail (13–15). The perinuclear theca is a condensed layer of selected cytoplasmic proteins that are sandwiched between the nuclear envelope and the inner acrosomal membrane apically and between the nuclear envelope and the plasma membrane caudally. It is assumed to play a pivotal role in acrosomal-nuclear docking, nuclear shaping, and sperm-oocyte interactions (16, 17). ODFs surround the axoneme in the middle and principal pieces of the sperm tail. In the middle piece, nine ODFs associate with the corresponding microtubule doublets of the axoneme, whereas in the principal piece, ODF3 and ODF8 are fused to the two longitudinal columns of the FS, which are bridged by FS ribs and together surround the seven remaining ODFs (14, 18).
The function of the ODFs and FS is not fully elucidated, but it seems to be related to the control of flagellar motility (13, 19) and protection against shearing forces during epididymal transit (20). However, evidence for an active rather than a merely structural role of the ODFs and FS in sperm function is increasing, supported by the fact that, among their constituent proteins, there are several molecules displaying either enzymatic or regulatory functions. For example, in the FS, glyceraldehyde 3-phosphate dehydrogenase produces ATP via the glycolytic pathway. This is critical for the transition to hyperactivated motility as well as capacitation (21). Moreover, the initiation and maintenance of sperm motility are regulated by a cascade of phosphorylation/dephosphorylation events (reviewed in Ref. 22). In this respect, AKAP82 (A kinase-anchoring protein of 82 kDa), also located in the FS, presumably tethers cAMP-dependent protein kinase A, directing and specifying the actions of the kinase in close proximity to the sperm's axonemal machinery (reviewed in Ref. 23). Both the ODFs and FS are composed of many different polypeptides, of which only a few have been cloned and characterized to date. ODF1 (also known as RT7 and ODF27) (24–26), ODF2 (also named ODF84) (27–29), ODF3 (30), TPX-1 (31), and SAK57 (32) are among the characterized ODF proteins. A similar number of FS proteins have been described in mammalian spermatozoa: AKAP4 (also named p82, FSC1, and FS75) (33–38), AKAP3 (FS95) (37–39), TAKAP80 (40), glutathione S-transferase (41), glyceraldehyde 3-phosphate dehydrogenase-S (42), type I hexokinase (43), ropporin (44), rhophilin (45), FS39 (46), and CABYR (47). In addition, some other proteins found in the sperm tail, such as SHIPPO1 and OPPO1 (48, 49), may contribute to the molecular makeup of the FS and ODFs.
In this context, we have recently described the identification and characterization of SPTRX-1 (spermatid-specific thioredoxin-1), a novel member of the thioredoxin family of proteins that transiently associates with the longitudinal columns of the FS during sperm tail assembly (8, 50). A second member of the thioredoxin family named SPTRX-2 has also been identified in the human sperm tail and is composed of an N-terminal thioredoxin domain followed by three NDP kinase domains (9). We report here the cloning of mouse and rat Sptrx-2, their patterns of development during spermiogenesis, and their exact localizations within the sperm tail. SPTRX-2 incorporates into both the ribs and longitudinal columns of the FS in the last steps of spermatid development and remains as an integral FS component in mature epididymal spermatozoa. Its expression pattern contrasts with the transient nature of SPTRX-1 expression during FS formation (50). Finally, we identify SPTRX-2 as a novel sperm autoantigen after vasectomy.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Northern Blot Analysis and in Situ Hybridization—A mouse multiple-tissue Northern blot and mouse RNA Master blots with poly(A)+ RNA from different tissues were purchased from Clontech. The mouse Sptrx-2 open reading frame was labeled with [
-32P]dCTP (Rediprime random primer labeling kit, Amersham Biosciences) and hybridized at 60 °C overnight in UltrahybTM solution following the protocol provided by Ambion Inc. The blot was also hybridized with mouse 
-actin as a control. The blots were scanned and quantified with the Gel Pro Analyzer program (Media Cybernetics). For in situ hybridization, mouse and rat testes were frozen on dry ice, sectioned with a Microm HM500 cryostat at 14 µm, and thaw-mounted onto Polylysine glass slides (Menzel). The sections were stored at -20 °C until used. Four different probes (based on mouse and rat Sptrx-2 cDNA sequences, respectively) were used. Because all produced similar results when used separately, they were usually used simultaneously to intensify the signal. Several control probes with the same length and similar GC content and specific activity were used to determine the specificity of the hybridization. In situ hybridization was carried out as described previously (52).
