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Regular Article |
a Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada
b Center for Biotechnology, Department of Biosciences at NOVUM, Karolinska Institutet, S-14157 Huddinge, Sweden
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, spermatid, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The study of the function of ODF and FS has been hampered by the properties of their polypeptide constituents, because they are resistant to solubilization in ionic detergents due to a high content of disulfide bridges. Incorporation of molecular biology techniques into the field of spermatology has succeeded in identifying some of the proteins that organize the ODF and FS. The mammalian ODF is composed of numerous polypeptides [4, 5], of which only several have been cloned and characterized, such as ODF1 (also known as RT7 and ODF27) [6–8], ODF2 (ODF84) [9–11], Tpx-1 [12], and Sak57 [13]. The number of FS proteins appears to be in the same range as those of the ODF [4, 14], and to our knowledge, only a few have been described to date: AKAP4 (p82, Fsc1, FS75) [15–20], AKAP3 (FS95) [19, 21], TAKAP80 [22], glutathione S-transferase [23], glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) [24], type I hexokinase [25], ropporin [26, 27], rhophilin [28], and FS39 [29].
The function of both ODF and FS is assumed to be the provision of elastic rigidity and directionality to sperm tail movements as well as protection against the shearing forces generated during sperm transit through epididymis [30]. However, evidence is accruing for a more active role of ODF and FS in sperm motility. For example, in the FS, GAPDS produces ATP via the glycolytic pathway, which is critical for the transition to hyperactivated motility as well as capacitation [31]. Moreover, the initiation and maintenance of sperm motility are regulated by a cascade of phosphorylation/dephosphorylation events [32]. In this respect, AKAP of the FS tethers cAMP-dependent protein kinase A, directing and specifying the actions of the kinase in close proximity to the sperm's axonemal machinery [33].
We have recently reported the cloning and characterization of spermatid-specific thioredoxin-1 (Sptrx-1), a novel member of the thioredoxin family of proteins and the first one with a tissue-specific distribution in human germ cells [34]. The Sptrx-1 is composed of an N-terminal domain consisting of a 15-amino-acid residue motif repeated 23 times, followed by a C-terminal domain typical of thioredoxins, and is able to reduce disulfide bonds in the presence of thioredoxin reductase, a well established enzymatic assay for thioredoxin activity [35]. Expression of Sptrx-1 is restricted to the postmeiotic phase of spermatogenesis, being first detected at the round/elongating spermatid stage in human testis sections. In human ejaculated sperm, we have demonstrated that Sptrx-1 localizes mainly to the cytoplasmic droplet and residual cytoplasm of the sperm tail [34].
To gain more insight regarding the function of Sptrx-1 during spermiogenesis, we have carried out a complete developmental analysis of Sptrx-1 expression in rodent testis and spermatozoa by light and electron microscopy. The results obtained show that Sptrx-1 exclusively localizes to the two longitudinal columns of the FS during sperm tail assembly. However, this association is only transient, because the protein dissociates from the mature FS and is degraded in the spermatid cytoplasm. To our knowledge, this is the first report of a spermatid-specific protein with such a particular expression profile. The possible functions of Sptrx-1 in light of this localization are discussed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The open reading frame encoding human Sptrx-1 was cloned into the BamHI-EcoRI sites of the pGEX-4T-1 expression vector (Pharmacia Biotech, Uppsala, Sweden) and used to transform Escherichia coli BL21(DE3). A single positive colony was inoculated in 1 L of LB medium plus ampicillin and grown at 37°C until A600 = 0.5. The production of the fusion protein was induced by addition of 0.5 mM isopropyl-ß-D-thiogalactoside, and growth was continued for another 3.5 h. Overexpressing cells were harvested by centrifugation and frozen until use. The cell pellet was resuspended in 40 ml of 20 mM Tris-HCl, 1 mM EDTA, and 150 mM NaCl plus protease-inhibitor cocktail at the concentration recommended by the manufacturer (Sigma Aldrich, Stockholm, Sweden). Lysozyme was added to a final concentration of 0.5 mg/ml with stirring for 30 min on ice. Next, 1% (w/v) sarkosyl was added, cells disrupted by 10-min sonication, and the supernatant cleared by centrifugation at 15 000 x g for 30 min and loaded onto a glutathione sepharose 4B column (Pharmacia Biotech). Binding to the matrix was allowed to occur for 2 h at room temperature. Thrombin (5 U/mg fusion protein) was used to remove glutathione S-transferase (GST) by incubation overnight at 4°C. The resulting protein preparation was then subjected to ion-exchange chromatography using a HiTrap Q column (Pharmacia Biotech), and human Sptrx-1 was eluted using a gradient of NaCl. Protein concentration was determined from the absorbance at 280 nm using a molar extinction coefficient of 7690 M-1 cm-1.
