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Biology of Reproduction 59, 69-76 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Spermatid Perinuclear Ribonucleic Acid-Binding Protein Binds Microtubules In Vitro and Associates with Abnormal Manchettes In Vivo in Mice1

Jill M. Schumacher3,a, Karen Artzt4,a, , and Robert E. Braun2,a

a Department of Genetics, University of Washington, Seattle, Washington 98195


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatid perinuclear RNA-binding protein (SPNR) is a microtubule-associated RNA-binding protein that localizes to the manchette in developing spermatids. The RNA target of SPNR in vivo is unknown, although we have previously suggested the possibility that SPNR is involved in the translational activation of the protamine 1 mRNA in elongated spermatids. To increase our understanding of SPNR's association with the manchette, we sought to determine SPNR's subcellular localization in several mouse mutants that show reduced fertility or sterility and that have structurally abnormal manchettes. We show here that despite the highly abnormal manchettes and microtubule aggregates formed in azh, hop-sterile, tw2, and tw8 mutants, SPNR remains associated with the manchettes. Localization of SPNR to the abnormal manchettes suggests that SPNR is tightly bound to the manchette. SPNR could bind manchette microtubules directly, or it could bind indirectly via an interaction with a microtubule-associated protein (MAP). We sought to determine whether SPNR binds microtubules in vitro, and if so, whether it requires a MAP. We show by Western analysis that the endogenous SPNR protein can be pelleted with murine testis microtubules in a taxol-dependent manner in vitro. A recombinant version of SPNR produced in bacteria can also be pelleted with testis microtubules, as well as microtubules polymerized from purified bovine brain tubulin, an association that is salt-sensitive. These results suggest that SPNR, in addition to its function as an RNA-binding protein, is also a bona fide MAP.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Formation of a spermatozoon from a diploid spermatogonial progenitor cell involves reduction of the chromosomal content during meiosis and several major structural changes to the cell during spermatid differentiation. These changes, which include compaction of the chromatin, changes in nuclear morphology, development of the acrosome, and formation of a flagellum, require the expression of testis-specific genes and the formation of germ cell-specific organelles. One such organelle, a specialized microtubule array called the manchette, materializes in the differentiating spermatid and is transiently associated with the spermatid nucleus for several days [1]. Formation of the manchette is first detected in step 8 spermatids as a "grass-skirt" of microtubules emanating from a nuclear ring of rodlike material on the caudal surface of the nuclear envelope [2–4]. In steps 9–16, the manchette extends caudally around the developing flagellum although never reaching its end. The manchette is attached to the nucleus at distinct angles, reflecting changes in nuclear shape [5]. The manchette is disassembled in elongated spermatids before they are released from their Sertoli cell associations into the lumen of the seminiferous tubule.

Two possible and not mutually exclusive functions for the manchette are to provide the mechanical force necessary to mediate nuclear shaping and to act as a "track" for the movement of cellular components into the extending cytoplasm [2, 4]. Nuclear shaping is under genetic control [6], and drugs that affect the manchette, including taxol, cytoxan, and 5-fluorouracil, also affect sperm head shape [4]. Several mutations in the mouse that result in reduced fertility or sterility are characterized by highly abnormal manchettes and altered head shape [3, 7–10], suggesting that the manchette functions in nuclear shaping. It has also been suggested that the manchette could serve to transport cytoplasmic constituents caudally, an event that coincides with manchette appearance [2]. Manchette microtubules have been shown to be associated with vesicles and the endoplasmic reticulum [2, 11], and immunocytochemistry studies in the rat have shown that the microtubule motor proteins kinesin and dynein are both found on the manchette [12]. Other proteins have also been localized to the manchette, including the microtubule-associated protein (MAP) Tau [13], a protein kinase [14], and the intermediate filament protein Sak57 [15].

We have previously described an RNA-binding protein in mammalian spermatids whose association with the manchette suggests that it may be involved in the transport or translation of selected mRNAs [16]. The gene encoding the spermatid perinuclear RNA-binding protein (SPNR) was cloned in a screen for RNA-binding proteins that could bind to the 3' untranslated region (UTR) of the testis-specific protamine 1 (Prm1) mRNA in vitro [16]. The Prm1 gene encodes a small, highly basic protein that is involved in DNA packaging in sperm heads [17]. Synthesis of the PRM1 protein is under posttranscriptional control in spermatids [17, 18] and is controlled by sequences in its 3' UTR [19]. Separate elements within the 3' UTR appear to be involved in controlling both translational repression in round spermatids and translational activation of the Prm1 message in elongated spermatids [20]. Translational delay of the Prm1 mRNA is essential for spermatid differentiation, as premature translation of the Prm1 message leads to precocious nuclear condensation and an arrest in spermatogenesis [21].