Production of Recombinant SPTRX-2 Protein and Affinity-purified Antibodies—The open reading frame encoding human SPTRX-2 was cloned into the BamHI-EcoRI sites of the pGEX-4T-1 expression vector (Amersham Biosciences) and used to transform Escherichia coli BL21(DE3). A single positive colony was inoculated in 1 liter of LB medium plus ampicillin and grown at 37 °C to A600 = 0.5. The production and purification of the fusion protein were performed as described previously (9). Protein concentration was determined from the absorbance at 280 nm using a molar extinction coefficient of 47,330 M-1 1 cm-1. Purified glutathione S-transferase-human SPTRX-2 was used to immunize rabbits (Zeneca Research Biochemicals). After six immunizations, sera from rabbits were purified by ammonium sulfate precipitation. Affinity-purified antibodies were prepared using a cyanogen bromide-activated Sepharose 4B column (Amersham Biosciences) to which 0.5 mg of recombinant SPTRX-2 had been coupled using the procedure recommended by the manufacturer. Antibody specificity was tested by Western blotting using recombinant SPTRX-2 and testicular cell extracts.
Mouse/Rat Testicular and Epididymal Sample Preparation, Immunocytochemistry, and Electron Microscopy—Adult male Sprague-Dawley rats and CD-1 mice were anesthetized, and the testes and epididymides were fixed by perfusion through the abdominal aorta and heart, respectively, with 0.8% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer containing 50 mM lysine (pH 7.4), with 4% paraformaldehyde (mice only), or in Bouin's fixative (for light microscopy). Tissues destined for Lowicryl (SPI Supplies, West Chester, PA) or LR White (Polysciences, Inc., Warrington, PA) embedding (for electron microscopy) were immersed in the respective fixatives for 2 h at 4 °C, washed three times with phosphate buffer, and incubated with phosphate buffer containing 50 mM NH4Cl for 1 h at 4 °C. Tissues were subsequently washed with buffer, dehydrated in graded methanol up to 90%, and infiltrated and embedded in Lowicryl K4M or LR White. Thin sections were mounted on Formvar-coated nickel grids for immunogold labeling. Rat and mouse tissues fixed in Bouin's fixative were washed extensively with 75% alcohol before being completely dehydrated in ethanol and embedded in paraffin. For light microscopic immunocytochemistry, 5-µm paraffin sections were deparaffinized and hydrated through a graded series of ethanol concentrations before immunoperoxidase localization with anti-SPTRX-1 antibody by standard procedures (14). For electron microscopic immunocytochemistry, ultrathin Lowicryl or LR White sections on Formvar-coated nickel grids were immunogold-labeled according to the procedure of Oko et al. (53). Staging of the cycle of the seminiferous epithelium and determining the steps of spermiogenesis were done according to the classifications of Leblond and Clermont (54).
For immunofluorescence, mouse testes were isolated, cut into small pieces (5 x 5 x 5 mm), and fixed by immersion in 4% paraformaldehyde. After vigorous repeated washing with phosphate-buffered saline and sucrose, the tissues were embedded in paraffin using conventional histochemical techniques and sectioned. Four-millimeter-thick sections were mounted on slides, dewaxed by xylene and ethanol changes, treated for autofluorescence reduction in 1% picric acid in a steamer, and blocked for 30 min in 5% normal goat serum (Sigma) in phosphate-buffered saline. In the same trials, mouse epididymides were removed, and the spermatozoa were released by gentle mincing into Tyrode's albumin lactate pyruvate/HEPES medium. Spermatozoa were collected by a 5-min centrifugation at 350 x g and attached to poly-L-lysinecoated coverslips (22 x 22 mm). A labeling solution composed of phosphate-buffered saline with 1% normal goat serum and 0.1% Triton X-100 was used for antibody dilutions, incubations, and washes.
