Background
The fibrous sheath (FS), a unique cytoskeletal structure specific to the sperm, surrounds the axoneme and outer dense fibers and consists of two longitudinal columns connected by closely arrayed semicircular ribs. The FS is located only in the principal piece, a region devoid of mitochondria, and it assembles in a distal to proximal direction during spermiogenesis. The FS has been proposed to function as a protective girdle for the axoneme [
1,
2], influence the degree of flexibility, plane of flagellar motion, the shape of the flagellar beat and as a scaffold for enzymes involved in signal transduction, including protein kinase A by anchoring to AKAP3 [
3,
4] or AKAP4 [
5,
6], the Rho signaling pathway through ropporin [
7] and rhophilin [
8]. It has also been implicated in calcium signaling because it contains CABYR [
9,
10], a polymorphic, testis-specific calcium binding protein that is tyrosine [
9] as well as serine/threonine phosphorylated [
11] during in vitro sperm capacitation. At least nine glycolytic enzymes, including glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glyceraldehyde 3-phosphate dehydrogenase-2 (GAPDH-2) [
12,
13], hexokinase 1 (HK1) [
14,
15], isoform of aldolase 1 (ALDOA), lactate dehydrogenase A (LDHA) [
16], triose phosphate isomerase (TPI), pyruvate kinase, lactate dehydrogenase-C (LDH-C), and sorbitol dehydrogenase (SDH) [
16], have been localized to the human and/or mouse FS. Moreover, a unique ADP/ATP carrier protein, SFEC [AAC4] is co-localized with several glycolytic enzymes in the FS [
17]. The presence of four ion channel proteins, CatSper 1-4, in the membrane of the principal piece [
18] overlying the FS in which CABYR is found, has led to hypotheses that CABYR plays a role in calcium signaling. Thus, observations indicate that the FS plays important roles in energy metabolism, ATP generation for sperm motility, calcium signaling, and as a scaffold for signaling molecules in addition to its role as a structural girdle surrounding the outer dense fibers and axoneme. A model has been proposed in which the FS represents a highly ordered complex, somewhat analogous to the electron transport chain, in which adjacent enzymes in the glycolytic pathway are assembled to permit efficient flux of energy substrates and products, possibly as a nucleotide shuttle between flagellar glycolysis, protein phosphorylation and mechanisms of motility [
17].
Both mouse and human CABYRs are polymorphic proteins. Four murine CABYR variants, orthologous to human CABYR forms I, III, IV and VI, have been identified, which consist of two coding regions, CR-A and CR-B. The murine pattern is similar to that of human CABYR cDNAs of which six isoforms, three involving CR-B, are known. In the mouse, two stop codons (TGA and TGA) followed by six in-frame nucleotides separate CR-A from CR-B resulting in 453 and 199 aa reading frames, respectively [
19].
While both the genomic structure and RNA splicing of murine CABYR have been reported, information on CABYR dynamic expression in mouse spermatogenesis is lacking. Moreover, because of the complexity of this polymorphic protein, there are no data on how CABYR isoforms associate and assemble into the FS.
It has been identifed that CABYR, ROPN1, ASP and SP17 share high sequence conservation with the PKA regulatory subunit's (RII) dimerization/docking (R2D2) domain, which binds the amphipathic helix region of AKAPs [
20,
21]. Moreover, CABYR has been shown to co-precipitate from sperm lysate with AKAP3 [
22,
23], and recently, evidence has been provided that the aforementioned proteins interact with a variety of AKAPs and that the interaction of CABYR with AKAPs appears to be much more limited [
24]. However, further research on the relationships between CABYR and other FS proteins besides of AKAPs will facilitate the understanding of the basic physiology of FS. Interestingly, in our previous study, a novel protein named FSCB was found to co-imunoprecipitate with CABYR in sperm lysates [
25].
In the present study, the capacity of the multiple CABYR isoforms to associate into dimers and oligomers, and the relationships between CABYR and other FS proteins such as the AKAPs and ropporin were studied in the mouse. Full-length murine CR-A and CR-B were cloned and expressed as recombinant proteins CABYR-A and CABYR-B. Antibodies to each protein isoform were produced and employed in 2-D diagonal gel electrophoresis and immunoblotting to determine which CABYR isoforms are involved in CABYR assemblies. Co-immunoprecipitations with polyclonal or monoclonal antibodies to CABYR variants, AKAP3 and AKAP4 were performed and 2-D gel immunoblots were analyzed to identify CABYR partner proteins, interactions of which were studied further by yeast two-hybrid analyses.
