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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 2  |  Issue : 4  |  Page : 191-200

Monoclonal antibodies reveal novel localization of SPAG11E in spermatozoa and the antifertility potential of SPAG11E motifs


1 Clinical Laboratory (Hospital of the SIPPR) and NHC Key Laboratory of Reproduction Regulation, Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China
2 Core Facility of Basic Medical Sciences, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, China
3 Shanghai Key Laboratory of Molecular Andrology, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China

Date of Submission10-Nov-2018
Date of Web Publication11-Jan-2019

Correspondence Address:
He-Guo Yu
Shanghai Institute of Planned Parenthood Research, 2140 Xietu Road, Shanghai 200032
China
Hua Diao
Shanghai Institute of Planned Parenthood Research, 2140 Xietu Road, Shanghai 200032
China
Yong-Lian Zhang
Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2096-2924.249890

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  Abstract 


Objective: SPAG11E is the first β-defensin that has been reported to activate Ca2+ uptake and sperm motility. However, the exact subcellular localization and interaction of SPAG11E with sperm remain controversial because of the lack of qualified antibody tools. SPAG11E is also a potential male antifertility target because SPAG11E fragment conjugated with a carrier protein exhibits male contraceptive vaccine potential. However, the fine B-cell epitope motifs of SPAG11E have not been analyzed, which hampered further exploration of the potential target.
Methods: Polyclonal and monoclonal antibodies (mcAbs) of mature SPAG11E were raised and qualified with Western blotting. Subcellular localization of SPAG11E was revealed by Western blotting, immunohistochemistry staining, and electron microscopy. B-cell epitopes of rat SPAG11E were mapped by Western blotting using polyclonal and mcAbs. Based on the conservation of the identified epitope motifs between rat and mouse SPAG11E, antifertility potential of the epitope motifs was evaluated by the offspring of the males compromised with specific mcAbs.
Results: SPAG11E antibodies of high quality were obtained and all B-cell epitope motifs of rat SPAG11E were mapped, in which conserved epitope motifs of SPAG11E in various species were discovered. The epitope motifs recognized by mcAbs were identified respectively. With mcAbs, rat SPAG11E was proved to be expressed in the caput region of epididymis. A novel finding was that SPAG11E was located in the flagella and nuclei of sperm as revealed by immunoelectron microscopy. In addition, the males treated with mcAbs (3#-1 and 10#B4) showed apparently fewer offspring.
Conclusions: SPAG11E revealed a β-defensin with novel localization in sperm flagellum and nucleus with qualified antibodies. All B-cell epitope motifs of rat SPAG11E were determined, and the antifertility potential was proved by corresponding mcAbs.

Keywords: Antibody; Contraception; Epitope; SPAG11E; Sperm


How to cite this article:
Wang ZK, Yang YT, Chen XY, Hu SG, Zhu P, Xu WX, Ma L, Yu HG, Diao H, Zhang YL. Monoclonal antibodies reveal novel localization of SPAG11E in spermatozoa and the antifertility potential of SPAG11E motifs. Reprod Dev Med 2018;2:191-200

How to cite this URL:
Wang ZK, Yang YT, Chen XY, Hu SG, Zhu P, Xu WX, Ma L, Yu HG, Diao H, Zhang YL. Monoclonal antibodies reveal novel localization of SPAG11E in spermatozoa and the antifertility potential of SPAG11E motifs. Reprod Dev Med [serial online] 2018 [cited 2019 Jan 17];2:191-200. Available from: http://www.repdevmed.org/text.asp?2018/2/4/191/249890

Zhi.Kai Wang and Yi.Ting Yang equally contributed to this article.





  Introduction Top


β-defensins are endogenous cysteine-rich antimicrobial peptides (AMPs) with pleiotropic activities that contribute to host defense, signaling,[1] coat color,[2] cell apoptosis,[3] sperm functions,[4] and carcinoma suppression.[3],[5] A total of 43, 52, and 39 β-defensins have been reported to show regional specificity in rat, mouse, and human epididymis, respectively.[6],[7],[8] The presence of a vast array of β-defensins in the male reproductive tract is an intriguing phenomenon that warrants further investigation. Therefore, this protein family has been attracting growing interest owing to their functional diversity.