Immunofluorescence processing of paraffin sections was performed on a histology tray under humid and dark conditions. Samples were first incubated for 40 min with anti-SPTRX-2 serum (diluted 1:100) and then washed by submersion for 5 min and incubated with a mixture of red fluorescent TRITC-labeled goat anti-rabbit IgG (diluted 1:80; Zymed Laboratories Inc., South San Francisco, CA) and 2.5 µg/ml 4,6-diamidino-2-phenylindole (Molecular Probes, Inc. Eugene, OR). After washing, the samples were mounted on slides (whole mounted spermatozoa on coverslips) or covered with coverslips (tissue sections on slides) in Vectashield mounting medium (Vector Labs, Inc.). Negative controls were performed by the replacement of the first antibody with nonimmune rabbit serum or with anti-SPTRX-2 serum saturated with recombinant SPTRX-2. Samples were examined in a Nikon Eclipse 800 microscope. Images were captured with a CoolSnap HQ RTE/CCD 1217 digital camera using MetaMorph software.
Western Blot Analysis of Sperm Autoantibodies and the FS—Adult male Lewis rats (225–275 g; Charles River Laboratories, Wilmington, MA) received a bilateral vasectomy as described previously (55). Sera were collected prior to vasectomy and 3 months after the operation. Sperm were prepared from Lewis rat cauda epididymides by backflushing fluid through the vas deferens (55).
For two-dimensional gel electrophoresis, sperm were pelleted by centrifugation and solubilized in sample buffer containing 7 M urea, 2 M thiourea, 0.2% Nonidet P-40, 100 mM dithiothreitol, and 0.2% pH 3.5–10 Ampholine (Amersham Biosciences). Aliquots of the extracts containing 150–200 µg of protein were applied to a Protean IEF cell (Bio-Rad) with pH 3–10 immobilized pH gradient strips for the first dimension isoelectric focusing. After active rehydration of immobilized pH gradient strips with sample buffer at 50 V, they were focused at 250 V for 15 min and stepped up to 8000 V until 30,000 total V-h were reached (
6 h). SDS-PAGE on 8–15% gels in the second dimension was performed as described previously (56), and proteins were stained on the gels with Coomassie Blue or were electroblotted onto nitrocellulose membranes (56). For Western blot analysis, membranes containing sperm proteins were washed and blocked with milk. Primary pre- or post-obstruction sera (diluted 1:250) were incubated with the membranes overnight at 4 °C and exposed to the secondary antibody (diluted 1:5000), horseradish peroxidase-conjugated goat anti-rat IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Other two-dimensional blots of sperm proteins were incubated with a 1:25,000 dilution of affinity-purified rabbit antiserum raised against recombinant SPTRX-2. These blots were subsequently exposed to horseradish peroxidase-conjugated goat anti-rabbit IgG (diluted 1:5000), washed, and treated with the peroxidase substrate 3,3',5,5'-tetramethylbenzidine and 0.01% H2O2 (Kirkegaard & Perry Laboratories, Gaithersburg, MD).
For one-dimensional Western blots of recombinant SPTRX-2, the protein was solubilized in Laemmli buffer (57). Three micrograms of protein were loaded per lane for staining with post-vasectomy serum, and 50 ng were loaded per lane for use with anti-SPTRX-2 serum. SDS-PAGE was conducted as described previously (58); and after transfer to nitrocellulose, the membranes were exposed to post-vasectomy rat serum (diluted 1:250) or to anti-SPTRX-2 serum (diluted 1:100,000), followed by the appropriate secondary antibody and development of the reaction product as described for two-dimensional analysis. Controls consisted of pre-vasectomy rat serum, preimmune rabbit serum (for anti-SPTRX-2 antibody), and omission of the postvasectomy serum. Isolation procedures, SDS-PAGE (under reduced conditions), Western blotting, and immunoblotting of sperm head and tail fractions including the FS were done as described by Yu et al. (50).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A comparison of the mouse and rat SPTRX-2 protein sequences with the PROSITE Database (59) identified, along with the above-mentioned thioredoxin and NDP kinase domains, several potential phosphorylation sites and a PEST sequence for ubiquitin/proteasome-dependent degradation centered at residue 242. This result is identical to that obtained with human SPTRX-2 and supports a common regulatory mechanism for the three orthologs.