Purified GST-hSptrx-1 was used to immunize rabbits. After six immunizations, serum from rabbits was purified by ammonium sulfate precipitation. Affinity-purified antibodies were prepared using a cyanogen bromide-activated Sepharose 4B column, onto which 0.5 mg of recombinant Sptrx-1 N-terminal domain had been coupled using the procedure recommended by the manufacturer (Pharmacia Biotech). Specificity of the antibodies was tested by Western blot analysis using recombinant Sptrx-1 and testis cell extracts.
Mouse Rat Testis and Epididymis Sample Preparation, Immunocytochemistry, and Electron Microscopy
Adult male Sprague-Dawley rats and CD mice (Charles River, St-Constant, QC, Canada) were anesthetized and the testis and epididymides were fixed by perfusion through the abdominal aorta and heart, respectively, either with 0.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer containing 50 mM lysine (pH 7.4; for electron microscopy) or in Bouin fixative (for light microscopy). Human testes obtained from autopsies at the Kingston General Hospital were cut into pieces and fixed by immersion in Bouin fixative. Tissues destined for Lowicryl embedding (for electron microscopy) were immersed in the respective fixatives for 2 h at 4°C, washed three times in phosphate buffer, and incubated with phosphate buffer containing 50 mM NH4Cl for 1 h at 4°C. Tissues were subsequently washed in buffer, dehydrated in graded methanol up to 90%, and infiltrated and embedded in Lowicryl K4M (Chemische Werke Lowi, Waldkraiburg, Germany). Thin sections were mounted on Formvar-coated nickel grids (Polysciences Inc., Warrington, PA) for immunogold labeling. Bouin-fixed tissues were washed extensively in 75% alcohol before being completely dehydrated in ethanol and embedded in paraffin. Paraformaldehyde-fixed tissues were washed in phosphate buffer, dehydrated, and embedded in paraffin.
For light microscopic immunocytochemistry, paraffin sections (thickness, 5 µm) were deparaffinized and hydrated through a graded series of ethanol concentrations before immunoperoxidase localization with anti-Sptrx-1 antibody by standard procedures [2]. For electron microscopic immunocytochemistry, ultrathin Lowicryl sections on Formvar-coated nickel grids (Polysciences Inc.) were immunogold labeled according to the procedure of Oko et al. [36].
Staging the cycle of the seminiferous epithelium and determining the steps of spermiogenesis were done according to the classifications of Leblond and Clermont [37].
Isolation of Sperm Heads, Tails, and FS
Sperm obtained from human ejaculates and rat epididymides were washed twice by centrifugation at 500 x g at 4°C in 25 mM Tris-buffered saline (TBS; pH 7.5) containing 0.01 M PMSF and then sonicated on ice to separate sperm heads and tails. The suspension was then washed twice by centrifugation, and the resulting pellet was resuspended in TBS containing 80% sucrose and centrifuged at 200 000 x g for 1 h in a 60-Ti angle rotor (Beckman, Mississauga, ON, Canada). Sperm head and tail pellets obtained from the outside and inside walls, respectively, of the rotor tubes were resuspended in sucrose, and the whole centrifugation step was repeated to gain >99% purity of respective fractions (see [2] for details). The tail fraction was then used to isolate the FS according to previously published protocols used in our laboratory [2–4].