Methods
Cloning and sequencing of mouse CR-A and CR-B cDNA
Mouse CABYR gene-specific primers were designed to create an Nco I site at the 5' end and a Not I site at the 3' end of PCR amplicons encompassing the full length coding region A (CR-A) (nucleotide numbers 73-1431 encoding 453 amino acids) or coding region B (CR-B) (nucleotide numbers 1449-2039 encoding 193 amino acids) using the mouse CABYR form I (GenBank accession number AF359382) and form III (accession number AF359383 cDNA sequences). Primers for mouse CR-A (forward primer: 5'-CAT GCC ATG GTT TCT TCA AAG CCC AGA CTT-3'; reverse primer: 5'-ATA GTT TAG CGG CCG CAA CCT GTT CAG GAG CAG CTT CCC C-3') and CR-B (forward primer: 5'-CAT GCC ATG GCA ACA AGC GAA GCA GGA CAA CCA-3'; reverse primer: 5'-ATA GTT TAG CGG CCG CAG GTT CTG CTC TGC GGA CAT GGG C-3') were obtained from GIBCO BRL (Life Technologies, Carlsbad, CA). PCR was performed with 0.2 ng of mouse testicular Marathon-ready cDNA (BD Biosciences-Clontech, San Jose, CA) in a 50 μl assay system in a MJ Research (Waltham, MA) thermal cycler (PTC-200 DNA engine) using a program of one 6 min cycle at 94°C followed by 35 cycles of denaturation, annealing and elongation at 94°C for 1 min, 60°C for 1 min and 68°C for 3 min, respectively. The 1359 bp CR-A and 591 bp CR-B amplicons were separated on a 1% NuSieve (FMC Bio-Products, Rockland, ME) agarose gel, subcloned into the pCR 2.1-TOPO vector (Invitrogen, San Diego, CA), and sequenced in both directions to confirm authenticity, using vector-derived and insert-specific primers.
Expression and purification of murine CABYR-A and CABYR-B recombinant proteins and antibody production
Mouse CR-A and CR-B were cloned into the pET28b+ expression vector and transformed into E. coli BLR (DE3) host strain (Invitrogen, San Diego, CA), and recombinant CABYR-A and CABYR-B, each with six His residues at the C-terminus, were expressed. Female guinea pigs were immunized with purified recombinant CABYR-A or CABYR-B protein (100 μg/animal) in Freund's complete adjuvant. Animals were boosted three times at intervals of 14 days with 50 μg of recombinant protein in incomplete Freund's adjuvant. Guinea pigs were sacrificed after confirming the presence of serum antibody by Western blot analysis using recombinant CABYR-A, CABYR-B and mouse sperm proteins. These studies were conducted under protocols 1574 and 2545 approved by the Institutional Animal Care and Use Committee at the University of Virginia in accord with the humane use of animals in research.
In our preliminary studies, it was found that the rat anti-human recombinant AKAP3 polyclonal antibody which was previously reported [
4] can recognize mouse AKAP3 protein (data not shown). Therefore, the rat anti-human recombinant AKAP3 polyclonal antibody was used in the present study of mouse proteins. Mouse anti-mouse monoclonal AKAP4 antibody was purchased from BD Biosciences Pharmingen (San Diego, CA).
Non-reducing and reducing 1-D SDS-PAGE of mouse sperm proteins
Mouse cauda epididymal sperm were collected from 12-15 week old mice (ICR strain, Harlan Sprague-Dawley, San Diego, CA). 5 × 10
8 sperm/ml were routinely solubilized in Celis lysis buffer [
26] consisting of 9.8 M urea, 2% NP-40, and a complete protease inhibitor cocktail (Roche), with (reducing condition) or without (non-reducing condition) 100 mM DTT or 5% β-mercaptoethanol (β-ME), by constant shaking at 4°C for 30-60 min. Samples were heated at 100°C for 5 min, insoluble material was removed by centrifugation at 16,000 ×g for 5 min, and the supernatants were applied to 1-D SDS-PAGE.