To determine the genomic organization, the mouse and rat Sptrx-2 cDNA sequences were BLAST-searched against their respective data bases at NCBI. The results obtained indicate that the mouse Sptrx-2 gene is present in the supercontig NT_039578
[GenBank]
and that the rat Sptrx-2 gene is found in the supercontig NW_043107; and in both cases, the genomic organization is identical to that of the reported human SPTRX-2 gene (9), being composed of 17 exons and spanning 52 kb, with all of the introns conforming to the GT/AG rule. The mouse Sptrx-2 gene maps at chromosome 13A3.3 between markers D13Mit154 and D13Mit79, very close to the mouse Sfrp4 locus (60), although running in the opposite direction (Fig. 2). The rat Sptrx-2 gene is also found in close proximity to the rat Sfrp4 locus, which has been map at chromosome 17q12.1. These positions are syntenic with that of the human gene at chromosome 7p14.1 (9).
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Murine Sptrx-2 mRNA Is Exclusively Expressed in the Testis—Multiple-tissue Northern blots were used to determine the size and tissue distribution of mouse Sptrx-2 mRNA using the open reading frame as a probe. Mouse Sptrx-2 mRNA was detected in the testis only as a single band of 2.5 kb (Fig. 3A), which is in good agreement with the size of the cloned cDNA plus addition of the poly(A)+ tail (GenBankTM/EBI accession number AF548543
[GenBank]
). In contrast to our findings on human SPTRX-2 mRNA (9), mouse RNA blots did not require as long an exposure time to identify a hybridization signal of comparable intensity. To evaluate the possibility that mouse Sptrx-2 mRNA could be expressed during fetal development or in other tissues, we also screened an RNA dot blot containing poly(A)+ RNA from 18 adult tissues and four fetal stages. Of the tissues examined, a hybridization signal was observed only in the testicular mRNA (Fig. 3B). To further investigate the expression pattern of murine Sptrx-2 mRNA, in situ hybridization was performed in mouse and rat testicular sections. Mouse Sptrx-2 mRNA was first detected in some (but not all) seminiferous tubules of testes from 3-week-old mice, whereas the vast majority of the tubules from 4-week-old or adult mice were labeled. In contrast, testes from 2-week-old pre-pubertal mice were not labeled and neither were the control sections of adult liver (Fig. 3C). Similarly, analysis of adult rat testicular sections showed strong labeling in all seminiferous tubules (Fig. 3D). In dipped sections of mouse testis, a strong signal was seen in the middle part of the seminiferous epithelium (Fig. 3E). At higher magnification, the strongest labeling corresponded to round spermatids, with a low-to-moderate signal in late primary spermatocytes and secondary spermatocytes (Fig. 3F). No signal was evident in the remaining testicular tissue or when control probes were used. This expression pattern is consistent with the in situ data reported for human SPTRX-2 mRNA (9).
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SPTRX-2 Protein Is Expressed during the Final Steps of Spermiogenesis—We have previously determined that human SPTRX-2 protein is mostly expressed in round and elongating spermatid stages of spermatogenesis (9). However, the temporal sequence of expression was difficult to determine at the histological level because of the intermingling of stages within the human seminiferous tubular cross-sections. Therefore, we performed a detailed SPTRX-2 expression analysis in rat and mouse testicular sections, in which the stages of the seminiferous epithelium are easily discernible within each seminiferous tubular cross-section. As shown in Fig. 4A, SPTRX-2 immunostaining became prominent in the cytoplasmic lobe of step 15 spermatids in stage I of the seminiferous epithelium cycle (see Fig. 10 for orientation of rat spermatid steps), remained so until step 18, and diminished in step 19. On the other hand, tail immunoreactivity gradually increased through steps 15–18 and peaked in step 19 spermatids. By step 19 (stage VII), it was evident that immunoperoxidase labeling was confined to the distal part of the tail, i.e. the principal piece (Fig. 4B). Preincubation of anti-SPTRX-2 antibody with recombinant SPTRX-2 eliminated the immunostaining (Fig. 4C) and hence confirmed the specificity of the immunoreactivity of antibodies raised against human SPTRX-2 in rat testicular tubular sections. Mouse testis showed an expression pattern similar to that in rat testis (data not shown).
