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Testis |
a Department of Cell Biology and the Center for Research in Contraceptive and Reproductive Health,
b The W.M. Keck Biomedical Mass Spectrometry Laboratory
c Molecular Biology Computing Support ITC-ACHS, University of Virginia, Charlottesville, Virginia 22908
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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defensin, epididymis, immunology, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several investigators have identified sperm-specific autoantigens and isoantigens in humans and other species [6–10]. Recently, probing two-dimensional (2-D) Western blots with ASA-positive human sera from infertile men and women was successful in identifying novel isoantigens/autoantigens in human sperm [11]. In the Lewis rat model, circulating antisperm antibodies were induced when the reproductive tract was obstructed at the level of the vas deferens or the corpus epididymidis [12, 13], and after rats were immunized with rat spermatozoa [14, 15]. The resulting postobstruction sera and hyperimmune sera reacted on 1-D or 2-D Western blots with subsets of sperm proteins, which were thereby identified as autoantigens, isoantigens, or both [16]. Microsequencing analysis subsequently revealed the outer dense fiber proteins odf1 and odf2 [17], sperm mitochondria-associated cysteine-rich protein (SMCP) [18], and the testis-abundant protein termed asparaginase-like protein (ALP) [19] as major rat sperm autoantigens. Less attention has been given to the antigenicity of epididymal proteins, although the abundant epididymal protein DE has been characterized as an isoantigen and an autoantigen in rats [20].
The epididymal microenvironment is important in the development of sperm, which undergo a series of morphological, physiological, and biochemical changes that are tightly regulated by androgens [21]. It is well documented that androgen-regulated glycoproteins are added to, deleted from, or masked in the sperm membrane during passage through the epididymis [22–24]. Because a major role of the epididymis is to provide a specific intraluminal microenvironment [25, 26], antibodies to epididymal proteins could compromise sperm maturation and storage, as well as fertilizing ability.
The aim of the present study was to identify and characterize proteins from rat epididymal fluid that were isoantigenic, autoantigenic, or both, and that also associated with sperm. Therefore, we compared 2-D immunoblots of epididymal fluid proteins with blots of sperm extracts. One protein was selected for further study on the basis of its antigenicity and presence in both epididymal fluid and sperm extracts. This led to identification of a novel sperm-associating gene product of epididymal origin with a motif resembling either or both defensin-like or lectin-like proteins.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Retired male breeders of the Lewis rat strain were obtained from Charles River (Wilmington, MA). They were maintained on a 14L:10D cycle in a temperature-controlled room with food and water ad libitum. Investigations were conducted with the approval of the Animal Research Committee of the University of Virginia School of Medicine and in accordance with the Guide for the Care and Use of Laboratory Animals and other relevant publications.
Isolation of Caudal Sperm and Epididymal Fluid Protein Extraction
Rat spermatozoa were isolated from cauda epididymidis as previously described by back flushing through the vas deferens with M-199 medium [16]. Epididymal proteins were extracted by the cold acetone precipitation method. Briefly, the supernatant M-199 medium (containing cauda epididymal fluid) was collected after pelleting the spermatozoa and was centrifuged at 400 x g for 15 min at 4°C to ensure complete removal of any remaining spermatozoa. This supernatant was added to cold acetone at a 1:10 ratio and allowed to stand overnight at -20°C. Acetone-precipitated material was recovered by centrifugation at 1750 x g for 15 min. Precipitated proteins were dissolved in distilled water and dialyzed against several changes of distilled water at 4°C to remove salts. After dialysis, the soluble protein concentration was estimated by absorbance at A280 and A260 in a spectrophotometer, and then the sample was lyophilized. The epididymal proteins were stored at -70°C until 2-D gel electrophoresis.
Hyperimmune Sera
Hyperimmune rat antisperm serum was raised by immunization of male Lewis rats (n = 7) with isologous sperm as described earlier [12]. Hyperimmune serum generated in this way recognizes a similar set of sperm antigens as postvasectomy sera [16, 27]. The serum samples were stored at -70°C until used for 2-D Western blots of epididymal fluid and sperm extract.
Sperm Solubilization
Sperm were solubilized in Celis buffer [28] that contained 2% (v/v) NP-40, 100 mM dithiothreitol, 9.8 M urea, 2% (v/v) ampholines (pH 3.5–10), and a 10 µl/ml cocktail of protease inhibitors (Sigma Chemical Company, St. Louis, MO). Sperm extracts were prepared by solubilizing sperm (9 x 107/ml) in Celis buffer and shaking at 4°C for 20 min. Acetone-precipitated epididymal fluid proteins were dissolved in Celis buffer (4 mg/ml), and insoluble material was removed by centrifugation for 10 min at 10000 x g. Then both supernatants were used in isoelectric focusing gels.
Two-Dimensional Electrophoresis
For 2-D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2-D SDS-PAGE) of rat epididymal fluid proteins and sperm extracts, isoelectric focusing (IEF) in the first dimension was performed using gel and ampholine (isoelectric point ;obpI;cb of 3.5–6.5) compositions described previously [17]. Sperm extracts containing 150–200 µg of protein or 200–250 µg of solubilized epididymal fluid proteins were separated on 2-D (IEF and SDS-PAGE) gels and transferred to nitrocellulose paper [29]. Two-dimensional gels were stained with silver nitrate [30].
