Animals

The use of animals for the studies reported herein was approved by the Queen's University Institutional Animal Care and Use Committee.

Plasmid Constructions for Testing the Functionality of SubH2bv's bNLS

Full-length coding sequence of bovine SubH2Bv cDNA was subcloned from pBK-CMV vector (Stratagene, La Jolla, CA) into the EcoRI-KpnI sites of the vector pEGFP-C (Clontech, Palo Alto, CA) to generate an in-frame fusion gene of SubH2Bv-EGFP. Primers were designed to generate the artificial EcoRI and KpnI sites at the 5′ and 3′ ends, respectively, and were 5′-GGA ATT CAT GGC CAG AAA CGT CAC CAA GAG GA-3′ and 5′-GGG GTA CCT GGA GTT TAG GAG CGG ACA TAT CG-3.′

To create mutants of the fusion gene, primer-directed mutagenesis of the SubH2Bv-GFP construct was performed using long-distance inverse PCR [33–35]. Primers were designed with artificial Sal1 sites on the 5′ ends. The sequences of the primers used were as follows: mut 1A 5′-AGA GTC GAC AAC AAGCGC TGC AGA GGA C-3′, mut1B 5′-CGA GTC GAC GGT GAC GTT TCT GCC AT-3′, mut2A 5′-AGA GTC GAC TCA CAT TCC AGC TCT GAA TC-3′, mut2B 5′-CGA GTC GAC TTT GTA GAT TGC TTT TTG GTG-3.′ The PCRs were performed using an Expand Long Template PCR Systems (Roche Biomolecular, Germany), and the single-band PCR products were band prepped using a QIA prep Gel Extraction Kit (Qiagen, Mississauga, ON), digested with Sal1, and blunt-end ligated overnight at room temperature. The resulting construct was transformed and mini-prepped for analysis.

To obtain a construct with only the NLS portion of the SubH2Bv gene fused to EGFP, a C-terminus deletion mutant of SubH2Bv-GFP fusion gene was also created by PCR. The primers were designed to exclude the C-terminus of SubH2Bv and included a restriction site for ApaI at the 5′ ends. The sequences of the primers were 5′ATA GGG CCC TGA TTT CTT TTT GTA GAT TGC-3′ and 5′GAT ATG TCC GACT CCT AAA CT-3.′ The single-band PCR product was purified, digested with ApaI, ligated overnight at room temperature, and transformed.

The sequences of all constructs created were verified by sequencing with ABI PRISM Dye Terminator Cycle Sequencing Kit with AmpliTaq DNA Polymerase (Cortec, Kingston, ON).

Cell Cultures

The established cell lines of NIH 3T3 mouse fibroblast and HEK 293 human embryonic kidney cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum at 37°C. Cells were maintained in a water-saturated incubator with 5% CO₂.

Transfections of DNA Construct in 3T3 Cells

Cells were grown on cover slips placed on the bottom of a six-well dish (9.4 cm² per well) to a density of 3 × 10⁵ cells per well the night before transfection. When the cells were 60%–80% confluent, transfection was performed as follows. The plasmid DNA (2.0 ug) was diluted in 100 ul serum-free growth medium and incubated with 10 ul SuperFect Transfection Reagent (Qiagen) to allow transfection complexes to form. The transfection complexes were then incubated with the cultured cells for 2–3 h, the excess was washed off with PBS, and the cells were allowed to grow in fresh growth medium for 24–48 h. After transfection, cells were either observed directly in the culture dish (phase contrast) or fixed, and cover slips were prestained with 4, 6-diamino-2-pheylindole (DAPI) fluorescent dye and viewed with a fluorescent microscope (Axiovert 200; Zeiss, Toronto, ON).