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SPTRX-2 Is Incorporated into the Spermatid FS and Is an Integral FS Component of Mature Spermatozoa—To identify those structures in the developing spermatid tail with which SPTRX-2 associates, immunofluorescence microscopy was first performed on mouse testis and epididymal spermatozoa. As shown in Fig. 5 (A–C), SPTRX-2 was readily detectable in late spermatids in mouse testicular tissue sections, similar to the rat developmental analysis described above. In mouse epididymal sections, SPTRX-2 was found in the tails of spermatozoa (Fig. 5, D and E), and isolated spermatozoa clearly showed labeling in the tail principal piece compatible with localization to the FS (Fig. 5, F–H). During epididymal transit, SPTRX-2 was also found in the cytoplasmic droplet before and after it was shed from the distal mid-piece of the tail (Fig. 5, F and H) in what appears to be a common route for elimination of residual FS proteins (77). Pre-absorbed antibody with the recombinant protein was used as a negative control and demonstrated the specificity of the labeling (Fig. 5, I and J).
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To characterize in detail the developmental localization of SPTRX-2, a complete survey of the spermatid steps was carried out by immunogold electron microscopy of the rat testicular tissue sections (Fig. 6, A–E). Immunogold labeling first appeared in the tails of steps 14 and 15 spermatids and was randomly scattered throughout the cytoplasm of the axoneme (Fig. 6A). The development of the principal piece in these steps and earlier is characterized by the assembly of FS precursor material (FS anlagen). It assembles in a distal-to-proximal direction along the plasmalemma of the tail and precedes the assembly of the ribs and columns of the FS that follow in its wake (61). In these steps, neither the anlagen nor the more distally located mature FS was immunolabeled (Fig. 6A). From a structural point of view, the assembly of the FS is almost completed in step 16; and therefore, it is surprising that the labeling first began to appear over the FS at this step of spermiogenesis (Fig. 6B). During steps 17 and 18, the immunogold labeling progressively increased over both the ribs and longitudinal columns of the FS, and a much lesser labeling was found free in the cytoplasmic lobe surrounding the flagellum (Fig. 6C). In step 19 spermatids, labeling of the FS reached its peak (Fig. 6D). From observing the immunolabeling of tangential sections through the principal piece at this step, it was apparent that SPTRX-2 was incorporated deeply through the ribs and longitudinal columns of the FS (Fig. 6E). Confirmation that SPTRX-2 is an integral component of the mature FS came from the analysis of spermatozoa in the cauda epididymis, where immunogold labeling of SPTRX-2 was found throughout the FS (Fig. 7A). Preincubation of anti-SPTRX-2 antibody with recombinant SPTRX-2 blocked this signal (Fig. 7B). Immunoblots of isolated FS from mature spermatozoa verified SPTRX-2 as an authentic constituent of the FS (Fig. 8). Interestingly, SPTRX-2 was resistant to extraction by urea under reducing conditions, a property of FS proteins that facilitates selective isolation of the FS from other sperm tail components.
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Components of the sperm accessory structures (ODFs and FS) of mammalian spermatozoa are detected frequently as autoantigens in screens of sperm proteins with post-obstruction sera (62). Because rat SPTRX-2 was found to be a component of the FS, we asked whether it is recognized by antibodies in post-obstruction sera generated by performing vasectomies on rats (55). On two-dimensional Western blots of rat sperm extracts, the region between 65 and 84 kDa at pI 5.0–5.7, where SPTRX-2 is expected to migrate, was strongly stained both by anti-SPTRX-2 serum (Fig. 9A) and by post-vasectomy serum (Fig. 9D). Comigration of protein spots stained by anti-SPTRX-2 and post-vasectomy sera suggested that SPTRX-2 is an auto- and/or isoantigen in rats. To confirm this, Western blot analysis was conducted on one-dimensional polyacrylamide gels of human recombinant SPTRX-2 protein (Fig. 9, C and F). Anti-SPTRX-2 and post-vasectomy sera both reacted with a recombinant protein band at 68 kDa as well as with bands at lower molecular masses that are believed to be breakdown products of SPTRX-2. When blots of recombinant SPTRX-2 were stained with a panel of 13 post-vasectomy rat sera, 9 of the 13 sera (69%) showed a positive reaction: four bound a 68-kDa protein; two reacted with bands at 68 and 17 kDa; and five stained the putative breakdown product of 17 kDa.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The murid SPTRX-2 protein has a domain organization identical to that of its human counterpart (9): an N-terminal thioredoxin domain followed by three NDP kinase domains, the first of which is incomplete, lacking the typical active site of this class of enzymes. Interestingly, this domain organization is found not only in mammals, but also in lower organisms such as the intermediate chain-1 protein from the sea urchin sperm axoneme (64) and the ascidian Ciona intestinalis intermediate chain-3 flagellar protein (65). In contrast, a search of animal model data bases including Drosophila and zebrafish failed to identify the corresponding orthologs. Moreover, whereas these proteins are part of the flagellum axoneme in sea urchins and ascidians, mammalian SPTRX-2 is located in the FS, a cytoskeletal structure that envelopes the sperm axoneme. It is thus tempting to speculate that, during evolution, the SPTRX-2 ancestor shifted from an axonemal to a periaxonemal localization. Because we have recently identified another protein in human axonemal structures (sperm flagella and lung cilia) that is composed of one N-terminal thioredoxin domain followed by only one NDP kinase domain, it is possible that this protein might substitute functionally for the SPTRX-2 ancestor in the axoneme (10). Such an axonemal-to-FS transition has been proposed recently due to the fact that the DnaHc8 gene, which codes for a mouse testis-specific axonemal dynein heavy chain, might also code for an unusual isoform that could play a role in FS morphogenesis (66).