Two-Dimensional Immunoblots
For 2-D Western blots, the transferred membranes were washed for 5 min in PBS pH 7.4 and blocked with 5% dry milk in 0.05% Tween-20 in PBS pH 7.4 for 1 h at 22°C. Blots were incubated with preimmune or postimmune rat sperm hyperimmune serum diluted 1:2000 at 4°C overnight with shaking. The secondary antibody (Jackson ImmunoResearch, West Grove, PA), goat anti-rat immunoglobulin G (IgG) conjugated to horseradish peroxidase (HRP), was diluted 1:5000 in PBS containing 0.05% Tween-20 and the blots were incubated for 1 h at 22°C. The peroxidase reaction product was visualized by enhanced chemiluminescence (ECL) using the manufacturer's protocol (Amersham, Buckinghamshire, UK). The blots were scanned with a flatbed scanner (Umax, Fremont, CA) at a resolution of 300 dpi, and the resulting digitized images were analyzed manually.
Microsequencing of a 
28-kDa Isoantigen
After identification of a 28-kDa protein as an isoantigen on 2-D immunoblots, the corresponding spot was cored from a 1.5-mm-thick silver stained 2-D SDS-PAGE gel. The material was collected in a sterile, siliconized microcentrifuge tube and submitted to the University of Virginia W.M. Keck Biomedical Mass Spectrometry Laboratory for analysis. The sample was processed as described previously [17]. MS/MS spectra were automatically batch-searched using Sequest (ThermoFinnigan, San Jose, CA) against the nonredundant (NR) and expressed sequence tag (EST) databases. Any peptide not matching either database was de novo sequenced and synthesized for confirmation.
Lewis Rat Epididymal and Testicular cDNA Library Construction
Poly(A)+ mRNA was extracted from Lewis rat testes and epididymides using the FastTrack 2.0 kit (Invitrogen, Carlsbad, CA). The concentration of the isolated mRNA was estimated on the basis of absorbance at 260/280 nm. These preparations were used in cDNA library construction and Northern blot analysis.
Two micrograms of rat epididymal poly(A)+ mRNA were used for the construction of Marathon adapter-ligated cDNA using a Clontech kit (Palo Alto, CA). To summarize, the first strand of cDNA was constructed by using oligo(dT) priming with avian myeloblastosis virus reverse transcriptase. The RNA strand was digested and the second strand of cDNA was synthesized in a mixture of E. coli DNA polymerase I, RNase H, and E. coli DNA ligase. The blunt DNA ends were created by the addition of T4 DNA polymerase and cDNA adapters were ligated to both the ends of the cDNA by adding T4 ligase. Adapter library quality was tested by using control G3PDH primers with adapter primers in 3' and 5' rapid amplification of cDNA ends and polymerase chain reaction (RACE PCR) control reactions.
RACE-PCR
Two completely degenerate deoxyinosine containing primers (sense, 5'-AAR-TGG-TAY-CAR-ACI-GAY-CCI-GCI-ACI-GGI-AA-3'; antisense, 5'-TTI-CCI-GTI-GCI-GGR-TCI-GTY-TGR-TAC-CAY-TT-3', where I = deoxyinosine, R = A + G, and Y = C + T) were designed from a peptide (#5) obtained by mass spectrometry (K W Y Q T D P A T G K), and were synthesized by Gibco BRL (Grand Island, NY). Rapid amplification was performed by 3' RACE PCR using sense degenerate deoxyinosine primer (100 pM) and adapter (AP-1) primer (5 pM) with 0.25 ng of adapter ligated Marathon rat epididymal cDNA as template in a 25-µl volume reaction. Thermal cycling was performed in an MJ Research (Watertown, MA) thermal cycler (PTC-200 DNA engine) for 35 cycles with the hot start method. The first 12 cycles decreased the annealing temperature by 1.3°C from 68° to 54°C, after which the annealing temperature was held at 45°C. The denaturation step (94°C for 30 sec) and the polymerization step (68°C for 2.3 min) were the same throughout the 35 cycles. The PCR amplified product was separated on a 2% agarose gel (Gibco BRL). A 0.3-kilobase (kb) DNA fragment was isolated using a QIAprep Miniprep plasmid DNA isolation kit (Qiagen, Valencia, CA) and was cloned into pCR2.1-TOPO vector (Invitrogen). Sequencing was performed by the University of Virginia Biomolecular Research Facility. A partial 3' RACE clone was obtained that contained a 0.3-kb open reading frame (ORF) with a poly(A) tail. The 5' end of the DNA was then amplified by 5' prime RACE PCR using another 3' gene-specific antisense primer (5'-CAG-CAG-TGG-TGG-CCG-CTG-CAC-CTG-TG-3') plus AP-1 primer with the same template. The entire 0.5-kb full length of cDNA was cloned into pCR 2.1-TOPO vector. This full-length clone revealed 449 base pairs (bp) containing an ORF of 333 bp encoding 111 amino acids. Nucleotide and amino acid sequences were analyzed by using the Genetic Computer Group (Madison, WI) analysis program.