Isolation of Pachytene Spermatocyte and Round and Elongated Spermatids

Cells were prepared according to Romrell et al. [36] and separated using a counterflow centrifugation method based on the cell's sedimentation velocity and flow rates against a vertically directed stream of liquid. DMEM was prepared with the addition of Na Lactate and Na Pyruvate and added to rat testes that had been cut and prepared on ice to expose the seminiferous tubules. Collagenase (0.5 mg/ml) was added to the sample for 15 min at 32°C, and the sample was allowed to settle afterwards. The tubules were washed three times with Krebs media, and the wash supernatant was also spun to maximize yield and retrieve any lost sample. Resuspending the sample pellet in Krebs media with trypsin (0.125 mg/ml) and DNase I (40 μg in 40 ml), the mixture was allowed to stand for 15 min at 32°C, but no longer to avoid cellular degradation. The resulting cell suspension was mixed well and checked to ensure the sample was well homogenized with no cell clusters. Trypsin inhibitor was added and the sample was filtered into a centrifuge tube, spun at room temperature for 10 min at 400 × g, rinsed with Krebs media, and spun again. Krebs media and DNase were added to the pelleted sample, which was loaded into a syringe for elutriation. Pachytene cells (220 fraction), round spermatids (90 fraction), and elongated spermatids (50 fraction) were separated in the elutriator, which was set at 18°C for 100 min at 475 × g using a Beckman J-6B centrifuge (elutriation rotor 5.2; Mississauga, ON). The rotor speed was not changed; only the buffer flow rate was adjusted (between 40 and 200 ml/min).

Isolation of Bovine Sperm Heads

As outlined in Oko and Maravei [10], bull epididymes were collected and separated into cauda and caput sections. A Tris-buffered saline (TBS) stock solution (25 mM Tris-HCl [pH 7.5], 0.9% NaCl) was prepared with 0.2 mM phenylmethylsulfonylfluoride (PMSF). The tissue was minced and filtered on ice with TBS-PMSF, and the filtrate was spun for 10 min using an angle rotor at 2500 × g at 4°C. The sperm pellet was washed three times in TBS-PMSF (under the same conditions), resuspended in the stock solution, and sonicated (Vibrocell Sonicator, 50W model; Sonics and Materials Inc., Danbury, CT). Three rounds of sonication were carried out in 15-sec intervals at 50 W on ice to avoid protein degradation. Phase contrast microscopy was used to verify that greater than 99% cleavage at the sperm head-tail junction had been attained. The sperm pellet was suspended and thoroughly mixed in 80% sucrose gradient and ultracentrifuged at 200 000 × g for 90 min in a Ti55 angle rotor (Beckman) to separate the head and tail components. The pellet on the centrifugal side of the tube was collected with TBS-PMSF and checked using phase contrast microscopy to ensure that the sample contained more than 99% sperm heads. Additional sonication and ultracentrifugation was performed as required. The isolated and sonicated sperm heads (SSpH) were again washed three times with TBS-PMSF at 2500 × g at 4°C and prepared for extraction.

Protein Extraction

The sonicated sperm heads were incubated with 0.2% Triton-X-100 for 1 h at room temperature with agitation, or overnight in the cold room at 4°C, to extract membrane-associated proteins. The Triton-extracted suspension was centrifuged at 2500 × g at 4°C to separate the extracted proteins from the remaining sperm head pellet. The isolated Triton extract was dialyzed with MWCO 6–8 kDa Spectra/Por membrane dialysis tubing (Spectrum Laboratories, Rancho Dominguez, CA) against four changes of distilled water. Alternatively, the samples were concentrated with MWCO 10 kDa ultrafiltration Centriprep tubes (Amicon, Beverly, MA) and spun at 3000 × g at 4°C for 30-min intervals using a fixed angle rotor. The extracts were aliquoted into 1-ml eppendorfs and lyophilized for SDS-PAGE analysis. The Triton-extracted sperm head pellet was then successively extracted with 1 M KCl to extract ionic-bound PT polypeptides [37] and 0.1 M NaOH to extract the alkali-soluble PT proteins [10].