In addition to the axoneme, the mammalian sperm tail contains unique structural components such as ODFs and FS, which are synthesized during spermiogenesis and are involved in the regulation of sperm motility (14). By electron microscopic immunocytochemistry, we have determined that SPTRX-2 is an integral component of the sperm FS. Fig. 10 illustrates the expression pattern of SPTRX-2 during spermiogenesis in comparison with the pattern found for the majority of sperm FS proteins to date (14, 35, 77), including the transiently expressed thioredoxin SPTRX-1 (50). As shown, SPTRX-2 is incorporated into the FS after the bulk of FS proteins have already assembled to form it. Interestingly, the incorporation of SPTRX-2 into the FS during steps 17–19 is concomitant with the gradual disappearance of SPTRX-1 from the longitudinal columns of the FS. This replacement of one thioredoxin for another in the FS indicates a specific developmental/maturational function for each of these thioredoxins. Equally intriguing is that the FS has the appearance of a very electron-dense structure by step 17, yet is still penetrable by proteins. In this context, it is possible that not until the incorporation of SPTRX-2 into the FS is intensive disulfide bonding of this structure initiated. Alternatively, the S–S cross-linking could be delayed even further after SPTRX-2 incorporation into the FS and could occur during final sperm maturation in the epididymis.
Protein spots stained by anti-SPTRX-2 and post-vasectomy rat sera comigrated on two-dimensional Western blots of sperm extracts, and identification of SPTRX-2 as an autoantigen was subsequently confirmed by demonstrating that post-vasectomy rat serum bound recombinant SPTRX-2. Several characteristics of SPTRX-2 may be related to its antigenicity in vasectomized animals. Like other proteins expressed only during spermatogenesis, SPTRX-2 may be autoantigenic because it is not produced until after puberty, thus avoiding identification as a self-protein during the earlier development of the immune system (67). In addition, SPTRX-2 is associated with the FS, and components of the sperm cytoskeleton such as the ODFs are known to be prominent post-obstruction autoantigens (62), perhaps because the cytoskeleton is among the most degradation-resistant components of spermatozoa after obstruction, thus furnishing a supply of slowly released antigen to stimulate the immune system.
Both thioredoxins and NDP kinases are regarded as catalytic molecules, the first one being involved in redox reactions as a general disulfide reductase (1) and the second one catalyzing the transfer of 
-phosphates between nucleoside and deoxynucleoside di- and triphosphates (68). Surprisingly, we have not been able to detect any thioredoxin or NDP kinase enzymatic activity using recombinant SPTRX-2 expressed in bacteria (9). To our knowledge, neither activity has been detected in isolated sea urchin and ascidian intermediate chain-1 proteins (64, 65), suggesting that additional factors are required.