Northern Blot Analysis
Total mRNA was isolated from Lewis rat testis and from initial, caput, corpus, and cauda epididymal segments using Trizol reagent as recommended by the manufacturer (Gibco BRL). Samples of total mRNA from different regions of the epididymis (10 µg/lane), the entire epididymis and testis poly(A)+ RNA (as mentioned earlier) (5 µg/lane), along with RNA standards (4 µg; Millennium marker, Ambion, Austin, TX), were separated on a 1% agarose gel containing formaldehyde [31], transferred to a Duralon-UV membrane, (Stratagene Corp., La Jolla, CA) and UV cross-linked to the membrane with a Stratalinker (Stratagene). In addition, a commercial rat multitissue Northern (MTN) blot (Clontech) containing 2 µg of poly(A)+ RNA per lane from eight different rat tissues—heart, brain (whole), spleen, lung, liver, skeletal muscle, kidney, and testis—was probed. Because there was no epididymal RNA lane on the MTN blot, 2.5 µg of rat epididymal poly(A)+ mRNA was spotted as a positive control.
The probe for Northern analysis was prepared from nucleotides 1 to 333 and was labeled with 32P using the random-primed method (Prime-a-Gene Labeling System labeling kit; Promega, Madison, WI). The blots were incubated with ExpressHyb Hybridization solution (Clontech) for 1 h at 65°C. The probe was thawed, heated at 95°C for 5 min, mixed with 10 ml of fresh ExpressHyb solution added to the blot, and hybridized for 2 h at 65°C. Blots were washed twice for 15 min each with 2x saline-sodium citrate (SSC) containing 1% SDS and twice for 30 min each in 0.1x SSC containing 0.5% SDS at 65°C. The blots were exposed to autoradiographic film for 24 h and then continuously for 2 wk.
Bacterial Expression of E-3
Primers (sense, 5'-CTC-TCT-CCA-TGG-ACT-GGT-ACG-TTC-GAA-AGT-GTG-CAA-ACA-AAT-TGG-GC-3'; antisense, 5'-CTC-TCT-CTC-GAG-GGG-TGC-AGC-AGT-GGT-GGC-CGC-TGC-ACC-TGT-3') were designed to encompass the E-3 cDNA corresponding to 90 amino acids from 21 through 111 (i.e., without signal peptide). The sequence was amplified by PCR using rat epididymal Marathon cDNA as a template. The PCR product was cloned into the NcoI site at the 5' end and the XhoI site at the 3' end of the ORF of pET 28b(+) (Novagen, Madison, WI). The recombinant pET-E-3 construct with a six His-tag on the C-terminus was transformed into the Nova Blue (DE3) strain of Escherichia coli. A single colony bearing pET-E-3 was inoculated into Luria-Bertaine broth medium and induced with 1.0 mM isopropyl-thio-ß-D-galactopyranoside (IPTG) at 0.5 optical density (O.D. at A600), and cells were harvested 5 h later after reaching an O.D. of 1.0. His-tagged recombinant protein was purified using His-binding resin according to the manufacturer's protocol (Novagen), followed by Prep-Cell electrophoresis (BioRad, Hercules, CA). The presence of recombinant protein in purified fractions was confirmed by anti-His reactivity. Prior to immunization, the identity of the expressed recombinant protein was confirmed by microsequencing with tandem mass spectrometry.
Immunization with Recombinant E-3 Protein
White Leghorn chickens were immunized s.c. in the breast region with 100 µg of Prep-Cell purified recombinant E-3 protein (rE-3) emulsified in Freunds complete adjuvant (FCA), and were boosted four times at intervals of 21 days with 50 µg of protein emulsified in Freunds incomplete adjuvant (FIA). Ten days after the third booster, the animals were bled and the serum/plasma was collected. Prior to immunization, preimmune sera were collected and all the samples were stored at -20°C until used. Eggs were collected daily for 10 days after the third and fourth booster, and stored at 4°C until used. The specific antibody reactivity was determined in serum as well as in egg yolk (IgY). Control animals were administered normal saline (NS) alone in FCA and FIA according to the same immunization schedule.
Extraction of Egg Yolk IgY Antibodies
The purification of egg yolk IgY was performed according to the manufacturer's protocol using the Eggcellent (Pierce, Rockford, IL) chicken IgY purification kit. The final precipitated IgY was collected by centrifugation at 10000 x g for 15 min at 4°C, and dissolved in PBS equal to the original volume of yolk. The protein concentration of this preparation was estimated by absorbance at 280 nm, and the purity was checked by SDS-PAGE. Purified egg yolk IgY was filter-sterilized (using 0.42 µm Amicon filters), and sodium azide (0.02%) was added as a preservative prior to storage at 4°C.