Immunolocalization studies have shown that SPNR is associated with the manchette and that its appearance temporally coincides with the translational activation of the Prm1 mRNA [16]. SPNR contains two copies of a double-stranded RNA-binding motif that is found in several other proteins [22, 23]. Members of this protein family include Drosophila Staufen [24], murine protamine RNA-binding protein (PRBP) [25], and human protein kinase activated by RNA [26]. SPNR, and other family members that contain the double-stranded RNA-binding domain, bind double-stranded RNA and highly structured single-stranded RNAs in vitro [22, 26, 27]. Despite the lack of sequence-specific binding in vitro, it is clear that certain members of the family do interact with specific RNA targets in vivo [28, 29]. We have suggested that SPNR interacts with the Prm1 mRNA in vivo, although in vitro RNA-binding studies with portions of the SPNR protein failed to reveal sequence-specific binding to any RNAs, including the Prm1 3' UTR [16].

It is possible that SPNR binds microtubules directly, or that its association with the manchette is indirect and that it binds to manchette microtubules via an MAP like a microtubule motor protein. Revealing the nature of SPNR's interaction with the manchette may provide insight into its function during spermiogenesis. We sought to determine whether SPNR binds microtubules in vitro and whether the binding is direct or indirect. We also determined the subcellular localization of SPNR in several mouse mutants that have highly abnormal manchettes and unusual head morphology.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice

Azh mice were obtained from The Jackson Laboratories (Bar Harbor, ME). Hop-sterile (hop) mice were provided by Dr. Mary Ann Handel, University of Tennessee, Knoxville, TN. Mice carrying the tw8 and the tw2 haplotypes were from the T/t complex colony at the University of Texas at Austin. All animal experiments were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.

Immunocytochemistry

Testes were fixed overnight in Carnoy's fixative (60% ethanol, 30% CHCl3, 10% glacial acetic acid). The tissues were embedded in paraffin blocks and cut into 6- to 8-µm sections. The sections were deparaffinized with xylene, rehydrated using standard procedures, and incubated with 1:100 to 1:10 dilutions of affinity-purified primary antibody in PBS for 2 h at 22°C. Antibodies to the SPNR protein have been previously described [16]. Tubulin antibodies (Sigma, St. Louis, MO) were used at a dilution of 1:2000 in PBS. Tissue sections were treated with biotin-conjugated or fluorescently labeled secondary antibodies (Zymed Laboratories, South San Francisco, CA, and Vector Laboratories, Burlingame, CA). Biotinylated antibodies were visualized by light microscopy using a horseradish peroxidase-streptavidin conjugate and aminoethyl carbazole (Zymed and Vector). Fluorescent antibodies were visualized by confocal microscopy. Confocal images were processed with Adobe Photoshop (Mountain View, CA). Preimmune sera and secondary antibody alone were used as negative controls.

Microtubule Purification from Mouse Testes

Microtubules were purified from mouse testes by a taxol-dependent method modified from Vallee [30]. Testes were dissected from sexually mature Swiss Webster male mice, and the tunicas were removed. Approximately 400 µg of tissue was resuspended on ice in 1 ml microtubule (MT) buffer (0.1 M Pipes, pH 6.6, 1 mM EGTA, 1 mM MgSO4) with protease inhibitors (1 µg/ml aprotinin, 1 µg/ml pepstatin, 3.4 µg/ml PMSF, 10 µg/ml soybean trypsin inhibitor, 1 µg/ml Na-p-tosyl-L-arginine methyl ester hydrochloride, 1 µg/ml leupeptin) and sonicated for 15-20 sec followed by incubation on ice for 15 min. The lysate was cleared by spinning at 30 000 x g at 4°C for 15 min; this was followed by a 30-min spin at 100 000 x g at 4°C. Taxol (Sigma) was added to the supernatant at a final concentration of 20 µm, guanosine triphosphate (GTP; Sigma) was added to a concentration of 1 mM, and the tube was placed at 37°C for 15 min. Polymerized microtubules were pelleted through a 10% sucrose cushion (prepared in MT buffer supplemented with protease inhibitors, with taxol and GTP added to 20 µm and 1 mM concentrations, respectively) at 30 000 x g, for 15 min, at 37°C. The microtubule pellet was rinsed in MT buffer and resuspended in 125 µl MT buffer supplemented with taxol (20 µm final concentration), GTP (1 mM final concentration), and protease inhibitors (as above). The tube was placed at 37°C for 15 min, and microtubules were pelleted as described above. The pellet was resuspended in MT buffer and subjected to another round of purification (as above). The third microtubule pellet was resuspended in MT buffer containing protease inhibitors. Aliquots of the starting extract, the supernatants, and the microtubule pellets were analyzed by SDS-PAGE [31] followed by Coomassie staining or Western analysis.