SDS-PAGE and Western Blotting

Lyophilized extracts and isolated cells were solubilized in reducing sample buffer (2% SDS: 5% β-mercaptoethanol) and boiled for 5 min. The samples were run on 4.5% stacking and 10%–12% polyacrylamide separating mini-gels, as described by Laemmli [38], for 90 min at 120 V and compared against molecular weight standards. Low-molecular-weight markers (LMW-SDS; Amersham Biosciences, Piscataway, NJ) were used for KPNA and SubH2Bv immunoblots, while a high molecular weight marker (HMW-SDS; Amersham Biotechnology) was better suited for KPNB. The gel extract profiles were electrophoretically transferred to nitrocellulose membranes (0.45-μm pore size; Schleider and Schell, Keene, NH) or polyvinylidene difluoride microporous membranes (0.45-μm pore size; Millipore, Mississauga, ON) at 250 mA for 120 min using a Hoefer Transphor apparatus (Hoefer Scientific Instruments, San Francisco, CA) as prescribed by Towbin et al. [39]. The markers were identified with 0.1% ponceau rouge, and the membranes were washed with 25 mM PBS (pH 7.4) with 0.1% Tween-20 and blocked with 10% nonfat dry milk. The primary antibody, prepared in 2% nonfat dry milk, was incubated for 1 h at room temperature with agitation, or overnight in the cold room at 4°C, and detected with a conjugated secondary antibody in 2% nonfat dry milk. After a 2-h incubation period at room temperature with the secondary antibody, the immunoreaction was visualized with enhanced chemiluminescent substrate (Pierce, Rockford, IL).

Antibodies

KPNA1/6 (C-20) was a commercially purchased polyclonal anti-karyopherin α antibody against the C-terminal domain of KPNA1 and A6 of human origin raised in goat (Santa Cruz Biotechnology Inc., Santa Cruz, CA). The detection by KPNA1/6 was compatible with mouse, rat, bovine and humans and used at a dilution of 1:500 for immunoblotting and 1:30 for immunoperoxidase labeling. The secondary antibody for this peptide was horseradish peroxidase conjugated rabbit anti-goat IgG (H+L) (Bio-Rad Laboratories, Mississauga, ON) used at a dilution factor of 1:10 000.

KPNB1 (H-300) was a polyclonal anti-karyopherin β1 antibody purchased from Santa Cruz Biotechnology Inc., used at 1:500 for immunoblotting and 1:30 for immunohistochemistry. KPNB1 reacts to residue 1–300 of human KPNB's N-terminal and was raised in rabbit. The secondary antibody for this protein was goat anti-rabbit IgG (H + L) (Vector Laboratories Inc., Burlingame, CA) diluted at 1:10 000. Karyopherin β1 monoclonal antibody was raised in mouse (BD Biosciences, Franklin Lakes, NJ). The monoclonal was used at 1:500 and is reactive in dog, humans, mouse, and rat and detected using conjugated goat anti-mouse IgG (H+L) horseradish peroxidase (Bio-Rad Laboratories, Mississauga, ON) at a dilution factor of 1:10 000.

Anti-SubH2Bv antibodies were affinity purified in our lab according to a protocol adapted from Oko and Maravei [10]. Rabbit antiserum raised against sperm PT extracts was used to affinity purify antibodies from Western blot-immobilized SubH2Bv. The affinity-purified SubH2Bv antibodies were concentrated two times for Western blot analysis and another 10 times for immunoperoxidase labeling of paraffin-embedded tissue. Goat anti-rabbit IgG (H + L) was used as the secondary antibody (Vector Laboratories, Inc.) to detect the protein.