Affinity Purification of Anti-rE-3 Chicken Antibodies
Anti-E-3 chicken polyclonal antibodies were purified using the Olmstead method [32] with modifications. Briefly, a 100-mm circular nitrocellulose paper disc was soaked in PBS for 5 min, and then incubated with purified rE-3 antigen 1 mg/10 ml in PBS containing 0.05% Tween-20 (PBS-T) for 2 h at 22°C. The paper disc was blotted, washed, and blocked with 5% dry milk in PBS-T for 1 h. After blocking, the membrane was incubated overnight at 4°C with primary antibody (anti-rE-3 polyclonal IgY) diluted 1:1000. The remaining "absorbed" solution was collected and stored at 4°C. The membrane disc was washed with PBS-T and PBS, eluted with 8.5 ml of elution buffer (Pierce) for 5 min at 22°C, and immediately neutralized by the addition of 0.85 ml of 1 M Tris-HCl (pH 8.0). The sample was dialyzed against PBS at 4°C, and concentrated to 1 ml by Amicon filtration. The resulting affinity-purified antibodies were stored at 4°C prior to use in immunofluorescence studies.
Western Blot Analysis with Chicken Anti-rE-3 Antibodies
Region-specific spermatozoa were collected from the testis and from different epididymal segments (initial, caput, and corpus) by mincing tissue segments to liberate sperm in M-199 medium. After counting the number of sperm, aliquots of equal numbers of sperm were pelleted by centrifugation at 400 x g for 8 min at 4°C and stored at -20°C. Spermatozoa from the cauda epididymidis were collected as described earlier by flushing through the vas deferens. Epididymal fluid supernatants of corpus and caudal fluid (devoid of spermatozoa) were pooled and proteins were precipitated with cold acetone (as mentioned earlier) prior to use in 2-D and 1-D Western blots. Epididymal fluid and sperm extracts were applied to IEF gels that were expanded at the acidic end (pI of 2.5–5.5) by using an ampholine composition (v/v) of 30% 3.5–5 pH, 20% 5–7 pH, and 50% 2.5–5 pH. Subsequent steps in 2-D analysis followed the procedures described above. For 1-D gel analysis, samples of epididymal fluid proteins, sperm proteins, or rE-3 were extracted with Laemmli buffer [33] by boiling for 10 min, followed by centrifugation at 10000 x g for 10 min to remove the cell debris. Extracts were separated on 10% Tris-tricine SDS-PAGE [34] at 12 mAmp overnight and electroblotted onto 0.22 µm nitrocellulose (NitroPure; Osmonics Inc; Westborough, MA) at 1.0 amp for 1 h. The blots were stained with Ponceau S to visualize the loading of proteins in each lane. The membranes were blocked with 5% (w/v) fat-free milk in PBS-Tween (10 mM PBS pH 7.4, 0.05% Tween-20, PBS-T) for 1 h at 22°C, and were incubated with anti-rE-3 chicken serum diluted at 1:10 000 in PBS-T overnight at 4°C. Immunodetection was achieved either with HRP- or alkaline phophatase-conjugated rabbit anti-chicken IgG secondary antibody (Sigma) at a dilution of 1:20000 in PBS-T. The blots were washed three times with PBS-T followed by PBS for 5 min each and also visualized with a chromogenic substrate TMB (3,3',5,5'-tetramethylbenzidine and 0.01% H2O2) (Kirkegaard and Perry Laboratories, Gaithersburg, MD) or BICP/NBT substrate (Sigma). The peroxidase reaction product was visualized by ECL using the manufacturer's protocol (Amersham, Buckinghamshire, U.K.).
Indirect Immunofluorescence Localization of E-3 in Air-Dried Rat Sperm
Swim-up spermatozoa were prepared by layering 0.5 ml of washed rat spermatozoa in 2 ml of M-199 medium containing polyvinyl alcohol (PVA) and sodium pyruvate for 1 h at 37°C in 5% CO2. The motile sperm were air-dried onto poly-L-lysine coated slides (prepared in the laboratory) and stored at -20°C until used for localization studies. The preparations were initially incubated for 5 min with PBS and blocked with 10% normal rabbit serum (NRS) in PBS-T for 30 min. The slides were then incubated overnight at 8°C with anti-rE-3 polyclonal antibodies (1:1000), absorbed antibody (1:1000), or affinity-purified antibody (undiluted), washed three times with PBS-T, and incubated with fluorescein isothiocyanate-conjugated rabbit anti-chicken IgG (Sigma) at 1:200 dilution in PBS-T for 30 min at 22°C. The slides were washed thrice with PBS-T followed by PBS at intervals of 5 min, incubated for 5 min with equilibration buffer followed by Slow Fade (Molecular Probes, Eugene, OR), and mounted with cover slips. Images were captured with an Axioplan fluorescence microscope (Zeiss, Oberkochen, Germany) at 40x.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our previous studies of sperm antigens were directed mainly toward proteins that migrated at pIs of 4.5–7.4 in 2-D gels, because isoantigens and autoantigens appeared to be most abundant in this region in sperm extracts [17]. In the present study we expanded the acidic ends of the gels to achieve better resolution of proteins with pIs in the range of 3.5 to 6.5. This region was expected to include highly glycosylated proteins, possibly including some of epididymal origin. Indeed, immunostaining of 2-D blots using rat hyperimmune sera raised against rat sperm revealed several immunoreactive acidic proteins (pI 3.5–4.5) in both epididymal fluid and sperm extracts (Fig. 1, A and B). Immunoreactivity of these spots with pooled hyperimmune sera and the absence of staining by pooled preimmune sera (Fig. 1, C and D) indicated that these proteins were isoantigens.