Binding of His-SPNR to Mouse Testis and Bovine Brain Microtubules

The entire protein coding region of the Spnr was cloned in-frame with the N-terminal His-tag present in the pET15b vector (Novagen, Madison, WI). A 200-ml culture of pLysS Escherichia coli (Novagen) transformed with the His-SPNR vector was grown at 37°C to an OD600 of 0.5. Cells were induced with 1 mM isopropylthiogalactoside for 1 h and harvested by low-speed centrifugation. The bacterial pellet was resuspended in 2 ml MT buffer (0.1 M Pipes, pH 6.6, 1 mM EGTA, 1 mM MgSO4) supplemented with protease inhibitors as described above. The extract was sonicated on ice and spun at 4°C for 30 min at 100 000 xg. The supernatant was recovered and used for all subsequent binding experiments.

To assess binding to mouse testis microtubules, microtubule pellets from the first round of purification (described above) were resuspended in 120 µl His-SPNR E. coli extract. The mixture was incubated at 37°C for 15 min, and microtubules were pelleted by spinning at 30 000 x g for 20 min at 37°C. The pellets were resuspended in MT buffer and subjected to another round of purification. Aliquots of the starting extract, the supernatants, and the microtubule pellets were analyzed by SDS-PAGE, followed by Coomassie staining or Western analysis with anti-His-tag antibodies.

Two hundred micrograms of bovine brain tubulin that had been purified by phosphocellulose chromatography (kindly provided by Joe Howard, University of Washington, Seattle) was added to 100 µl His-SPNR E. coli extract. Taxol was added to a final concentration of 20 µm, GTP was added to a concentration of 1 mM, and the mixture was incubated at 37°C for 15 min. The microtubules were pelleted as described above. The pellets were resuspended in MT buffer and subjected to another round of purification. Aliquots from each supernatant and pellet were run on SDS-PAGE gels, followed by Coomassie staining or Western analysis with anti-His-tag antibodies. Controls included the omission of tubulin and the addition of 1 M NaCl to the starting extract.

Western Analysis

Protein extracts were mixed with Laemmli buffer [31], boiled for 5 min, and electrophoresed on 8% SDS-PAGE gels. The gels were blotted to nitrocellulose overnight at 4°C using an electroblotter run with water-cooling at 200 mA in a Tris-glycine buffer (25 mM Tris, 192 mM glycine, 20% v:v methanol, pH 8.3). The blots were dried and blocked in 5% dry milk for 30 min at 22°C. Primary antibodies were diluted in 5% dry milk and incubated with blots overnight. The blots were washed twice in 5% dry milk + 0.05% Tween 20 for 20 min each and then in 5% dry milk for 20 min. They were then probed with either anti-rabbit or anti-mouse secondary antibodies conjugated to biotin; this was followed by washing as described above and by incubation with streptavidin-horseradish peroxidase conjugate. Labeled proteins were visualized by chemiluminescence. Chemiluminescent reagent was prepared by dissolving 20 mg 3'-aminophthalhydrazide and 5 mg 4-iodophenol in 0.5 ml dimethyl sulfoxide and adding 17 ml distilled H2O, 2.5 ml NaCl, and 5 ml 0.1 M Tris, pH 8.5. Right before use, 62.5 µl H2O2 was added. Western blots were incubated in the reagent for 1-2 min and exposed to x-ray film.