Recent reports indicate that β-defensins are important in sperm maturation, capacitation, protection, and penetration of cervical mucus.[4],[9],[10],[11] Deletion of nine β-defensin genes in the mouse results in male sterility derived from reduced sperm motility and increased sperm fragility.[12] Moreover, recent findings disclosed potential dual role of defensins in the regulation of infection and control of sperm maturation. Their antimicrobial activity might also be combined with the ability to interact with cell membrane receptors and to modulate ion transport.[11]

SPAG11E (also named BIN1B; GeneID: 246305) is the first β-defensin that was reported to initiate progressive motility in immotile immature sperm.[4],[13] Actually, the quality of anti-SPAG11E rabbit antibody utilized in the previous studies was not good enough either in Western blotting or immunohistochemistry staining assays. The widespread of SPAG11E on the head of sperms from caput, corpus, and cauda regions of rat epididymis revealed by immunofluorescence staining was controversial because the polyclonal antibody could not recognize SPAG11E in tissue extract in Western blotting assays and immunohistochemistry (IHC) staining. Furthermore, the expression pattern did not conform to the specific caput expression of SPAG11E confirmed by Northern blotting. Although one might argue that diffusion of SPAG11E along the lumen fluids was the cause, nonspecific cross-recognition of the polyclonal antibody could not be excluded.

SPAG11E is also a novel epididymal target for male contraception.[14],[15] Downregulation of SPAG11E expression in lipopolysaccharide-induced epididymitis is associated with decreased sperm motility.[16] Its human homolog, SPAG11B isoform D (SPAG11B/D), is hypothesized to modulate signaling pathways of host defense and male reproduction.[17] Immunization of male Wistar rats with a synthetic peptide segment of SPAG11E (MCRSGERKGDICSDP-conjugated with KLH), specifically interfered with sperm motility and resulted in a compromised fertilizing capacity of sperm without any observable orchitis or epididymitis.[18] All these observations and other reports[14] indicate that SPAG11E might be a novel target for the development of posttesticular male contraception. However, detailed epitope information as well as the subcellular localization of SPAG11E on sperm remains unclear, which hampered the exploration and the development of SPAG11E as a potential target of posttesticular male contraception.

A safe and effective peptide vaccination requires an immunogen that does not carry T-cell epitope.[19],[20] One strategy is to develop peptide vaccines based on minimal B cell epitopes,[20] since it is known that T cell epitopes capable of inducing a CD4+ T-cell response are longer (9–30 residues),[21],[22],[23] whereas linear B cell epitopes are shorter (6–8 residues).[24] Thus, for any peptide of more than eight residues, minimal motif mapping should be carried out to remove potentially damaging T cell epitopes.[25]

A simple, cheap, reliable, and adaptable epitope mapping method that has already been successfully applied in the minimal motif analysis of human zona pellucida protein-4[25] might also be applicable for SPAG11E. To clarify the B-cell epitope motifs of SPAG11E, the binding patterns of SPAG11E on rat sperm and their contraceptive potential, the 8-mer epitope motifs of SPAG11E were mapped and monoclonal antibodies (mcAbs) were raised. The contraceptive potential of SPAG11E epitope motifs was also evaluated by comparing the fertility of male mice that were treated with different mcAbs. We also determined the binding patterns of SPAG11E to rat epididymal sperm and identified the novel localization of SPAG11E in the sperm flagellum and nucleus by immunoelectron microscopy (EM) using mcAbs.