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Individual serum from each of seven hyperimmunized animals bound epididymal fluid proteins, including an elongated band or a streak designated as E-3 (Fig. 1A) at 28 kDa and pI 3.5 that was stained by 6 of 7 (86%) hyperimmune sera. Apart from the acidic antigens, scattered spots and constellations of epididymal proteins toward the neutral end of the gel (pI 6.5) were likewise immunoreactive.
In the case of sperm extract blots, pooled hyperimmune sera recognized multiple isoantigens (Fig. 1B) as previously reported [16]. With attention focused on the acidic end of the Western blot corresponding to the region in which most of the reactivity was detected for epididymal proteins, we observed several immunoreactive sperm proteins in common with epididymal proteins (arrows in Fig. 1B). A group of proteins with molecular weights greater than 60 kDa was stained and the streak (E-3) at 28 kDa and pI 3.5 in sperm extracts reacted with 6 of 7 (86%) hyperimmune sera.
Microsequencing of E-3 by Mass Spectrometry
To obtain further information on the isoantigen designated E-3, the corresponding spot was cored from a silver-stained 2-D SDS-PAGE gel of epididymal fluid proteins (circled area in Fig. 2), and amino acid sequence was obtained by tandem mass spectrometry. Ten peptides were observed (Table 1). Peptides 1–3 were identical except for the type of cysteine modification. Peptide 5 contained a single additional amino acid not found in peptide 4. Peptides 7–10 were heavily glycosylated and provided no amino acid sequence. The analysis also revealed that the tyrosine in peptides 4 and 5 was partially phosphorylated. Searches were performed on the MS/MS spectra for these peptides using Sequest (ThermoFinnigan) against the nonredundant and EST databases. No matches were found, indicating these peptides derived from a potentially novel protein. Later comparison to the E-3 cDNA allowed peptide 6 to be fully deduced (partial sequence initially obtained).
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Cloning and Sequence Analysis of E-3: A Novel Epididymal Fluid Protein
A completely degenerate inosine-containing forward primer was designed from peptide 5 (Table 1). Using rat epididymal adapter ligated cDNA as template, the first round 3' RACE PCR amplified a partial sequence of 0.3 kb. In a second round of 5' RACE PCR, another reverse primer from the C-terminus was designed and a PCR amplimer of 0.5 kb was cloned and sequenced. The resulting cDNA revealed a stop codon at 352–354 bp and a polyadenylation signal (ATTAAA) at 396 bp, 11 bp upstream from the poly(A) tail. The full-length nucleotide sequence, obtained and deposited in GenBank (accession number AF329091) consisted of 449 bp and a complete ORF of 333 bp that encoded a protein of 111 amino acids (Fig. 3). The deduced sequence for the protein had an in-frame start codon at nucleotides 19–21 that conformed to a Kozak consensus [35]. The original immunoreactive spot had a molecular mass 28 kDa and pI 3.5. However, the cloned sequence encoded 111 amino acids with a predicted molecular mass of 11 kDa and pI of 9.4. This difference could be due to glycosylation, phosphorylation, and aggregation of the native protein. In any case, it is important to emphasize that peptides 1–6 that had been obtained by microsequencing (Table 1) were embedded in the deduced sequence (underlined in Fig. 3), demonstrating that correct cDNA for the protein initially cored from the gel was cloned.
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Sequence Analysis
The E-3 protein appeared to represent a novel gene, which did not match any known proteins from the databank, and analysis of the predicted amino acid sequence of E-3 revealed an N-terminal signal peptide (amino acids 1–21) with a consensus cleavage site. Further analysis with PredictProtein site [36] demonstrated the presence of four putative phosphorylation sites, and four O-glycosylation sites at the C-terminus of the protein (as shown in Fig. 3). Maxhom multiple sequence alignment [37] indicated that E-3 had 3-D homologues among certain lectin family proteins. E-3 had conserved cysteine residues and 28%–30% identity over 70 amino acids at the C-terminus with 3 lectin proteins in the WGA family (Fig. 4A).