Immunohistochemistry

Testis from bull, mouse, and rat were perfused in Bouin fixative, embedded in paraffin, and prepared for immunolabeling according to the protocol published by Oko [13]. Each 5-μm-thick section on a glass slide was deparaffinized with xylene and then hydrated through a graded series of ethanols. Once hydrated, the sections were subjected to antigen retrieval if needed by microwaving in a 0.01 M sodium citrate solution, pH 6 (for details, see [40]). To minimize nonspecific binding, the sections were blocked with 10% normal goat serum (NGS) for 15 min. The primary antibody was then added, and the tissue samples were incubated for 1.5 h at room temperature or overnight at 4°C. Subsequently, the sections were washed with TBS containing 0.1% Tween-20, blocked with 10% NGS, and incubated with biotinylated secondary antibody for 30 min prior to incubation with Immunoperoxidase avidin-biotin reagent (ABC; Vectastain Elite ABC Kit; Vector Laboratories). To elicit the immunoperoxidase reaction, 0.5% diaminobenzidene tetrahydrochloride in TBS containing 0.1 M imidazole and 0.03% H₂O₂ was used. The tissue was then counterstained, dehydrated into xylene, and mounted with permount (Fischer Chemicals, Fairlawn, NJ).

Construction and Expression of Recombinant SubH2Bv

Bull recombinant SubH2Bv and primers were prepared from SubH2Bv's cDNA (GenBank Accession No. bankit36366 AF315690) and amplified using Qiagen Taq PCR kit (Qiagen). Two primers were designed by Cortec Technologies—a 24-bp primer with a nucleotide sequence of 5′-GAGCT′CAATGGCCAGAAACGTCAC-3′, which included a 5′-GAGCT′C-3′ SAC 1 site, and a 23-bp primer sequence 5′-GC′GGCCGCGGAGCGGACATATCG-3′, with an incorporated 5′-GC′ GGCCGC 3′ NOT 1 site. The ′ indicates the cut site for the restriction enzymes.

A mastermix containing 5 μl of 10× PCR buffer, 1 μl dNTP, 10 μl of 25 mM MgCl₂, 5 μl of each primer, and 0.4 μl of Taq DNA polymerase was prepared and added to 23.1 μl of ddH₂O and dispensed into PCR tubes. The SubH2Bv cDNA was added and placed in the thermal cycler that alternated between 94°, 52°, and 72°C. In this instance, annealing was optimized at 52°C. A DNA band of approximately 381 bp was visualized in a 2% agarose gel stained with ethidium bromide. The PCR products were cloned with the Qiagen Cloning Kit (Qiagen) into pDrive Cloning Vectors. A ligation-reaction mixture was added, and the suspension was incubated for 2 h at 16°C.

Transformation was carried out with Qiagen EZ competent host cells (Qiagen), and ampicillin LB agar plates were prepared (1 μl/ml LB). Ligation reaction mixture was added to each tube of competent cells, incubated on ice for 5 min, then heat-shocked with a hot water bath for 30 sec at 42°C. SOC medium (Sigma-Aldrich, Oakville, ON) was added to each tube, and the transformation mixture was plated and incubated. Colonies were selected and tested on 2% agarose for the insert.

The inserts and expression vectors were linearized for subcloning with SAC 1 and NOT 1 restriction enzymes, incubated for 2 h at 37°C. The inserts were subcloned and expressed in pet21b (+) expression vectors from Novagen (Madison, WI) for 30 min and transformed in E. Coli BL21 DE3 [PlyS] cells (Novagen) using the above protocol.

Protein Purification and Affinity Pull-Down

His-tagged recombinant SubH2Bv (recSubH2Bv) was immobilized on Ni-NTA-charged beaded agarose (Qiagen) to bait KPNA obtained from 0.1% Triton-extracts of isolated sperm heads. Two ml of E. coli cell culture was collected, sonicated, and spun down to produce the pellet containing the insoluble recombinant peptide and denatured with 8 M urea (100 mM NaH₂PO₄, 10 mM Tris, pH 8) overnight in the cold room (4°C). The suspension was spun and cleared, and the supernatant with the solubilized recSubH2Bv was incubated with 10 μl of Ni-NTA agarose for 4 h at 4°C. The Ni-slurry was washed five times with TBS containing 10 mM Imidazole (pH 8) and spun. The immobilized SubH2Bv on Ni beads was then incubated with 500 μl of Triton extract, obtained from isolated sonicated sperm heads for 4 h in the cold room, spun, and washed five times with TBS with 10 mM Imidazole. The proteins were eluted twice with 2× reducing sample buffer and detected on Westerrn blots for SubH2Bv and KPNA.