  Methods Top


Expression of recombinant antigens

As we have shown that double-copy β-defensin protein expressed in  Escherichia More Details coli was effective for raising β-defensin antibodies,[26] both SPAG11E[27] and double-copy SPAG11E (tandem repeat SPAG11E) were expressed without signal peptides. The plasmids pET28 (Novagen, Madison, Wisconsin, USA) and pXXGST[20] were used to express recombinant tandem peptide antigen and epitope peptides, respectively, as described previously.[20],[26] Two pairs of primers were designed for tandem Spag11e amplification: P1, 5′-CATATGGGAATCAGAAACACC-3′(Nde I site underlined); P2, 5′-AGATCTTCTGTTTTTAATGGA-3′ (Bgl IIsiteunderlined); P3, 5′-GGATCCGGAATCAGAAACACC-3′(Bam HI site underlined); and P4, 5′-GTCGACCTATCTGTTTTTAATGGA-3′ (Sal I site underlined). Recombinant tandem SPAG11E antigen and human SPAG11B/D protein were produced and purified as previously described.[26],[28]

Plasmid pTWIN1 (New England Biolabs, USA) was used to produce β-defensin peptides as described previously.[27] The β-defensins and their accession numbers were as follows: rBD14 (GenBank ID: AY621346), rBD15 (GenBank ID: AY621347), rBD42 (GenBank ID: AY621369), rBD1 (GenBank ID: AF068860), mBD15 (GenBank ID: 246082), and mBD30 (GenBank ID: DQ012040). Only mature peptides were cloned and expressed.

Preparation of polyclonal and monoclonal antibodies

BALB/c mice (female, 6-week-old) were purchased from SIPPR-BK Animal Co. Ltd., Shanghai, China. All animal experiments were done according to the laboratory animal care protocols approved by the institutional animal care committee. The protocols for mouse antiserum and mcAb production are described in the laboratory manual.[29] Briefly, the purified recombinant SPAG11E antigen peptide (0.5 mL and 0.8 mg/mL) was mixed with an equal volume of complete Freund's adjuvant (Sigma Aldrich, Inc., USA), until emulsion was formed. BALB/c mice (female, 6-week-old) were immunized by intraperitoneal injections (0.2 mL per mouse) of the emulsion with disposable syringes. The following four injections every 2 weeks to boost antibody production were the same as the primary injection, except that incomplete Freund's adjuvant (Sigma Aldrich, Inc., USA) instead of complete Freund's adjuvant was used. Three days before cell fusion, protein antigen with no adjuvant was directly inoculated to give a final boost. For titer determination, primary antibody was added to wells preincubated with 100 μL of SPAG11E antigen (5 μg/mL) at 4°C for 12 h following the indirect ELISA method.[29]

The fusion of splenocytes with myeloma cells (sp2/0) was mediated by polyethylene glycol 4000 (PEG4000). Cell cloning was performed with 3–4 times of successive limiting dilution of hybridomas in HAT medium. Antibody titer was determined with the indirect ELISA method described in the manual.[29] Positive clones were subcultured and injected to induce ascitic fluid.[29] The class and subclass of a mcAb were determined by indirect ELISA with theMouse Monoclonal Antibody Isotyping Reagents(Sigma-Aldrich, Inc., USA).

Epitope mapping of SPAGE11E

The oligo DNA fragments coding for epitope peptides (with BamHI and SalI restriction ends) were synthesized (Shanghai SBS Genetech Technology Co. Ltd.) and annealed to double strands. To analyze the epitope motifs recognized by antibodies, the epitope peptides (16- or 8-mer) were fused to the C terminus of a truncated GST protein (GST188) by inserting the coding DNA of epitope peptides into thermo-inducible plasmid pXXGST-1 at Bam HI and Sal I sites downstream of the GST188 gene.[20],[30] The oligo DNAs are listed in Supplementary Materials. For epitope fusion protein expression and epitope mapping, refer to references[20],[30] and Supplementary Materials.

Protein extraction of epididymal tissues and spermatozoa

Adult Sprague–Dawley rats purchased from SIPPR-BK Animal Co. Ltd., Shanghai, China, were sacrificed, and the epididymides were separated into three segments: caput together with the initial segment, corpus, and cauda. One aliquot of the tissues was homogenized on ice in RIPA (Pierce Biotechnology, Inc., USA) containing 10 μg/mL each of aprotinin and leupeptin. The lysates were centrifuged at 12,000 ×g at 4°C for 15 min, and the supernatants were stored at −80°C. From the other aliquots, spermatozoa were released by cutting with scissors and placed into prewarmed phosphate-buffered saline (PBS, pH 7.4) at 37°C. Spermatozoa were allowed to swim out by incubating the tissues in PBS at 37°C for 15 min, with 5% CO2. The spermatozoa were collected and washed with prewarmed PBS by centrifuging at 500 ×g for 5 min 3 times. For Western blotting analysis, spermatozoa were lyzed in 2% sodium dodecyl sulfate (SDS).