For reprobing with an additional antibody, the blots were stripped with 2.2 M glycine and 0.5 M NaCl for three washes of 20 min each, then washed in PBS with 10% BSA for 20 min, and finally washed two times, 20 min each, in PBS alone. The blots were blocked with 5% dry milk in distilled H2O and reprobed with primary antibody as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunocytochemical localization of the SPNR protein in wild-type, azh/azh, hop/hop, tw8/tw8, and tw2/tw2 mutant testes is shown in Figure 1. Confocal micrographs of SPNR (fluorescein isothiocyanate [FITC], green) and tubulin (Texas red) immunofluorescence in wild-type and mutant testes are shown in Figures 2–4. Spermatogenesis in the mouse has been divided into 12 developmental stages based on the presence of particular ages of developing germ cells within a given seminiferous tubule [32, 33]. Spermiogenesis, the haploid phase of spermatogenesis, has been divided into 16 different steps, depending on histological criteria that change as the haploid spermatids develop, including the size and position of the acrosome and nuclear shape [33–35]. Descriptions of SPNR immunostaining refer to spermatids at these various steps of development.



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  		FIG. 1. Immunocytochemical localization of SPNR in wild-type (WT), azh, hop, tw8, and tw2 testes. Testis sections were treated with rabbit polyclonal affinity-purified anti-SPNR antibody, washed, treated with a biotinylated goat anti-rabbit secondary antibody, and then incubated with a streptavidin-horseradish peroxidase conjugate. See text for a description of arrows and arrowheads. Bar = 10 µm.



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  		FIG. 2. Confocal micrographs of SPNR and tubulin immunolocalization in wild-type testes. Testis sections were treated with rabbit polyclonal affinity-purified anti-SPNR and/or mouse monoclonal anti-{alpha}-tubulin primary antibodies. Sections were then treated with an anti-rabbit FITC-conjugated and/or anti-mouse Texas red-conjugated secondary antibody. A and D) SPNR; B and E) tubulin; C and F) merged SPNR and tubulin staining.

azh/azh

Males homozygous for the azh mutation produce normal numbers of sperm but are very weakly fertile [3, 7]. As these previous studies have described, the reduced fertility is apparently due to gross defects in nuclear head shape. Nearly all of the sperm heads are very long and cylindrical, much different from the short, compact, and hook-shaped nuclei of normal murine sperm. The manchette forms properly in step 9 spermatids, but by step 11 the microtubules in azh/azh mutants continue to lie parallel to instead of protruding from the caudal end of the nucleus at the normal 45° angle. The extending manchette continues to be conical in shape, remaining long and straight, rather than assuming the proper angular shape of the wild-type manchette. As the spermatids elongate, the mutant manchettes tend to split off from the end of the nucleus and "float" into the cytoplasm. In addition to these defects, large aggregates of microtubules are often found in the spermatid cytoplasm, with no resemblance to a manchette-type organization.

SPNR was localized by immunocytochemistry (Fig. 1) and by immunofluorescence (Fig. 3) to Carnoy's-fixed sections of testis from azh/azh males. Confocal microscopy of azh/azh sections labeled with anti-SPNR (Fig. 3, A and D) and anti-tubulin antibodies (Fig. 3, B and E) showed that as in the wild-type testis (Fig. 2), SPNR is coincident with tubulin. SPNR was first detected on the forming manchette in step 9 spermatids from azh/azh males (Fig. 3A), just as in wild type. Immunostaining followed the formation of the abnormal manchette up until its disassembly in step 15 spermatids. As the spermatids elongated, SPNR remained attached to mutant manchettes that had detached from the end of the nucleus (arrow in Azh panel, Fig. 1) and was also localized to large ectopic microtubule aggregates (Fig. 3F, arrow). As in wild type, SPNR immunostaining disappeared in step 15 spermatids coincident with the disassembly of the manchette, leaving the distorted head shape as the only recognizable abnormality in the mature sperm.