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When E-3 was first cloned, FASTA searches showed no significant match with known proteins. Later, however, basic local alignment search tool and FASTA searches revealed a 100% identity to the 2D6-antigen sequence (accession number AJ309150). In addition, partial matches of the N-terminal portion of E-3 were found to a "novel human protein" (45%) (Accession number CAC17684), human ESP 13.2 (43%) (accession number NP112193), and a "putative novel human protein" (36%) (accession number DJ1018D12) over 60 amino acids. ESP 13.2 from the macaque showed a 36% match to E-3 (accession number CAC27133) over 90 amino acids. ESP 13.2 and EP2 have some similarity with defensins, and recently, defensin-like proteins have been reported in the rat [38], primate [39], and human epididymis [40, 41]. Therefore, we further explored possible similarities of the E-3 sequence to defensins. The alignment of E-3 sequence with known defensins was carried out based on the presence of cysteines in E-3. The alignment was forced to match six cysteines in defensins. These constitute a motif (Fig. 4B) that is characteristic of defensins [42]. In addition, computer analysis of predicted secondary structure in mature E-3 (without a signal sequence) yielded a terminal alpha helical barrel followed by three beta sheets as depicted (Fig. 5). This succession of alpha helix and 3 beta sheets resembles the crystal structure of 41 amino acids from human ß defensin-2a (hBD-2a) as determined by NMR (PDB #1fd4) [43]. Based on structural similarity and amino acid homology and the predictions above, we created a hypothetical model to depict the potential relationships between E-3, defensin, and WGA lectin (Fig. 6).
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Recombinant E-3 Expression
The cDNA sequence encoding a 90 amino acid (22–111) mature E-3 protein (without signal peptide) was cloned into the bacterial expression vector pET 28b+. Nova blue (DE3) cells were used as host cells. When the bacteria were induced with IPTG for 6 h, bands of 30 kDa and 14 kDa were produced, and confirmed by Western blotting with Ni-NTA-peroxidase conjugate, which identified the recombinant protein with a C-terminal 6 His-tag. The identity of the expressed and purified recombinant E-3 protein was confirmed by microsequencing, which recovered E-3 peptide sequences (data not shown).
Secretory and Sperm-Associated E-3 Protein in Epididymal Fluid
The chicken anti-rE-3 antibodies were characterized using serum IgG in 2-D Western blot analysis. On 2-D Western blots of epididymal fluid proteins (Fig. 7A), anti-rE-3 antibody specifically stained the E-3 streak at 28 kDa between 2.5–3.5 pI (indicated by arrows in Fig. 7A) as well as a few minor spots. Preimmune sera did not stain any spots among epididymal fluid proteins (Fig. 7C). Similarly, anti-rE-3 antibody stained an acidic streak at 28 kDa; 2.5–3.5 pI in sperm extract suggesting that E-3 protein secreted by the epididymal epithelium associates with sperm (Fig. 7B). Preimmune serum weakly stained a few spots (Fig. 7D), but did not stain the acidic streak at 28 kDa.
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The 28-kDa streak originally observed, which reacted with hyperimmune sera (Fig. 1) in epididymal fluid and sperm extract, comigrated with the spots stained by anti-rE-3 polyclonal serum. For further confirmation that the antiserum recognized native E-3, the spot stained with anti-rE-3 antibodies was cored from silver-stained gels of epididymal fluid protein or sperm extract and subjected to mass spectrometry. Microsequencing revealed E-3 peptides in both instances, confirming that the rE-3 polyclonal antibody recognizes E-3 in both epididymal fluid and sperm. Apart from the E-3 reactivity in the acidic region at 28 kDa, the other spots bound by polyclonal antisera may represent different iso forms or posttranslational modification of E-3 or E-3 related proteins.
Tissue- and Region-Specific Expression and Identification of E-3 mRNA and Protein in the Rat Epididymis
To determine the site of expression of the E-3 gene, poly(A)+ mRNA was isolated from Lewis rat testis and epididymis. Total mRNA was extracted from the initial segment, caput, corpus, and cauda segments of the epididymis, and a rat multiple tissue Northern (MTN) blot-containing poly(A)+ RNA from eight different tissues was obtained. Strong hybridization to a 0.5-kb band appeared only in the epididymis and not in the testis (Fig. 8A, left panel). Further, analysis of the regional expression of E-3 (Fig. 8A, center panel) revealed that the 0.5-kb E-3 message was expressed predominantly in the corpus with lower level of expression in the cauda of the epididymis and no detectable message in the caput and initial segment. On the MTN blot (Fig. 8A, right panel), hybridization of E-3 probe was negative for all tissues except for the positive control of epididymal poly(A)+ mRNA. These results suggest that E-3 is an epididymal-specific gene that is predominantly expressed in the corpus and cauda of the epididymis.
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The association of E-3 protein with sperm in different regions of the epididymis was studied by 1-D Western blotting as shown in Figure 8B. Anti-rE-3 antisera (1:15000) stained a specific band at 27 kDa in blots of sperm extracts from the corpus (occasionally also a 31-kDa band in sperm extract from corpus), and slightly lower than 27 kDa in the cauda epididymis (Fig. 8B, postimmune sera), but the antiserum did not bind proteins from caput, initial segment, nor did it stain testicular sperm. Preimmune sera did not cross-react on Western blot (Fig. 8B, preimmune sera). These results suggest that the E-3 mRNA and protein is predominantly expressed and secreted in the corpus of the epididymis, and that it associates with luminal spermatozoa.