Immunohistochemical staining and Immunofluorescence staining of SPAG11E

Immunohistochemical staining of tissues was performed according to a previously described protocol.[26] All slides were incubated with 3% (v/v) H2O2 in PBS for 10 min before secondary antibody addition to avoid nonspecific peroxidase reaction.

For immunofluorescence staining, spermatozoa were resuspended in 4% paraformaldehyde (w/v) for 1 h. After being washed with PBS 3 times, spermatozoa were resuspended in PBS, placed on polylysine-coated slides, and air-dried at 37°C for 5 h. For xylene treatment, the slides were immersed in clean xylene (prewarmed at 58°C) for 30 min and incubated for 15 min at room temperature. Then, the slides were processed through a gradient of 100%, 90%, 75%, and 50% ethanol followed by PBS. The slides were washed with PBS 3 times (5 min/wash). To make the process comparable to that of immunohistochemical staining, we incubated the slides with 3% (v/v) H2O2 in PBS for 10 min, although H2O2 was not necessary for immunofluorescence staining of the slides. Finally, the specimens were washed with distilled water 3 times (5 min/wash). The slides were blocked for 1 h at 37°C with 10% goat serum in PBS. The slides were then incubated with primary antibody (diluted 1:200) at 4°C overnight and then washed with PBS 3 times. The corresponding secondary antibody was applied (TRITC-or FITC-conjugated goat anti-mouse, 1:200 diluted in PBS containing 10% goat serum) and incubated at 37°C for 1 h. The slides were washed 3 times with PBS and mounted in 80% glycerol. Images were captured with an Olympus BX-52 microscope.

Sequential extraction of sperm-associated protein

Western blotting was used to analyze the localization of SPAG11E on sperm. Sperm-associated protein was extracted with buffers of different stringency. Caput spermatozoa of rat were collected by mincing tissues on ice in Dulbecco's PBS, pH 7.4, containing the protease inhibitor 10 mmol/L iodoacetamide, 0.5% aprotinin, and 0.1% phenylmethylsulfonyl fluoride (PMSF, PBS-PI). The pelleted spermatozoa were washed in 1 mL of PBS and centrifuged at 500 ×g for 10 min. The pellets were gently resuspended in 300 μL of PBS-PI, and sperm concentrations were determined.

The epididymal spermatozoa were sequentially extracted with low salt (PBS-PI with 5 mmol/L EDTA, pH 7), high salt (PBS-PI with 5 mmol/L EDTA, 0.5 mol/L NaCl, pH 7), 0.1% Triton X-100 (PBS-PI with 0.1% Triton X-100, 5 mmol/L EDTA, pH 7), 2% SDS, and Laemmli buffer containing 5% SDS and 1% mercaptoethanol as described in the previous publication.[31]

Briefly, the spermatozoa resuspended in 100 μL of PBS-PI were centrifuged again to remove supernatant and then the sperm pellets were resuspended in 100 μL of low-salt buffer (PBS-PI with 5 mmol/L EDTA, pH 7) and extracted for 30 min at room temperature. The resulting supernatant solution, following a 500 ×g spin for 10 min, was designated as the low-salt extract. The sperm pellet then underwent a high-salt extraction (PBS-PI with 5 mmol/L EDTA, 0.5 mol/L NaCl, pH 7) followed by a Triton extraction (PBS-PI with 0.1% Triton X-100, 5 mmol/L EDTA, pH 7) under the same conditions. The sperm pellet was then extracted in 100 μL of 2% SDS for 5 min at 95°C followed by centrifugation at 10,000 ×g for 5 min, and the final sperm pellet was resuspended in 100 μL Laemmli buffer.