2-D IEF-SDS/PAGE of mouse sperm proteins
200 μl sperm extract by Celis lysis buffer with 100 mM DTT (1 × 108 sperm/ml) was applied to the first dimension after addition of 2% v/v Ampholines (pH 3.5-10). Isoelectric focusing (IEF) was performed using a Protean IEF cell and an 11 cm tray (Bio-Rad, Hercules, CA, USA). Nonlinear strips (11 cm, pH 3-10) were rehydrated at 50 V for 12 h and then linearly increased to 8,000 V for a total of 30,000 V·h. For second dimension SDS-PAGE, the IPG strips were first incubated in equilibration buffer containing 37.5 mM Tris-HCl (pH 8.8), 6 M urea, 4% w/v SDS, 20% glycerol, and 100 mM DTT for 20 min. The equilibrated IPG strips were then transferred onto Criterion 4-15% linear gradient gels (Bio-Rad, Hercules, CA, USA) and electrophoresis was carried out at room temperature using a Criterion gel system (Bio-Rad, Hercules, CA, USA). Immunoprecipitates with various antibodies were separated by 2-D IEF-SDS/PAGE and identified by Western blot and mass spectrometry.
2-D nonreducing/reducing diagonal electrophoresis
Diagonal SDS-PAGE was performed as described by Sommer, et al [
27] employing SDS-PAGE gradient gels (4-15%) in large format (20 × 16 cm, Protean II XL, Bio-Rad) and standard electrophoretic settings, using 1.0 mm or 1.5 mm thick gels for the first and second dimensions, respectively. In all assays, 100-200 μg samples of Celis-extracted sperm lysates (without β-ME and DTT) were diluted 1:1 with 2× sample buffer (without β-ME and DTT) and heated at 100°C for 5 min immediately preceding sample application to the first dimension. After the initial non-reducing electrophoresis, the entire lane containing the resolved proteins was excised, placed into containers with reducing buffer (containing 100 mM DTT or 5% β-ME), and incubated at 22°C for 15 min. The strips were then placed horizontally on top of the second dimension gradient gels (4-15%), overlaid with the SDS buffer containing β-ME to provide reducing conditions, and electrophoresis was carried out at room temperature. Proteins in the gels were either stained with Coomassie Brilliant Blue R-250 or silver nitrate for image analysis or were transferred to PVDF membranes for Western blotting.
Immunoblotting
Proteins were transferred from unstained gels to PVDF membranes using a Bio-Rad Trans Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions. Membranes were blocked with 5% nonfat milk in PBS for 1 h at room temperature, washed three times with PBS-Tween (0.05% Tween-20 in PBS) and incubated overnight at 4°C with 15 ml of a previously determined working dilution of guinea pig pre-immune or immune serum (anti-CRA serum at 1:3000, anti-CRB serum at 1:1000). After washing thrice, the membranes were incubated with HRP-conjugated goat anti-guinea pig immunoglobulin (Sigma-Aldrich Quimica S. A. Madrid, Spain), washed × 3, and immunoreactive spots were detected by ECL (Amersham Pharmacia Biotech, Sunnyvale, CA) as described [
8] or were visualized with the colorimetric reagent 3, 3', 5, 5'-tetramethylbenzidine (TMB) (Kirkegaard and Perry, Gaithersburg, MD).
Indirect immunofluorescence localization of CABYR-A in the seminiferous tubules and epididymis
Testes and epididymides were obtained from 12-15 week-old mice (ICR strain, Harlan Sprague-Dawley, San Diego, CA) and fixed in Bouin's solution overnight at room temperature. They were embedded in paraffin, serially sectioned at 5 μm. For staging of tubule sections, periodic acid-Schiff (PAS) staining was performed following the standard protocol.
For immunostaining, after de-paraffination and rehydration, adjacent sections were incubated in 2 N HCl for 20 min, washed 3 × 5 min in PBS with 0.05% Tween 20 (PBS-T), and incubated in blocking solution containing 10% normal goat serum (NGS) in PBS-T for 30 min. The preparations were then incubated with anti-CABYR-A antiserum or pre-immune serum (1:400) in PBS-T containing 1% BSA (PBS-T-BSA) for 1 h at room temperature or overnight at 4°C, washed 3 × 5 min in PBS-T, incubated with Cy3-labeled goat anti-guinea pig IgG (1:400; Jackson Immuno-Research, West Grove, PA) in PBS-T-BSA, washed 3 × 5 min in PBS-T, and mounted with Slow Fade (Molecular Probes, Eugene, OR). PAS stained sections were observed by light microscopy and each tubule cross section was scored for the stage of the cycle of the seminiferous epithelium according to the criteria of Russell, et al [
28]. Adjacent serial sections were observed by fluorescence microscopy to determine the corresponding immuno-staining pattern of the seminiferous epithelium. Red fluorescent images were recorded with a Zeiss digital camera and compiled using Openlab software (Improvision, Inc., Boston, MA). The immuno-staining to the caudal epididymal sperm is also conducted by the same method.