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  		FIG. 3. Confocal micrographs of SPNR and tubulin immunolocalization in azh (A–F) and hop (G–L) testes. Testis sections were treated with rabbit polyclonal affinity-purified anti-SPNR and/or mouse monoclonal anti-{alpha}-tubulin primary antibodies. Sections were then treated with an anti-rabbit FITC-conjugated and/or anti-mouse Texas red-conjugated secondary antibody. A, D, G, J) SPNR; B, E, H, K) tubulin; C, F, I, L) merged SPNR and tubulin staining. F) Arrow marks a microtubule aggregate. I) Arrow is pointing to degenerating spermatid; arrowhead highlights an abnormal elongated manchette. L) Arrowhead points to "caplike" staining pattern.

hop/hop

Male mice homozygous for the hop mutation are infertile [10]. As previously described [10], seminiferous tubules in hop/hop males have few elongated spermatids, and these cells have abnormal flagella. Manchette microtubules form at the appropriate time, but they overproliferate and begin to impinge on the integrity of the nucleus. The elongating spermatids appear to degenerate, forming large round cells that extend into the lumen of the seminiferous tubules. The presence of these cells in the lumen suggests that the elongating spermatids may be degenerating as they are being released from their Sertoli cell connection upon spermiation in stage VIII. No mature sperm are found in the epididymides of hop homozygotes.

Confocal micrographs of SPNR staining in hop/hop testes show that, as in wild type, SPNR forms as a caplike structure around the caudal end of the nucleus in step 9 spermatids, entirely coincident with tubulin staining (arrowhead, Fig. 3L). SPNR staining continued to overlap with tubulin staining as the spermatids formed long, abnormal manchette microtubules (arrowhead, Fig. 3I). An apparent overabundance of SPNR was found in degenerating elongating spermatids, filling the cytoplasm of these cells (arrow, hop panel, Fig. 1 and Fig. 3, G and J). Double immunostaining of these degenerating cells with both SPNR and tubulin antibodies showed that these cells also had diffuse tubulin staining that was sometimes punctuated by brightly staining regions where SPNR staining was also concentrated (arrow, Fig. 3L). These areas of intense staining may be microtubule structures that have not completely disassembled. Thus it appears that manchettes form properly in these cells but undergo an abnormal proliferation that is followed by premature depolymerization as the spermatids degenerate.

tw8/tw8

Males homozygous for the tw8 haplotype are sterile [8, 9]. As those studies describe, testes from tw8/tw8 males have many degenerating seminiferous tubules that are devoid of germ cells. Other tubules undergo grossly normal spermatogenesis; however, many spermatids have abnormally shaped heads and dumbbell-shaped nuclei [8, 9]. Some tubules also seem to be less organized than in wild type, having germ cells of apparently different stages within the same section of the tubule. The manchettes show subtle defects, mostly appearing less uniform than manchettes in wild type. Some manchettes appear to bundle around the caudal end of the nucleus with fewer numbers of microtubules extending into the cytoplasm. There also appear to be aggregates of ectopic microtubules that are unrelated to manchette formation. As in wild type, the manchettes are disassembled in step 15 spermatids.

SPNR staining in tw8 homozygotes appeared nonuniform, being highly localized to the caudal end of the nucleus and exhibiting less staining as the manchette extended into the cytoplasm (tw8 panel, Fig. 1). SPNR-positive microtubule aggregates were also evident (arrow, tw8 panel, Fig. 1). SPNR could also be seen in the cytoplasm of some elongating spermatids, as well as in a Sertoli cell at the basal lamina, apparently from the phagocytosis of an elongating spermatid (arrowhead tw8 panel, Fig. 1). Confocal micrographs of SPNR and tubulin in early-stage spermatids show that the two proteins colocalized with one another (Fig 4, A–C). As the spermatids elongated, SPNR (Fig. 4D) and tubulin (Fig. 4E) staining became patchy and discontinuous (Fig. 4F, merged image).



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  		FIG. 4. Confocal micrographs of SPNR and tubulin immunolocalization in tw8/tw8 (A–F) and tw2/tw2 (G–L) testes. Testis sections were treated with rabbit polyclonal affinity-purified anti-SPNR and/or mouse monoclonal anti-{alpha}-tubulin primary antibodies. Sections were then treated with an anti-rabbit FITC-conjugated and/or anti-mouse Texas red-conjugated secondary antibody. A, D, G, J) SPNR; B, E, H, K) tubulin; C, F, I, L) merged SPNR and tubulin staining. F) Arrow points to "patchy" staining pattern. I and L) Arrow points to ectopic manchette.

tw2/tw2

Male mice homozygous for the tw2 haplotype are sterile [8]. Although many of the seminiferous tubules degenerate and are devoid of germ cells, as seen in the tw8 haplotype, other seminiferous tubules have an abnormal phenotype that is distinct from that in tw8/tw8 testes (Fig. 1). Previous studies with tw2/tw2 testes have shown that the nuclear envelope becomes discontinuous in a small proportion of elongating spermatids, allowing direct contact between the cytoplasm and nucleoplasm [8]. These cells lack manchettes: either they never formed, or they underwent premature depolymerization. The majority of elongating spermatids contain large numbers of disorganized microtubules and the cells have abnormal head shapes. Abnormal spermatids, as in the tw8 haplotype, are phagocytized by the Sertoli cells prior to spermiation [8].