SubH2Bv Directs Nuclear Entry of EGFP into Somatic Cells

To determine the subcellular localization of SubH2Bv in somatic cells, a fusion gene of EGFP and SubH2Bv was constructed under the control of the immediate early promoter of CMV. The expression vector, pEGFP-SubH2Bv, was transfected into 3T3 mouse fibroblasts and visualized by confocal microscopy. The enhanced green fluorescent signal was detected mainly within the cell nuclei, as visualized by DAPI staining (Fig. 1A). Results obtained in the human embryonic kidney cells were identical (not shown).

FIG. 1.

Subcellular localization of GFP fused with either whole SubH2Bv (A) or the N-terminus of SubH2Bv (1MARNVTKRKNRCRGHQKAIYKKKS24), containing the intact bNLS (B). Subcellular localization of GFP alone (C). Subcellular localization of GFP fused with SubH2Bv containing mutants (#1 and #2) of the bNLS motif (D, E). In mutant #1 (1MARNVTVDKNRCRGHQKAIYKKKS … 122) the upstream basic cluster KR was substituted with VD (D). In mutant #2 (1MARNVTKRKNRCRGHQKAIYKVDS … 122) the downstream basic cluster KK was substituted with VD (E). Expression vectors of the fusion genes were transfected into 3T3 mouse fibroblast cells, and then the cells were cultured for 24–48 h before being stained with DAPI and examined by confocal fluorescent microsocopy. Bars = 10 μm.

SubH2Bv's bNLS Confers Nuclear Specificity

Amino acid sequence analysis revealed that SubH2Bv coded a series of amino acids in its N-terminus typical of the classical bNLS sequence, made up of two basic clusters tethered by a string of 10–12 amino acids [8].

To test the function of this predicted bNLS (a.a., 7–23), a fusion gene of the N-terminus (a.a., 1–24) of SubH2Bv and EGFP was constructed and transfected into 3T3 cells. Transfection of this C-terminal deletion construct enhanced nuclear localization of EGFP (Fig. 1B), indicating that SubH2Bv's bNLS was the predicted motif, conferring nuclear localization of SubH2Bv into somatic cells. This contrasted with EGFP control results, where the green fluorescence was visualized throughout the entire cell (Fig. 1C).

Mutation of the bNLS Obliterates SubH2Bv's Nuclear Entry

To confirm the functionality of the bNLS in SubH2Bv, fusion proteins containing selectively mutated basic residues of the bNLS within SubH2Bv were created and transiently transfected in the cell lines. The first mutated construct (mutant #1) contained two amino acid substitutions, resulting in the replacement of the upstream basic cluster of KR (a.a., 7, 8) with VD. The second mutant (mutant #2) also contained two amino acid substitutions, resulting in altering the downstream cluster of KKK (a.a., 22, 23) with KVD. Both mutated fusion proteins failed to efficiently enter the nucleus of the cultured cells and instead congregated as cytoplasmic clumps (Fig. 1, D and E). Altering either of the basic clusters in the bNLS obliterated nuclear entry in an identical manner, providing evidence that SubH2Bv, through its bNLS, can bind to the nucleocytoplasmic import receptor KPNA and acts as a karyophilic cargo molecule.