SDS-PAGE and Western Blot Analysis
The SDS-PAGE and electroblotting of protein onto polyvinylidene fluoride membrane were performed according to the method of Oko [2]. The blot was stained with amido black for protein profile. After destaining, the blot was blocked with 10% NGS (normal goat serum) in 25 mM TBS for 20 min, incubated with primary antibody in 5% NGS in TWBS (25 mM TBS with 0.05% Tween 20) at 4°C overnight, and immunoreactivity detected with horse radish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Vector, Burlingame, CA) diluted 1:10 000 (v/v) using enhanced chemiluminescent substrate (Pierce, Rockford, IL) with exposure to x-ray film. The blot was then stripped of antibodies using 100 mM ß-mercaptoethanol, 62.5 mM Tris-HCl (pH 6.7), and 2% SDS at 56°C for 30 min and reprobed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because human recombinant Sptrx-1 was used to produce the immune serum [34], we first analyzed its immunoreactivity in human testicular tissue sections. Immunoperoxidase staining revealed that Sptrx-1 expression was restricted to spermiogenesis, being most prominently expressed in elongated spermatids and with relatively weaker staining in the cytoplasm of developing round spermatids (Fig. 1A). 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. Hence, the expression of Sptrx-1 was analyzed in detail in rat and mouse testicular sections, in which the stages of the seminiferous epithelium are clearly delineated within each seminiferous tubular cross-section. Furthermore, performing a developmental analysis in rodents facilitates sample procurement and treatment. Previous work in our laboratories had shown that affinity-purified antibodies raised against human Sptrx-1 were able to identify by Western blot analysis a band of similar size in testis extracts from rat, mouse, and bull [34]. To further demonstrate the specificity of this cross-reactivity, testicular tubular sections from rat were incubated with affinity-purified antibodies directly or preadsorbed with human recombinant Sptrx-1. As shown in Figure 1, B and C, rat seminiferous tubules immunostained mostly in the tail region of elongating spermatids, which was abolished when using the preadsorbed antibody preparation (Fig. 1D). No staining was detected in the presence of preimmune serum (data not shown). Further demonstration of the specificity of the antibodies came from the immunogold labeling of the cytoplasmic droplet of rat spermatozoa, because preadsorbtion of the antibodies precluded any signal (see Fig. 5, A and B). Mouse testis showed a distribution pattern of Sptrx-1 labeling similar to that in the rat (not shown).
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Sptrx-1 Is Expressed During Spermiogenesis
Although the following description of Sptrx-1 expression pertains to rat testis, a similar pattern of expression was found in the mouse. As in humans, Sptrx-1 expression was restricted to the postmeiotic phase of spermatogenesis, but unlike humans, it first began in elongating spermatids (Fig. 1, B and C). The first detectable immunoreaction was seen in the spermatid tails during stages IX and X (steps 9 and 10) and was prominent in this region during stages XI–III (steps 11–16); the tail reactivity was also accompanied by a weaker immunostaining in the cytoplasmic lobe of these spermatids (Fig. 1, B and C). During the last steps of spermiogenesis (stages V–VII, steps 17–19), Sptrx-1 reactivity diminished drastically in the tail but appeared to increase or become concentrated in the shrinking cytoplasmic lobe (Fig. 1C). By the last step of spermiogenesis (stage VIII, late step 19), cytoplasmic reactivity was barely detectable in the resulting residual body but was still detectable in the cytoplasmic droplet (Fig. 1C).
Sptrx-1 Transiently Associates to the Longitudinal Columns of the FS During Tail Elongation
As shown by light microscopy, Sptrx-1 is detectable in the developing tail of the elongating spermatid but appears not to be associated with any tail structure after its completion. This expression pattern suggests that Sptrx-1 might assist the tail assembly process and that, once the tail is formed, the protein is discarded. To confirm the Sptrx-1 expression pattern obtained by light microscopy and to identify those structures of the spermatid developing tail to which Sptrx-1 associates, a complete survey throughout all the spermatid steps was carried out by immunogold electron microscopy on rat testis sections. Figure 2 demonstrates that Sptrx-1 expression peaks at step 15 in stage I of the cycle and is found associated to the two longitudinal columns (LC) of the FS, whereas very little immunogold labeling is found in the ribs or any other structure of the spermatid tail. Spermatids in steps 3 and 4, corresponding to stages III and IV, displayed no labeling in the sperm head or in the anlagen of the LC of the FS, which first became apparent during these early steps. However, in the older generation of spermatids (steps 16 and 17) in stages III and IV, the labeling of the LC of the FS was evident (Fig. 3). Spermatids in stage V followed a pattern similar to that of spermatids found in stage III or IV. However, the unlabeled anlagen of the LC was now more widespread throughout the distal part of the forming principal pieces of step 5 spermatids (not shown), whereas the Sptrx-1 immunogold labeling began to diminish in the LC of late step 17 spermatids (Fig. 4A). Stages VII and VIII of the cycle, corresponding to steps 7, 8, and 19, showed that the immunoreactivity had already disappeared from the LC of step 19 spermatids, whereas spermatids in steps 7 and 8 still lacked any labeling despite the LC anlagen being present in the distal part of the principal pieces (Fig. 4B). Progressing into step 9 spermatids in stage IX, now without an accompanying older generation of spermatids, a distal-to-proximal gradation in labeling was evident in the forming LC anlagen (Fig. 4C). It is important to note that LC anlagen had not yet formed in the proximal regions of step 9 principal pieces (not shown). Finally, by step 14, LC anlagen was immunogold labeled throughout the forming principal piece (Fig. 4D).