Light microscopy studies examining SPNR staining in tw2/tw2 testes showed the same abnormal proliferation of microtubules that was found in electron micrograph studies (arrow, tw2 panel, Fig. 1). Sertoli cell phagocytosis of SPNR-staining cells was also evident (arrowhead, tw2 panel, Fig. 1). SPNR was found abundantly in rounded cells that were reminiscent of the degenerating spermatids found in hop/hop seminiferous tubules (Fig. 1). Confocal micrographs showed that SPNR and tubulin colocalized on highly abnormal manchette structures, structures that even extended into the lumen of seminiferous tubules, entirely free of a nuclear association (arrows, Fig. 4, I and L). Thus, as in hop testes, spermatogenesis in the tw2 haplotype is characterized by an overproliferation of abnormal microtubules that is followed by premature depolymerization and cell degeneration.

Microtubule Binding In Vitro

The SPNR protein is coprecipitated with murine testis microtubules and its associated proteins. Extracts prepared from as few as four mouse testes (approximately 400 µg tissue) were incubated at 37°C in the presence of GTP and the microtubule-stabilizing drug taxol and then centrifuged at 100 000 x g to pellet microtubules. The pellets were washed, resuspended, and subjected to two further rounds of centrifugation and washing. A Coomassie-stained SDS-PAGE gel containing samples taken from the starting extract, each supernatant, and each pellet from a typical purification experiment is shown in Figure 5A. A great increase in tubulin concentration is seen in the first pellet, as well as an enrichment for high-molecular-weight MAPs. By the third pellet, very few of the proteins found in the starting extract were visible, except for tubulin and a few associated proteins. The enrichment for tubulin in this purification procedure was taxol dependent, as shown in Figure 5A. In the absence of taxol there was a slight enrichment of tubulin in the first pellet, but it was lost along with other proteins from the starting extract by the third round of purification. Western blots of SDS-PAGE gels identical to those stained with Coomassie were incubated with anti-SPNR and anti-kinesin antibodies (Fig. 5B). Both SPNR and kinesin were highly enriched in the microtubule pellets through three rounds of purification. This enrichment was also dependent on the presence of taxol, since there was a significant loss of each protein along with the loss of tubulin after further rounds of washing and centrifugation (Fig. 5B).



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  		FIG. 5. SPNR coprecipitates with testis microtubules. A) Aliquots of the starting testis extract, each supernatant, and each pellet from a typical microtubule purification performed with (lanes 2–8) and without (lanes 10–16) taxol were run on SDS-PAGE gels and stained with Coomassie blue. B) Western blots of duplicate gels to those shown in A were probed with anti-kinesin and anti-SPNR antibodies. Molecular weight standards (x 10-3) are on the left (lanes 1 and 9).

To test whether a recombinant His-tagged version of SPNR produced in E. coli could be purified with testis microtubules, as is the endogenous SPNR protein, testis microtubules were pelleted and resuspended in SPNR-containing E. coli extracts prepared as described in Materials and Methods. These microtubule-enriched extracts were subjected to two further rounds of centrifugation and washing, and aliquots from each supernatant and pellet were assayed by SDS-PAGE followed by Coomassie staining or Western blotting. As shown in Figure 6A, the addition of E. coli extracts did not interfere with the purification of tubulin, and taxol was still necessary for the continued enrichment of tubulin through three rounds of purification. Gels duplicate to those shown in Figure 6A were blotted onto nitrocellulose and incubated with an {alpha}-His-tag antibody specific for the exogenous E. coli-produced SPNR protein (Fig. 6B). The SPNR protein added to the first pellet remained in the microtubule pellet through two further rounds of purification, an association that was taxol dependent. Thus, the His-tagged SPNR protein behaves in a manner similar to that of the endogenous testicular protein.