Localization of E-3 on Rat Spermatozoa
Indirect immunofluorescent localization of noncapacitated and air-dried rat spermatozoa using anti-E-3 serum (diluted 1:1000) revealed staining of the entire sperm (Fig. 9). However, the intensity of immunostaining varied as follows. The polyclonal antibody reacted most intensely with the middle piece, followed by the remainder of the tail, which showed a punctate or uneven staining, while the head showed the least staining (Fig. 9A). Affinity-purified antibodies reacted in a similar pattern to the polyclonal serum staining the entire tail, with most intensity in the midpiece (Fig. 9B), but showed diminished staining in the head. Immune IgY, which had been adsorbed with rE-3 as a control, showed greatly diminished immunoreactivity with the midpiece and no reactivity with the principal piece and head (Fig. 9C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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28 kDa) kDa with a pI of 3.5. The E-3 cDNA contains the complete coding sequence (Fig. 3) as indicated by the presence of a Kozak consensus [35] sequence upstream of the most 5' ATG, and a stop codon at the 3' end. The deduced amino acid sequence of E-3 contains three microsequenced peptides in its ORF, which supports the contention that the original cored protein was cloned. Previously, other sperm-specific antigens were detected by various methods. In humans, sperm coating autoantigens including a sperm-specific FA-1 glycoprotein [6] were identified by using monoclonal antibodies [44, 45]. A novel 67-kDa peptide antigen was identified in mice [8], and studies in rabbits revealed a sperm autoantigen designated as RSA-1 [46]. Many rat sperm antigens have been identified as isoantigens or autoantigens by reaction with postobstruction, hyperimmune sera, or both on 1-D or 2-D rat sperm immunoblots [14, 16]. Although a few of these have been characterized further, the rat autoantigens previously identified in sperm extracts may have been either of testicular or epididymal origin. On the other hand, E-3 cDNA was cloned by using a gene-specific, degenerate inosine-containing primer in RACE PCR with rat epididymal Marathon cDNA as template. The E-3 protein in epididymal fluid was most likely secreted into the epididymal lumen by the epithelial cells because the mRNA and the E-3 protein are expressed predominantly in the corpus followed by the cauda epididymidis. The secreted E-3 protein in epididymal fluid thus appeared to associate with sperm during their transit through the epididymis. Various other proteins have been identified as epididymis-specific, and their synthesis is frequently restricted to specific regions of the epididymis [47–49].
The nature of the association between E-3 and the maturing spermatozoa is unknown. However, secreted E-3 protein does associate with spermatozoa as indicated by immunofluorescence, and protein was detected in corpus and caudal sperm extracts on Western blot. Moreover, surface localization of the 2D6 antigen, recently shown to be identical to E-3 [50], was demonstrated by electron microscopy. Using labeled 2D6 monoclonal antibody, immunogold particles were shown to bind the plasma membrane of the flagellum [51]. This association with spermatozoa might occur in several ways: 1) the lectin-like motifs may bind to carbohydrates on the sperm, 2) the putative defensin-like N-terminal alpha-helical peptide could help to anchor the protein into the membrane, or 3) a receptor might mediate association with the sperm.
Although the functional properties of E-3 are yet to be defined, its molecular characteristics and pattern of expression suggest that it might act in sperm maturation, sperm-egg binding, as a decapacitation factor, or as a defensin to protect sperm from bacterial infection. Moreover, information can be gleaned from previous studies because the E-3 sequence (accession number AF329091, December 13, 2000) is that of the antigen recognized by the 2D6 monoclonal antibody (accession number AJ309150, April 4, 2001) [50], which was investigated in the 1980s using immunologic approaches [52–54]. The 2D6 antigen is androgen-dependent, and the earlier studies also identified it as a secretory protein [53]. The 2D6 monoclonal antibody stained the flagellum of corpus and cauda sperm [51, 52], while the present study also detected E-3 staining of the flagellum, albeit with greater intensity in the midpiece. The 2D6 antigen was also detected on the heads of about 30% of caput spermatozoa [51] (not examined in the present study). Remarkably, staining spread to the egg plasma membrane following fertilization [55], suggesting a possible postfertilization function. Previous studies of proteins recognized by the 2D6 antibody revealed 31-, 28-, 23-, and 20-kDa moieties in epididymal fluid and 32- and 23-kDa peptides from spermatozoa [53]. These values compare favorably with the 31- and 27-kDa bands observed in 1-D gels of epididymal fluid and sperm in the present study with polyclonal antisera (data not shown). Thus, our observations agree with the previous studies using the 2D6 antibody and extend them to include cloning of the antigen (E-3), its expression in bacteria, generation of antisera to the recombinant protein, and Northern analysis of regional patterns of gene expression in the epididymis as well as analysis of the protein sequence, which provides insight into potential functions of the molecule.