Although the thioredoxins are traditionally regarded to be general protein-disulfide reductases (1), disulfide bond formation (rather than reduction) is a major requirement for the stabilization of the different components of the cytoskeletal structures that provide the scaffolding for the sperm head and tail skeleton (69). The presence of two novel thioredoxins (SPTRX-1 and SPTRX-2) that are associated with the sperm FS at different times during its assembly raises important questions regarding the requirement of this class of proteins in spermiogenesis. We have recently reported that recombinant SPTRX-1 can act both as a reducing and an oxidizing protein (8, 70). This may facilitate the formation of disulfide bonds among individual components of the FS as well as destabilize the misfolded and/or improperly incorporated sperm tail proteins. However, we have not been able to demonstrate any thioredoxin-dependent activity (either reducing or oxidizing) using recombinant SPTRX-2. Several reasons might account for this lack of activity in vitro, such as the requirement for additional cofactors not present in bacteria, post-translational modifications that might expose the active site upon conformational changes, etc. Nevertheless, given its FS localization and expression pattern during spermiogenesis, it is reasonable to speculate that SPTRX-2 might play a crucial role in disulfide bonding within the FS.
It is also conceivable that, although SPTRX-1 may fulfill a transitional function in sperm flagellar differentiation/biogenesis during spermiogenesis (50), SPTRX-2 may be necessary for post-testicular events such as epididymal sperm maturation, hyperactivation/capacitation, or even fertilization and zygotic development. In this regard, we have shown previously that the sperm tail FS, where SPTRX-2 resides, is one of the first sperm structures to be degraded in the zygotic cytoplasm upon fertilization. Solubilization of the FS precedes the degradation of paternal mitochondria and ODFs and coincides with the early stages of male pronuclear development (71). Extensive studies demonstrated the requirement of disulfide bond reduction for the successful processing of the sperm nucleus and sperm accessory structures during mammalian fertilization (72), and SPTRX-2 might help in regulating this process.
Furthermore, although we could not detect any kinase activity with recombinant SPTRX-2, its well conserved NDP kinase domain supports the idea that SPTRX-2 could be a phosphate donor for the phosphorylation of other FS proteins. Phosphorylation is the major regulatory mechanism in spermatozoa and underlies important processes during the acquisition of fertilizing capability by the spermatozoon, such as capacitation and hyperactivation (73). Interestingly, the major protein in the sperm FS is AKAP4, whose proposed function is to recruit/anchor protein kinase A to the FS to facilitate phosphorylation of proteins that regulate sperm flagellar motility (74). SPTRX-2 has several potential sites of phosphorylation by protein kinase A and other kinases and could be associated with the scaffold formed by AKAP4 and structural proteins of the sperm FS. The recent characterization of the mouse AKAP4 knockout phenotype suggests that AKAP4 does not affect the initial formation of the longitudinal columns and connecting ribs that characterize the mature FS, but AKAP4 does affect later steps of its assembly, leading to incomplete FS development (74). In this context, SPTRX-2 could then be incorporated into the fully differentiated FS as an associated protein of AKAP4, which remains in the mature FS and might play a role in later events undergone by the sperm by virtue of its proposed thioredoxin/kinase activity.
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FOOTNOTES |
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* This work was supported in part by grants from the Canadian Institutes of Health Research and National Sciences and Engineering Research Council of Canada (to R. O.) and by Swedish Medical Research Council Projects 03P-14096, 03X-14041, and 13X-10370 and grants from the Åke Wibergs Stiftelse and the Karolinska Institutet (to A. M.-V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
b Both authors contributed equally to this work.
d Supported by the Fundación Margit and Folke Pehrzon.
f Supported by the Research Fund of the Tampere University Hospital, Tampere University.
h Supported by NIDDK Grant P50 DK45179 and NICHD Grant HD U54-29009 from the National Institutes of Health.
j Supported by the Food for the 21st Century Program of the University of Missouri, United States Department of Agriculture Grants 2002-02069 and 99-35203-7785, and National Institute for Occupational Safety and Health Grant OH07324-01.
k To whom correspondence should be addressed. Tel.: 613-533-2858; Fax: 613-533-2566; E-mail: ro3{at}post.queensu.ca.
1 The abbreviations used are: ODFs, outer dense fibers; FS, fibrous sheath; TRITC, tetramethylrhodamine isothiocyanate; contig, group of overlapping clones.
2 Available at www.ncbi.nlm.nih.gov/.
3 Available at www.ncbi.nlm.nih.gov/Traces/trace.cgi.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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