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  		FIG. 6. Exogenous SPNR binds to testis microtubules. A) Microtubules pelleted from testis extracts with and without the addition of taxol were resuspended in His-tagged SPNR-containing E. coli extracts. Two additional rounds of pelleting and washing of the microtubules were performed. Aliquots of the starting testis extract, each supernatant, and each pellet were run on SDS-PAGE gels and stained with Coomassie blue. The E. coli lysate was added to the first pellet. B) Western blots of duplicate gels to those shown in A were probed with an anti-His antibody specific for the His-tagged SPNR protein. Molecular weight standards (x 10-3) are on the left.

To ascertain whether the association of SPNR with microtubules occurs by direct binding to tubulin, or via a MAP, MAP-free bovine brain tubulin was added to His-tagged SPNR E. coli extracts, and microtubule polymerization was induced by incubating the extract at 37°C in the presence of GTP and taxol. Microtubules were pelleted, washed, and repelleted. A Coomassie-stained SDS-PAGE gel containing aliquots of each supernatant and pellet is shown in Figure 7A. The vast majority of the added tubulin is pelleted under these conditions, and most of the starting material remains in the first supernatant. Figure 7B shows a corresponding Western blot probed with the {alpha}-His-tag antibody that recognizes the E. coli-produced SPNR. The SPNR protein present in the starting extract was pelleted with the MAP-free bovine brain microtubules through two rounds of purification. The pelleting of the His-tagged SPNR protein was entirely dependent on the presence of added tubulin, as shown in Figure 7B. As a control for the possible nonspecific "trapping" of SPNR in the microtubule pellet, the experiment described was repeated with and without the addition of NaCl to the starting extract. As shown in Figure 8A, the addition of NaCl at a concentration of 1 M did not interfere with the pelleting of tubulin through two rounds of purification. However, as shown by Western blotting with the {alpha}-His-tag antibody (Fig. 8B), the addition of NaCl resulted in all of the SPNR protein remaining in the first supernatant, not in the tubulin-enriched pellet. Thus, it appears that the pelleting of His-tagged SPNR from E. coli extracts with MAP-free bovine brain microtubules is due to a specific salt-sensitive interaction with tubulin and is not due to a nonspecific trapping event.



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  		FIG. 7. SPNR binds to MAP-free bovine brain microtubules. A) MAP-free bovine brain tubulin was added to His-tagged SPNR-containing E. coli lysates. Microtubules were polymerized with the addition of taxol and GTP. Microtubules were pelleted, washed, and repelleted. Aliquots from each supernatant and pellet were run on SDS-PAGE gels and stained with Coomassie blue. A control experiment without the addition of tubulin to the E. coli extract was also performed. B) Western blots of duplicate gels to those shown in A were probed with an {alpha}-His antibody specific for the His-tagged SPNR protein. Molecular weight standards (x 10-3) are on the left.



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  		FIG. 8. A) MAP-free bovine brain tubulin was added to His-tagged SPNR containing E. coli lysates with and without NaCl added to a concentration of 1 M. Microtubules were polymerized with the addition of taxol and GTP. Microtubules were pelleted, washed, and repelleted. Aliquots from each supernatant and pellet were run on SDS-PAGE gels and stained with Coomassie blue. B) Western blots of duplicate gels to those shown in A were probed with an {alpha}-His antibody specific for the His-tagged SPNR protein. Molecular weight standards (x 10-3) are on the left.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have shown by both light and confocal microscopy studies that the murine SPNR protein, which is associated with manchette microtubules in wild-type testes [16], is also present on the abnormal manchettes and microtubule aggregates found in the testes of four mouse mutants: azh, hop, tw8, and tw2. We never found localization of SPNR to be independent of microtubules in the testis. If cytoplasmic microtubules are present, SPNR is associated with them, regardless of the type of manchette structure present. In cases in which we saw an overproliferation of microtubules, we also saw an abundance of SPNR, suggesting that the expression or stability of SPNR is linked to the concentration of manchette microtubules. Variations in tubulin concentration along the length of the manchette, as in tw8 testes, is reflected by the same variation in SPNR staining. When microtubules are degraded in the degenerating spermatids of hop and tw2 testes, SPNR staining overlaps with that of free tubulin. We conclude that SPNR is tightly associated with manchette microtubules.