The supernatant of each extract was immediately centrifuged at 10,000 ×g for 5 min to remove residual sperm/cellular debris and then desalted into distilled water using Centricon-10 columns (Amicon Co., Danvers, MA, USA). The final concentrates were brought to 100 μL and subjected to SDS-PAGE followed by Western blotting analysis. The absolute number of remaining spermatozoa after each extraction was used to normalize the loading volume of each extract for Western blotting. Treatments of rat sperm with LL-pronase followed a previous publication[32] with minor modification on the treatment time.

Immunogold electron microscopy

The caput, corpus, and cauda epididymides were collected from 4-month-old adult Sprague-Dawley rats. The tissues were gently punctured and squeezed in 0.5 mL of warmed PBS in an incubator to allow the sperm to disperse throughout the medium for 10 min. The “swim-out” sperm were then centrifuged at 400 ×g for 5 min at 4°C and washed 3 times with cold PBS. Sperm pellets were embedded in LR White resin (London Resin Co. Ltd., UK) and cut into ultrathin sections. The sections were mounted on grids, incubated in PBS twice for 5 min, and then immersed in PBS/normal goat serum for 1 h. After incubation with primary antibody overnight at 4°C in a moist chamber, the sections were washed 6 times with PBS/bovine serum albumin (BSA) and then incubated with gold-tagged secondary antibody for 2 h at ambient temperature. The sections were washed 4 times with PBS/BSA and 4 times with PBS and then fixed with 1% glutaraldehyde in PBS for 15 min. After being washed with water 4 times, the sections were stained with 3% (w/v) uranyl acetate for 5 min and briefly washed again. The dried grids were visualized with a transmission electron microscope (Philips, CM120).

Animal assays for the antifertility effects of monoclonal antibodies

Hybridoma cell strains and sp2/0 cells were subcultured and diluted to 5 × 106/mL. Only BALB/c mice (male, 12–18-week-old) that proved to be fertile were utilized. Male mice were inoculated with hypodermic injections of hybridoma cell strains or sp2/0 cells at four different points (0.5 × 106/100 μL per point) on the back. Blood samples of mice were collected at the end of tails 2 days postinjection for ELISA analysis of antibody concentration. Tumor diameter of 3 mm measured at 7 days postinjection was designated as a threshold to evaluate inoculation success. Anti-SPAG11E antibodies in the sera and epididymis of mice with successful inoculation were analyzed by ELISA.

Male mice with successful inoculation were mated with adult females for 8 days. Females that formed vaginal plug after copulation were fed separately and their liter sizes were recorded. Successfully mated females were replaced by unpregnant females to make sure free copulation of one male versus three adult females per cage per day during the 8 days. Every male in this experiment was allowed to successfully mate with four females in total.


  Results Top


Preparation of polyclonal antiserum and monoclonal antibodies

mcAb raising and epitope mapping were key tools for the evaluation of SPAG11E-a potential epididymal target. Although a synthetic peptide segment of SPAG11E (MCRSGERKGDICSDP-conjugated with KLH) has been successfully utilized to raise SPAG11E antiserum, the exact epitope motifs remain unclear for SPAG11E. We immunized mice with recombinant SPAG11E as described in the methods to raise antibody. The mouse with the highest serum titer (1:160,000) was selected to construct hybridomas. Three clones secreting IgG1 (clones 3#-1, 10#A5, and 10#B4) and one secreting IgG2a (clone 14#F6) were obtained. To evaluate the specificity of the mcAbs, recombinant β-defensins (SPAG11E; RBD1, RBD14, RBD15, RBD42, MBD15, and MBD30) were expressed as previously described[27] and analyzed by Western blotting. All the mcAbs selectively recognized both the recombinant and the natural SPAG11E of the rat epididymal protein extract [10#B4 as an example, [Figure 1]]. As evidenced by the results of epitope motif mapping, the mcAbs (clones 3#-1, 14#F6, and 10#A5) cross reacted with recombinant human SPAG11B/D antigen [Figure 1]d, which indicates conserved B-cell epitope motifs among mammalian SPAG11E/BIN1B.
Figure 1: The specificity of mcAb 10#B4 was determined by Western blotting (a, c, and d) and dot blot (b) assays. The loaded recombinant active defensin peptides (SPAG11E, RBD14′, and RBD15′) in (a) was 2 ng per lane and the tandem repeat antigens (RBD1, RBD14, RBD15, RBD42, MBD15, and MBD30) loaded in (b) was 1 μg per dot. The loading amount of protein extract from different tissues (caput region, segments 1–8; corpus region, segments 9–14; and cauda region, segments 15–19) was 30 μg per lane (c). (d) The recognition abilities of monoclonal antibodies 10#A5, 3#-1, 14#F6, and 10#B4 (1:1000) for recombinant full-length human SPAG11B/D and tandem-repeat antigen SPAG11E. *Tandem-repeat antigen of SPAG11E (5 ng per lane) as a positive control. Pre: E. coli cell lysate before induction as a negative control to show the background of Western blotting for the designated antibodies; Post: E. coli cell lysate after induction with IPTG to show the specific recognition of the designated antibodies to recombinant SPAG11B/D; E. coli: Escherichia coli.