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Consistent with this developmental study, Sptrx-1 localization in epididymal rat spermatozoa showed a prominent deposit in the cytoplasmic droplet, with little labeling in any other compartment of the sperm tail (Fig. 5).
Sptrx-1 Is Absent in the Fully Formed Rat FS
To make sure that the lack of Sptrx-1 immunogold labeling in the fully formed FS was not caused by masked epitopes, Western blots of isolated FS and sperm tails were analyzed and compared for Sptrx-1 reactivity (Fig. 6). After probing with anti-Sptrx-1 antibody (Fig. 6B), the blot was stripped and reprobed with an antibody against another spermatid-specific thioredoxin (Fig. 6C), which we have recently identified as a mature FS structural component (unpublished results). Although Sptrx-1 immunoreactivity was detectable in the sperm tail of rat and human sperm, it was undetectable in rat FS and imperceptible in human FS (Fig. 6B). This observation appears to be justified, because the reprobed blot using the FS structural thioredoxin as control showed the intensity of immunostaining (FS protein load) to be similar between the isolated FS and tail lanes (Fig. 6C). It is also important to note that the molecular mass of the Sptrx-1 band in the sperm tail of the rat (Fig. 6B) was much smaller than the expected 90-kDa testicular form [34], indicating that Sptrx-1 had undergone partial proteolysis. In the human sperm tail, on the other hand, the Sptrx-1 band retained its expected size.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mammalian sperm tails contain, in addition to the axoneme, unique structural components named ODF and FS, which are formed during spermiogenesis and are involved in the regulation of sperm motility [3, 20]. Thus, to produce a functional tail, the synthesis and reorganization of the different polypeptides that compose the axoneme, ODF, and FS must be exquisitely coordinated. However, the regulatory mechanisms that ensure this process are just beginning to be understood. We have previously reported Sptrx-1 as a novel member of the thioredoxin family of proteins specifically expressed in the tail of elongating spermatids and spermatozoa [34]. The present study provides evidence that Sptrx-1 is not a structural component of the tail cytoskeletal elements but transiently associates to the LC of the FS during its assembly.
Figure 7 diagramatically summarizes our light- and electron-microscopic analyses of Sptrx-1 distribution throughout spermiogenesis. It shows that Sptrx-1 reactivity is transient, incorporating steps 9–19 of spermiogenesis (stages IX–VII of the cycle of the rat seminiferous epithelium). As soon as Sptrx-1 mRNA is translated in the cytoplasmic lobe of step 9–14 spermatids, its product is transported down the periaxonemal compartment of the sperm tail to associate with the LC anlagen, which gradually forms from the most distal region of the forming principal piece to its most proximal part during these steps (Fig. 7, brown lines). At step 15, the annulus, at the level of connecting piece, migrates distally to eliminate the narrow periaxonemal compartment in the midpiece region of the tail, and from this point onward, Sptrx-1 localization in the LC is restricted to the principal piece. By step 17, coincident with an almost fully formed FS, Sptrx-1 reactivity begins to diminish in the principal piece of the tail, and by step 19, it has completely dissociated from the mature FS. As Sptrx-1 dissociates from the principal piece, a residual buildup of this protein becomes apparent in the shrinking cytoplasmic lobe. By the time of spermiation, however, only a relatively small amount is left in the transient cytoplasmic droplet of the epididymal spermatozoa.
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The pattern of Sptrx-1 distribution during spermiogenesis mirrors the pattern and assembly of FS proteins documented by Irons and Clermont [40], Oko and Clermont [41], and Clermont et al. [42]. In these studies, it was shown that FS precursor material (FS anlagen), unreactive to antibodies directed at mature FS proteins and associated with the plasmalemma in the distal regions of the forming tail, precedes the assembly of FS proteins, which begins in steps 9 and 10 of rat spermiogenesis and reaches a peak in steps 15–17. The residual FS proteins left after their assembly are then eliminated via the cytoplasmic droplet. It was hypothesized by Clermont et al. [42] that the purpose of FS anlagen was to assemble the FS proteins. As far as we are aware, our present study is the first to identify a protein that associates with or contributes to FS anlagen (specifically with LC anlagen) and to provide a regulatory factor (discussed below) in FS assembly. Confirming the description by Irons and Clermont [40], LC anlagen is first observed in the very distal tail region of step 3 and 4 spermatids and gradually extends proximally along the tail until step 14. However, it is not until step 9 that Sptrx-1 begins to associate with the LC anlagen, and most importantly, Sptrx-1 appearance is coincident with the initiation of FS assembly.