Localization of CABYR-A in mouse sperm by immunoelectron microscopy
Caudal sperm from adult Swiss mice were retrieved and immunostained following the reference [
25]. Guinea pig antiserum against CABYR-A and the pre-immune serum was diluted by 1:100. 5 nm gold-conjugated secondary antibodies, goat anti-guinea pig IgG (Goldmark Biologicals, Phillipsburg, NJ) was diluted by 1:50. The stained sections were observed with a 100CX electron microscope (JEOL, Peabody, MA).
Co-immunoprecipitation of mouse sperm using anti-CABYR-A, anti-CABYR-B, anti-AKAP3 polyclonal antibody or anti-AKAP4 monoclonal antibody
To optimize immunoprecipitations of CABYR and partner proteins from mouse sperm extracts, two methods were used. Method 1: 2 × 108 epididymal sperm were suspended in 1 ml lysis buffer (10 mM Na2HPO4, 50 mM β-glycerophophate, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml aprotinin, 1 mM PMSF, 0.1% Tween 20) at 2-8°C, and the protocol described in the Immunoprecipitation Kit (Roche Applied Science, Indianapolis, USA) was followed. Immunoprecipitates on agarose pellets from one set of experiments, including the anti-CABYR-A and pre-immune control, were microsequenced by mass spectrometry. Another set of immunoprecipitates was retrieved by elution from protein A-agarose using 200 μl of Celis buffer followed by denaturation at 100°C for 3 min, 2-D gel electrophoresis, and silver staining or Western blotting. Method 2: Since AKAP3 and AKAP4 proteins were not thoroughly extracted using the lysis buffer described in method 1 above in initial studies, a modified immunoprecipitation strategy to extract low solubility proteins was followed. 8 × 108 sperm were resuspended in 2 ml Celis buffer with Complete Protease inhibitor cocktail without DTT, and the suspension was incubated for 0.5-1 h at 4°C on a rocking platform. Following centrifugation at 4°C, 12,000 ×g, in a tabletop microfuge for 10 min to remove debris, the supernatant was transferred into a dialysis cassette with 10 kDa cut off (Pierce) and dialyzed against 1 × PBS solution for 24h at 4°C with two changes of PBS. The dialyzed suspension was centrifuged at 4°C, 6,000 ×g for 10 min to remove the precipitates. The suspension was then transferred to four 1.5 ml tubes and the manufacturer's immunoprecipitation protocol followed as in method 1. Immunoprecipitates were retrieved by elution from the protein A-agarose pellet with 200 μl of Celis buffer or with 50 μl of 2 × Laemmli sample buffer, and proteins were denatured at 100°C for 3 min. The protein A-agarose was centrifuged at 12,000 ×g for 20 s at 15-25°C in a microfuge, and supernatants were transferred to a fresh tube for 2-D gel IEF-SDS/PAGE. Western blots were performed as described above.
Tandem mass spectrometry peptide sequencing
2-D gel spots from immunoprecipitates with immune sera were compared to those with pre-immune sera. Spots appearing in the experimental samples and not in the pre-immune samples were cut out of the 2-D gel using fine coring tools [
29], in-gel digested by trypsin overnight at 37°C, and microsequenced by tandem mass spectrometry at the W. M. Keck Biomedical Mass Spectrometry Laboratory of the University of Virginia. The data were analyzed by database searching using the Sequest search algorithm against the Non-redundant database.