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B-cell epitope mapping assays

Epitope motif analysis is valuable for the evaluation of both antigen and antibody. First, the 16-mer epitope fusion proteins [S1–S6; [Table 1]] were expressed in E. coli BL21 and analyzed with 15% SDS-PAGE, as shown in [Figure 2]a. SPAG11E mouse antiserum could recognize the epitopes located in four regions [S2, S3, S4, and S5; [Figure 2]b]; mcAbs 14#F6, 3#-1, and 10#A5 recognized S4 and S5, whereas 10#B4 recognized S3 [Figure 2]c. Second, an 8-mer peptide mapping was conducted to locate the exact epitope sequences within the three core regions [Table 2] and [Figure 2]d. The epitope sequence “MQRGHCRL” recognized by antiserum was specific in rat SPAG11E. It is interesting that epitope “SDPWNRCC” (recognized by antibodies 10#A5, 14#F6, and 3#-1) was a consensus sequence existing in mammals, whereas epitope “RSGERKGD” (recognized by antibody 10#B4) was only in rat and mouse SPAG11E/BIN1B [Supplementary Figure 1]. The epitope motifs conformed to the recognition patterns of corresponding mcAbs observed in Western blotting [Figure 1]d.
Table 1: Primary epitope mapping of rat mature SPAG11E with the 16-mer overlapping peptides fused to truncated GST tag

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Figure 2: Epitope mapping of the SPAG11E antigen protein by Western blotting. Recombinant 16-mer epitope fusion proteins were analyzed by SDS-PAGE (a) and Western blotting (b and c) assays. The cell lysate of clones S1 to S6, which produce the corresponding 16-mer epitope fusion proteins as shown in Table 1, was analyzed with antiserum (b) and monoclonal antibodies (c). All the epitope motifs in human (HumE), mouse (MouE), and rat (RatE) are presented in d, with consensus motifs highlighted by a box, and the detailed data of the epitope mapping are shown in Table 2. *Epitope motifs recognized by antiserum. C: Control; Pre: Cell lysate before induction; Po: Cell lysate after induction.

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Table 2: Immunorecognition of fusion proteins carrying variant epitope motifs by antibodies in Western blotting

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Localization of SPAG11E by immunohistochemical staining, immunofluorescence staining, and immunogold electron microscopy

Using the epitope-specific mcAbs, we determined the expression patterns of SPAG11E in rat epididymis and sperm from different epididymal regions. SPAG11E expression patterns in the rat epididymal sections revealed by the mcAbs were similar (data not shown). The expression level of SPAG11E was high in the caput region and gradually decreased from caput segment 8 to corpus segment 12 [Figure 3]a and [Figure 3]b. The same epitope motif was also recognized in human epididymal SPAG11 B/D [Figure 3]c.
Figure 3: Immunohistochemical staining of SPAG11E in rat epididymis (a and b) and SPAG11B/D in human epididymis (c) with antibody 3#-1 (1:500). The brown color indicates immunostaining of SPAG11E and SPAG11B/D in the segments of epididymis with blue counterstain (hematoxylin). Scale bars, 4 mm in A and 100 μm in b and c. The thickness of all the sections was 10 μm. CN: Preimmune antiserum as control; ini: Initial segments of human epididymis; cap: Caput region of human epididymis; cor: Corpus region of human epididymis; cau: Cauda region of human epididymis.