Human Sptrx-1 consists of two distinct domains: an N-terminal domain organized in 23 repeats of a 15-residue motif highly conserved among repetitions and a C-terminal domain typical of thioredoxins [34]. We have recently cloned and characterized the mouse Sptrx-1 gene and protein, which display a high homology with their respective human orthologues [43]. A phylogenetic analysis shows that Sptrx-1 gene has arisen from a retrotransposition of the mammalian ancestor of Trx-1 mRNA, which integrated into a different position within the genome and became transcriptionally active in testis fused to the repetitive N-terminal domain, which has no homology to any other protein or region of the human genome [34]. The Sptrx-1 displays some characteristics of FS proteins, such as several putative phosphorylation sites or being rich in aspartic acid and glutamic acid residues, although it is highly soluble in contrast to most FS proteins [29]. These characteristics might be a determinant of its physiological function.
Similar to the rest of mammalian thioredoxins, Sptrx-1 is able to reduce insulin disulfide bonds in the presence of thioredoxin reductase and NADPH [34]. However, we have been able to detect, in addition to the reducing activity, a mild but consistent oxidizing activity during several in vitro assays (unpublished results). Disulfide bond formation is a very important issue in sperm physiology. The nuclear and accessory structures of mammalian sperm tail become stabilized by extensive disulfide bonding during spermiogenesis and epididymal maturation (reviewed in [44]), and this stabilization is reversed during fertilization by the reduction of these disulfide bonds [45]. Both FS and ODF are characterized by a high content of disulfide bonds, which contribute to their stabilization and insolubility [46]. The mechanisms underlying this extensive disulfide bond formation are not known to date. The oxidizing activity of Sptrx-1, together with its spatial and temporal expression simultaneous to the FS assembly, clearly suggests that it might play a key role in the regulation of this process by favoring the formation of disulfide bonds during FS maturation. In addition, its reducing activity might be required to rectify noncorrect disulfide pairing and to generate the suitable ones between the LC and the ribs of the FS, similar to what occurs to the disulfide bond reshuffling activity of protein disulfide isomerase [47]. Thus, Sptrx-1 might constitute a nucleation point for the formation of the mature LC and of the ribs that connect to it by regulating formation of the proper disulfide bonds.
Male infertility is a heterogeneous phenotype, and evidence for a genetic basis underlying the majority of cases involving infertile males is increasing. Among those affecting the proper organization of the structures that form the sperm tail are ciliary defects (e.g., Kartagener syndrome or primary ciliary dyskinesia) characterized by alterations in ciliary movements ranging from dysmotility to complete immotility because of alterations of the normal disposition or number of axonemal microtubules [48, 49]. Another pathology associated to a sperm tail structure is the so-called dysplasia of the FS (DFS) characterized by a hypertrophy and hyperplasia of random FS constituents that form broad meshes without the orderly disposition in LC and transversal ribs that characterize normal FS, resulting in male sterility associated with severe or complete asthenozoospermia [50, 51]. The familiar component clustering of the DFS phenotype strongly suggests a genetic component of the disease, which is likely to be developmental rather than degenerative, because it apparently arises during the course of spermiogenesis [50]. The DFS features and Sptrx-1 expression pattern, taken together, suggest that a failure in regulating Sptrx-1 expression or activity might lead to a phenotype similar to that described for DFS. A malfunction of Sptrx-1 could impede correct formation of the transversal ribs connecting the two LC, and this material would then be randomly distributed along the principal piece of the sperm tail in a fashion that would resemble that of DFS. We are currently evaluating this hypothesis on a mouse knock-out strategy as well as sequencing the genes of some patients diagnosed with DFS, taking advantage of the fact that the Sptrx-1 gene is rather small (2 kilobases including the 5'-untranslated region intron) [34].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 613 533 2566; ro3{at}post.queensu.ca
3 These authors contributed equally to the manuscript
Received: 26 February 2002.
First decision: 15 March 2002.
Accepted: 10 June 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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