Although a number of different proteins have been implicated in linking transported and translationally controlled mRNAs to microtubules [36, 37], only a few have been shown to bind directly to microtubules [38, 39]. Previous studies have shown that an RNA-binding activity specific for sequences present in the 3' UTRs of a number of different mRNAs, including Prm2 and Tau, can be pelleted when protein extracts enriched for this RNA-binding activity are added to preassembled bovine brain microtubules [39]. We have shown that SPNR can be pelleted with testis microtubules in a taxol-dependent manner. In addition, a His-tagged version of SPNR produced in bacteria behaves similarly to the endogenous protein, also pelleting with microtubules through multiple rounds of purification. This E. coli-produced protein can also be pelleted with MAP-free bovine brain microtubules, an association that is salt sensitive. Since SPNR can be pelleted with microtubules in the absence of other MAPs, this experiment suggests that SPNR itself is a bona fide MAP.

SPNR is a member of the double-stranded RNA-binding protein family that includes Drosophila Staufen [24] and murine PRBP [25]. Numerous studies addressing the in vitro RNA-binding properties of different members of this family of proteins suggest that they are sequence-independent RNA-binding proteins that prefer double-stranded RNAs and highly structured single-stranded RNAs [22, 26, 27, 40]. Northwestern analysis with an SPNR-maltose-binding protein fusion showed that it too can bind to a number of different mRNAs in vitro [16]. Despite the apparent nonspecific binding properties of these proteins in vitro, genetic studies suggest that at least some members of this family interact with selected mRNAs in vivo [23, 28, 41]. It is possible that the subcellular localization of these RNA-binding proteins is what confers their apparent in vivo specificity for RNA targets. For instance, SPNR may only bind to its RNA target if it is associated with microtubules, a requirement that would help guarantee that only proper temporal and spatial mRNA interactions occur. SPNR's association with microtubules may also link it to other proteins that help confer RNA-binding specificity in vivo.

Translational control of the oskar mRNA in Drosophila oocytes may be regulated by the RNA-binding protein Bruno [41]. Mutation of the Bruno-responsive elements in the oskar 3' UTR results in the unregulated translation of the oskar mRNA. This unregulated translation requires the presence of functional Staufen protein [41]. Staufen is an RNA-binding protein that may be associated with the cytoskeleton and is involved in mRNA localization within the oocyte [42]. If Staufen is missing, the oskar mRNA is not translated, even in the absence of functional Bruno-responsive elements, implying that the Staufen protein is involved in the translational activation of the oskar mRNA [41]. We have recently shown that there are separable translational repression and activation elements for the Prm1 mRNA [20]. Transgenic studies with reporter mRNAs fused to various portions of the Prm1 3' UTR have identified individual sequence elements that are required for translational repression, as well as others that are necessary for the alleviation of this repression. As is the case for the Drosophila oskar message, we have identified RNA-binding proteins that may be involved in the translational repression of Prm1 [25, 43]. Likewise, SPNR shares many similarities with Staufen, including the presence of double-stranded RNA-binding domains and an association with the cytoskeleton. In the future it will be important to determine whether SPNR, like Staufen, is also involved in the translational activation of localized and stored mRNAs during spermatid differentiation. Clearly, defining SPNR's functional role in spermatogenesis will require a mutation at the SPNR locus itself, as well as further experiments aimed at directly assessing the role of manchette microtubules in RNA transport and translational activation.


   ACKNOWLEDGMENTS
 
The authors thank Mary Ann Handel for providing the hop mice and for critical reading of the manuscript. The authors thank Joe Howard and the members of his laboratory for their generous offers of reagents, equipment, and advice. Additional help from Bill Therkauf, Karen Butner, Paul Walden, Harry Higgs, and Ed Krebs' laboratory is also appreciated.


   FOOTNOTES
 
1 This work was supported by grants to R.E.B. from the March of Dimes Birth Defects Foundation and the National Institutes of Health (HD27215 and HD12629) and to K.A. from NIH (HD10668 and HD30658). Back

2 Correspondence: Robert E. Braun, Department of Genetics, Box 357360, 1959 NE Pacific, University of Washington, Seattle, WA 98195. FAX: (206) 543-0754; braun{at}genetics.washington.edu Back

3 Current address: ABL-Basic Research Program, NCI-FCRDC, Frederick, MD 21702. Back

4 Current address: The Cell and Molecular Biology Institute, The University of Texas at Austin, Austin, TX 78712. Back

Accepted: February 21, 1998.

Received: August 25, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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