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It is interesting that none of the mcAbs recognized SPAG11E on separated epididymal sperm with immunofluorescence staining. We hypothesized that the epitopes might be masked. We applied common approaches for epitope retrieval such as boiling water bath and microwave oven incubation, but unsuccessful. Considering that these antibodies recognized antigen in IHC staining, we introduced a xylene treatment step (by mimicking the treatment of tissue slides) to treat the sperm. With polyclonal antibodies, SPAG11E was stained on caput and corpus epididymal sperm heads. Using mcAbs combined with xylene treatment as described in Methods, we observed SPAG11E on caput epididymal sperm heads but not on corpus and caudal sperm [Figure 4], which is concordant with the observations in Western blotting and IHC assays. The rabbit polyclonal antibody had been described in a previous publication.[4] The binding of SPAG11E with sperm was resistant to sequential washing of different stringency with PBS, PBS with low salt, PBS with high salt, PBS with 0.1% Triton X-100, or LL-pronase treatment [Figure 5]. Because the LL-pronase had been claimed to be able to completely remove nonnuclear materials,[32] Western blotting analysis was conducted to examine the resistance of SPAG11E to LL-pronase treatment, which might give more hints respecting the localization of SPAG11E. The observed resistance of SPAG11E to LL-pronase treatment suggested that SPAG11E might colocalize with the sperm nucleus in the pellet. Thus, the intracellular localization of SPAG11E was further investigated by EM.
Figure 4: Localization of SPAG11E with rabbit and mouse polyclonal antibodies and mcAb 10#B4 in fluorescent immunochemical staining of the caput, corpus, and cauda sperm of rat. Nuclear staining was not done to avoid the appearance of pseudosignals. The profiles of sperm are shown in phase-contrast fields instead. Rabbit and mouse antibodies were labeled with FITC (green)-and TRITC (red)-conjugated goat IgG. The fields were observed under a ×100 objective lens. Bars, 5 μm.

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Figure 5: Western blotting analysis of SPAG11E colocalized with sperm by treatments with buffers of different stringency and LL-pronase. The detail of sequential protein extract of rat sperm at caput region with buffers designated from the left to right side (left panel) was described in methods, and the protein extract was analyzed by Western blotting with mcAb 10#B4. Rat sperm of caput region was treated with LL-pronase (right panel) for 15, 30, and 45 min to remove nonnucleus materials and equal amount of pellet protein was analyzed by Western blotting analysis using mcAb 10#B4. PI: Protease inhibitor; LL-pronase: l-α-phosphatidylcholine-pronase.

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At the ultrastructure level, gold particles were localized in the nucleus and dense fibers of sperm from the caput region [Figure 6] and were associated with the endoplasmic reticulum (ER) of epithelial cells in the caput epididymis (data not shown). However, no obvious nuclear localization of SPAG11E was observed in the nucleus of epididymal epithelial cells. The flagellum localization of SPAG11E could be observed in the longitudinal and transverse sections [Figure 6]c,[Figure 6]d,[Figure 6]e,[Figure 6]f. To confirm the novel localization of SPAG11E that we observed in rat sperm, we also observed SPAG11E/BIN1B in mouse sperm since mouse SPAG11E/BIN1B and rat SPAG11E/BIN1B has conserved amino acids. It was rational to observe the exact localization of SPAG11E/BIN1B in the sperm of transgenic mice that overexpress HA-tagged mouse SPAG11E/BIN1B[33] using anti-HA antibody to avoid artifacts as possible as we can, because SPAG11E/BIN1B of wild mice showed similar expression pattern like that observed in the rat,[33] and the HA-tagged mouse SPAG11E/BIN1B was also expressed in the caput and corpus of the epididymis,[33] which is consistent with the endogenic SPAG11E/BIN1B expression pattern in wild-type mice. In addition, HA tag epitope “YPYDVPDYA” was absent in wild-type mice, which makes anti-HA antibody a highly specific probe to localize HA-tagged SPAG11E/BIN1B and to tell it from the endogenic SPAG11E/BIN1B.
Figure 6: Immunogold staining of rat epididymal sperm with monoclonal antibody 3#-1. Control sections (a, c, and e) showing the sperm nucleus and flagella stained with secondary antibody only. Specific staining of the epitope was observed in the sperm nucleus (b) and outer dense fibers in the transverse (d) and longitudinal (f) sections. Scale bars, 500 nm. N: Nucleus.