Karyopherin α Is a Constituent of the Sonication-Resistant, Isolated Sperm Head Fraction

Because the SSpH fraction is enriched in the PT proteins SubH2Bv, RAB2A, Calicin and the four somatic histones, it was used to determine if a KPNA was also confined to this SSpH fraction by Western-/immunoblotting. Indeed, an anti-KPNA antibody (anti-KPNA 1/6) recognized a single 60-kDa band, consistent with the reported molecular mass of KPNA1 or A6 (KPNA1/6), in bovine (Fig. 2A) and rat (not shown). To determine its binding characteristics, sequential extracts of 2% Triton-X100, 1 M KCL, and 0.1 M NaOH were performed on the SSpH fraction. KPNA1/6 was extractable from SSpH with Triton X-100 and not present in subsequent KCL and NaOH extracts or in the final pellet (Fig. 2A). This suggests a hydrophobic association with either IAM and NE and/or proteins associated with the SAL-PT.

FIG. 2.

A) Western blot of SSpH and sequential extractions of SSpH (i.e., 2% Triton X-100, 1 M KCL, and 0.1 M NaOH) immunolabeled with anti-KPNA1/6 antibody. B) Western blot of elutriated rat testicular germ cells immunolabelled with anti-KPNA1/6 antibody. The cells including mature spermatozoa (sperm) were loaded at approximately equal numbers per cell lane. Values are kDa. Pach S, pachytene spermatocytes; R Sptd, round spermatids; E Sptd, elongated spermatids.

Karyopherin α Is Enriched in Isolated Spermatids

To demonstrate that a KPNA was present in spermatids during acrosomal biogenesis, germ cell fractions, consisting of pachytene spermatocytes, round spermatids, and elongated rat spermatids (estimated 85%, 85%, and 60% purity for each fraction, respectively), were purified by centrifugal elutriation. Immunoblots of these three germ cell fractions showed that KPNA1/6 was more abundant in round spermatids at the time of acrosomal morphogenesis and in elongated spermatids than in pachytene spermatocytes (Fig. 2B), confirming our suspicion of developmental immunohistochemical localization during spermatogenesis.

Karyopherin α Is Associated with Acrosome Formation During Spermiogenesis

In order to determine the localization of KPNA in round spermatids and obtain a chronology of this protein's predicted association with acrosomal morphogenesis, immunoperoxidase immunocytochemistry was performed on deparaffinized testicular sections using anti-KPNA1/6 antibody. KPNA, like SubH2Bv [8], was mainly localized to the spermatid cytoplasm, where it associated with the secretory vesicles forming the acrosome in all three species investigated (i.e., mouse, rat, and bull; Figs. 3 and 4 and Supplemental Fig. S1, available online at  www.biolreprod.org). In these testicular sections, immunoperoxidase staining clearly demarcated the surface of PA vesicles in step 2 of spermiogenesis as they began to fuse to form the AV and migrate to the nuclear envelope (Fig. 3, A and B). In step 3 spermatids, the fusion of the PA vesicles was complete, and the immunoperoxidase-reaction product now covered the surface of the AV as it attached to the NE of the spermatid (Fig. 4A). At the beginning of the cap phase of spermiogenesis (step 4), KPNA-immunoperoxidase staining initially expanded around the surface of the growing AV (Fig. 3C) but by step 5 had become diminished from the outer acrosomal membrane surface and concentrated between the inner acrosomal membrane and NE in the subacrosomal margin (Figs. 3D and 4B). From steps 6 to 8, KPNA antigenicity expanded with the increase in size of the subacrosomal margin as the acrosome capped the nucleus (Fig. 4C). During spermatid elongation, KPNA-immunoperoxidase staining was present over the sperm nucleus in the region covered by the acrosome (Fig. 4D). Normal rabbit serum was used as a control and showed either little or no immunoperoxidase immunoreactivity (see Supplemental Fig. S1).

FIG. 3.

Murid (rat and mouse) testicular sections immunoperoxidase stained with anti-KPNA1/6 antibody counterstained with methylene blue. KPNA-immunoreactive PA vesicles fuse with each other (arrows) in step 2 of spermiogenesis (A, B). The fusion of these vesicles results in a larger acrosomic vesicle (arrow), which by step 4 has firmly attached to the spermatid nucleus and is beginning to expand over it (C). KPNA immunoreactivity is clearly seen associated with the periphery of these vesicles. By step 5 in spermatids, the immunoperoxidase staining predominates in the subacrosomal margin (between the inner acrosomal membrane and NE) or the PT (D). A diagrammatic interpretation of KPNA's association (orange dots) with the developing acrosome in respective spermatids is displayed below in this and the subsequent figure. GA, Golgi apparatus; AC, acrosomal cap. Bars = 5 μm.