Yeast two-hybrid assay
Vectors, yeast, and major reagents were supplied as part of the Matchmaker Gal4 Two-Hybrid System 3 (Clontech, Palo Alto, CA). Gene segments were obtained by PCR using 0.2 ng of mouse testicular marathon-ready cDNA (BD Biosciences-Clontech) as the template and the primers listed in Table
1. The full-length open reading frame of CR-A (79-1431), a deletion construct of a truncated CR-A (TCR-A) without the RII-like domain (223-1431), and the full-length open reading frame of CR-B (1443-2039) were cloned into the pGADT7, while full-length open reading frames of AKAP3 (292-2883), AKAP4 (148-2664) and ropporin (1-636) were cloned into the pGBKT7 vectors using standard cloning procedures. Yeast strain AH109 was simultaneously co-transformed with two recombinant plasmids having different selection markers using the LiAc-mediated yeast transformation as described in the Yeast Protocols Handbook (PT3024-1; Clontech). In this two-hybrid system, the GAL4 BD (binding domain) binds to the GAL upstream activating sequence and, if the fusion proteins interact, the AD (activating domain) is brought into proximity with the promoters of four reporter genes (HIS3, ADE2, MEL1, and lacZ), thereby activating transcription and permitting growth on selection media (His- and Ade-) and the expression of α-galactosidase (MEL1 product) and β-galactosidase (lacZ product). Co-transformed yeast cells were isolated by growth on SD/-Leu/-Try plates at 30°C for 3 days. For medium stringency or high stringency selection, cells were then transferred to SD/-His/-Leu/-Trp or SD/-Ade/-His/-Leu/-Trp plates, supplemented with 20 μg/ml X-α-Gal (5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside), and allowed to grow at 30°C for 3-5 days to select for colonies that expressed interacting proteins.
Table 1
Construction of Gal4 fusions of mouse genes for yeast two-hybrid assay
pGADT7-mCRA | AF359382 | 73-1431 | 5'-CGGGATCCAGATGATTTCTTCAAAGCCC AGAC-3' 5'-CCGCTCGAG TCAAACCTGTTCAGGAGCAGCTT-3' | BamH I Xho I |
pGADT7-mTCRA | AF359382 | 223-1431 | 5'-CGGGATCCACGGGAATTCGTCTCTAGATATAA-3' 5'-CCGCTCGAGTCAAACCTGTTCAGGAGCAGCTT-3' | BamH I Xho I |
pGADT7-mCRB | AF359382 | 1443-2039 | 5'-CGGAATTCGCACTAGCAACAAGCGAAGCAGG-3' 5'-CCGCTCGAGTTAAGGTTCTGCTCTGCGGACA-3' | EcoR I Xho I |
pGBKT7-mAKAP3 | NM_009650 | 292-2883 | 5'-CGGAATTCATGGCGGATAGGGTTGACTG-3' 5'-ACGTCGACCAGGTTTGCCATCAGCCAGTCC-3' | EcoR I Sal I |
pGBKT7-mAKAP4 | NM_009651 | 148-2664 | 5'-CGGAATTCATG TCTGATGACA TTGACTGGT-3' 5'-ACGTCGACCAGGTTAGCGAGAAGCCAGTCC-3' | EcoR I Sal I |
pGBKT7-ropporin | AF178531 | 1-636 | 5'-CATGCCATGGGACCTCAGACAGACAAGCAAG -3' 5'-CGGAATTCTTATTCCAGCCGAACCCTAGGGT-3' | Nco I EcoR I |
α-Galactosidase quantitative assay
SD/-Leu/-Trp or SD/-His/-Leu/-Trp cultures were inoculated with a single, fresh yeast colony and incubated overnight at 30°C. The A600 was measured, and the supernatant was harvested by centrifugation at 14,000 ×g for 2 min. 40 μl of the supernatant was combined with 120 μl of fresh assay buffer [2:1 ratio of 0.5 M sodium acetate, pH 4.5, to 100 mM p-nitrophenyl-D-galactopyranoside (Sigma) ] and incubated at 30°C for 60 min. The reaction was stopped by addition of 840 μl of 0.1 M Na2CO3. Optical density was measured at 410 nm in a 1.5-ml cuvette, and α-galactosidase units were calculated from the formula: [milliunits/(ml × cell)] = A410 × 1000 μl × 1000/[16.9 (ml/μmol) × 60 min × 40 μl × A600] (Yeast Protocols Handbook). Each yeast colony was assayed in triplicate.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
YFL conceived of the study, carried out the oligomerization, immunoprecipitation and drafted the manuscript. WH carried out the yeast two-hybrid assay. YHK carried out the gene cloning and antibody production. AM participated in part of the immunoprecipitation study. LD participated in the immunofluorescence study. KK carried out the study by immunoelectron microscopy. CJF participated in the design of the study and helped to draft the manuscript. JCH participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.