We observed the induced expression of two bands, at 30 and 14-kDa, in the bacterial system. Both bands were identified with Ni-NTA, which binds the 6 His-tag at the C-terminus of recombinant protein, and in both cases microsequencing revealed E-3 peptides, suggesting that bacterially expressed recombinant E-3 protein of 14 kDa aggregated to form 30-kDa dimers (data not shown). On the other hand, on 2-D blots of epididymal fluid or sperm extract, anti-rE-3 serum recognized a 28-kDa "native" protein that comigrated with the streak initially cored for study. Because this 28-kDa spot is both larger and more acidic (pI 2–5–3.5) than the deduced protein sequence (11 kDa, pI 9.4), the data suggest that E-3 synthesized in the epididymis undergoes dimerization or posttranslational modification or both, leading to the 28-kDa form. It was interesting that sperm extracts contained almost exclusively the acidic 28-kDa E-3, whereas some smaller spots were also stained lightly in epididymal fluid (Fig. 8A). Small size differences in the native E-3 in fluid and sperm extracts from different regions of the epididymis may reflect changes in glycosylation of E-3 during processing, secretion, and association with sperm.
In our initial searches, E-3 had no recognizable homology with known sequences from the databank. Subsequently, we found a resemblance to other newly discovered proteins such as ESP13.2 from human and macaque [56], as well as two putative novel human proteins (accession numbers CAC17684 and DJ1018D12), which were detected by sequencing the human genome (Fig. 4B). E-3 has a motif consisting of six cysteines that appears to be present in other epididymal-specific proteins such as ESP 13.2 [56], EP2 [39, 57], ESC42 [58], and Bin1 b [38], as well as in ß-defensin [59] proteins produced by different epithelia from various species. Based on its predicted secondary structure, E-3 is similar to ß-defensins, having an alpha helical structure followed by three beta sheets, and by extension, one can speculate that other epididymal proteins have a similar configuration (as shown in Fig. 5). Recently, it has been shown that conservation of secondary and tertiary structure is more important in terms of functional aspects than is sequence similarity among human and murine ß-defensins [42].
These considerations support the hypothesis that E-3 is a novel epididymal member of the defensin family of proteins. Defensins are broad-spectrum cationic antimicrobial peptides that exist in many species from insects to mammals including humans [60]. Vertebrate defensins are classified as either 
or ß types based on differences in disulfide linkages. They are characterized by the presence of three intramolecular cysteine disulfide bonds, ß sheets, and a hairpin loop. Various epithelial cells, including those of the respiratory and intestinal tract, produce the ß-defensins, which are believed to help protect against infection [60, 61]. Thus, the presence of these defensin-like proteins in the male and female reproductive tracts in humans and rodents [38, 40, 41] may protect sperm and the epididymis from bacterial infections. Confirmation of this possibility for E-3 awaits tests of antibacterial activity against a variety of organisms. Recently it has been shown that ß-defensins also play multifunctional roles apart from antibacterial activity [60], including interaction with plasma membranes of Xenopus oocytes and chemoattractant to macrophages [62] indicating a possible role in sperm-egg interaction and fertilization. Thus, it is possible that E-3 (and perhaps other similar family members) may have assumed different roles in spermatozoa.
E-3 was also found to have a limited similarity, based on its putative 3-D structure, to isolectin agglutinin proteins belonging to the WGA family [63]. Each isolectin contains four chitin-binding domains, and binds to N-acetyl-D-glucosamine/N-acetyl-D-neuraminic acid. Phylogenetically, the lectins are more common in plants and invertebrates than in higher mammals, but some other lectin-like proteins have been identified in the male reproductive system. In sea urchin spermatozoa, the lectin-like protein known as bindin binds to fucose residues on the vitelline layer of eggs [64]. Among mammals, the rabbit sperm membrane autoantigen RSA is considered to be a lectin-like zona binding protein [65], and the demonstration that complex carbohydrates strongly inhibit RSA-zona binding underscores the important role for carbohydrate recognition in sperm-egg binding. Because the lectin and defensin domains are both represented in E-3, it is possible that E-3 represents a novel type of defensin that employs a lectin domain to target certain cell surfaces. This intriguing possibility may make E-3 unique among the defensins and confer unusual binding properties on E-3 that allow sperm to acquire the molecule for subsequent function in the female reproductive tract. The human cathelicidin, hCAP-18, provides another example of an antibacterial molecule that is produced in the epididymis, is present in seminal plasma, and associates with sperm via a portion of the protein from which the peptide (LL-37) with antibacterial activity is obtained by proteolytic cleavage [66].
In conclusion, E-3 is a novel and epididymis-specific secretory protein identified in Lewis rats. It is an isoantigen resembling either ß-defensins, a lectin, or both. It localized to the rat sperm flagellum. The results suggest that the E-3 gene is predominantly expressed in the corpus and cauda of the epididymis, and that the secreted E-3 protein associates with spermatozoa.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Jayasimha Rao, Department of Cell Biology, School of Medicine, University of Virginia Health System, P.O. Box 800732, Charlottesville, VA 22908-0732. FAX: 434 982 3912; jr7y{at}virginia.edu
Received: 5 June 2002.
First decision: 4 July 2002.
Accepted: 5 August 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, four O-glycosylation sites at the C-terminus are indicated by bold (T), and * represents the stop codon. The predicted signal peptide cleavage site is indicated by 
and the polyadenylation signal ATTAAA (located at position 396) is underlined. The deduced sequence of the predicted protein has a molecular mass of 