FIG. 4.

Bull testicular sections immunoperoxidase stained with anti-KPNA1/6 antibody. As in the murid, KPNA immunoreactivity is associated with the surface of the AV in step 3 spermatids and concentrated in the subacrosomal margin or PT of step 5 spermatids (A, B). During spermatid elongation, KPNA reactivity is retained over the sperm nucleus (N) in the region covered by the acrosome (C, D). AV, acrosomic vesicle; AC, acrosomal cap; SAL-PT, subacrosomal layer of perinuclear theca; MAN, microtubular manchette; ES, equatorial segment; PAS, postacrosomal sheath. Bars = 5 μm.

Sperm Karyopherin α and SubH2Bv Have a Binding Affinity

SubH2Bv's functionally active bNLS, coupled with near-identical sperm-localization and testicular-expression patterns of SubH2Bv [8] and KPNA1/6 (this study), suggested that a binding interaction exists between these proteins. To establish a protein-protein interaction between these proteins, recombinant His-tagged SubH2Bv was stabilized on Ni-agarose beads and used in an affinity pull-down assay to bait KPNA in germ cell extracts. Traditional immunoprecipitation could not be used because of the insoluble nature of SubH2Bv within the germ cell. Immunoblots of Triton X-100-extracts of SSpH (Tx, Fig. 5, lane 1) and Ni-conjugated, affinity-purified recombinant SubH2Bv (SubH2Bv-Ni, Fig. 5, lane 3) served as positive controls for KPNA and SubH2Bv, respectively, while immunoblots of Tx incubated with the Ni-beads (Tx-Ni, Fig. 5, lane 2) demonstrated that nonspecific binding of KPNA to the Ni-beads did not occur (Fig. 5). Furthermore, immunoblots of Tx incubated with the Ni-conjugated recombinant SubH2Bv (Tx-SubH2Bv-Ni;, Fig. 5, lane 4) showed a binding affinity between KPNA1/6 and SubH2Bv.

FIG. 5.

Affinity pull-down of sperm-extracted KPNA with recombinant SubH2Bv detected by immunoblotting using anti-KPNA1/6 and anti-SubH2Bv antibodies. SSpH were extracted using 0.1% Triton X-100 (Tx). Lane 1: Triton extract (Tx) alone showed labeling of a 60-kDa band as expected; lane 2: Tx purified over a Ni column (Tx-Ni) showed no labeling; lane 3: His-tagged recombinant SubH2Bv purified over a Ni column (SubH2Bv-Ni) showed labeling of a 15-kDa band as expected; lane 4: Tx pre-incubated with SubH2Bv and then purified over a Ni column (Tx-SubH2Bv-Ni) showed labeling of both KPNA1/6 and SubH2Bv.

Karyopherin β Is a Constituent of the SSpH and Present in Round Spermatids

Since the relationship between KPNA and Karyopherin β (KPNB) as transport heterodimers for bNLS-cargo was demonstrated, we further determined whether KPNB1 was present in the same sperm and testis locations as KPNA1/6. Immunoblots using a monoclonal antibody against KPNB1 identified KPNB1 as a detergent-extractable protein of SSpH and a constituent of isolated round spermatids (Fig. 6). This supported KPNB1's association with acrosomal morphogenesis, previously shown by Yang and Sperry [41].

FIG. 6.

Western blots of detergent extract of SSpH and elutriated round spermatid fraction (RS90) immunolabeled with anti-KPNB1 monoclonal antibody. Results were identical using KPNB1 (H-300), a polyclonal anti-KPNB1 antibody.