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As anticipated, HA-tagged mouse SPAG11E/BIN1B could be stained in the sperm flagellum of transgenic mice with anti-HA antibody [Figure 7] by immune EM. This observation supports the notion that rodent SPAG11E might have similar localization in sperm because 86.9% amino acid identity was observed between rat and mouse SPAG11E/BIN1B. Considering that human SPAG11B/D has a long N-terminal segment that interacts with its partners,[17] which is absent in mouse and rat SPAG11E, whether human SPAG11B/D has a similar localization remains to be addressed. It should be noted that not all caput sperm in the rat and transgenic mouse epididymal sections could be labeled with gold particles in the immunogold EM assays. The heterogeneity of sperm might be a reason; however, the nature of the “selective” staining remains to be investigated.
Figure 7: Immunogold staining of epididymal sperm of transgenic mice with anti-HA monoclonal antibody. Transgenic mice overexpressing HA-tagged SPAG11E were provided by Professor Jian Fei. Gold particles were observed in the transverse sections of the sperm flagellum (a) and the longitudinal sections of the sperm nucleus (b). Scale bars, 1,000 nm.

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Antifertility effects of SPAG11E monoclonal antibodies

In vivo inhibition of rat SPAG11E expression by antisense RNA has been shown to reduce the binding of SPAG11E to caput sperm and attenuate sperm motility and progressive movement in the rat.[4] Whether the antibodies corresponding to specific antigen epitope motifs had potential effects in the modulation of fertility was explored in mice now that there were conserved epitope motifs between mouse and rat SPAG11E. To make the experiments simple, mice with validated fertility were inoculated by injections of hybridomas or sp0/2 cells on the back. Success of inoculation was proved by the measured tumor diameters (≥3 mm) and antibody titers. Based on the epitope information, we observed that serum titters of mice inoculated successfully with hybridomas 3#-1 and 10#B4 could reach high level 7 days postinjection [Supplementary Figure 2]. Not only in serum, but also in epididymal tissues, antibodies could reach high levels at day 9 postinoculation [Supplementary Figure 3]. Mice with successful inoculation 7 days postinjection were mated with adult female mice as described in the methods. No mating behavior was affected. Based on the litter sizes, both mcAbs 3#-1and 10#B4 reduced male fertility by showing apparently lowered offspring number [Figure 8] and [Supplementary Table 1], Kruskal–Wallis test, P < 0.001].

Figure 8: Antifertility effects of SPAG11E monoclonal antibody 3#-1 and 10#B4. The fertile female mice copulated with male BALB/c mice that carried monoclonal antibodies 3#-1 or 10#B4 showed lowered litter size compared to females copulated with male mice that carried sp0/2 cell (Kruskal–Wallis test, P < 0.001) (males: n = 3/group; females: n = 4/male).

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  Discussion Top


In rat, SPAG11E was observed on all the sperm throughout the whole epididymis as stained with polyclonal antibody,[4] whereas the expression site of SPAG11E mRNA determined by Northern blotting was at caput region of epididymis. We evidenced here that the exact expression site of SPAG11E in rat was consistent with the mRNA expression[13] using qualified mcAbs in Western blotting, dot blots, IHC, and EM. The SPAG11E signals detected by IHC in the distal caput and corpus regions of epididymal tissues might arise from the diffusion of SPAG11E along the epididymal lumen.

It was surprising that no detectable signal of SPAG11E could be observed with our mcAbs by common conditions in the immunofluorescence assays, whereas in IHC assays, the antibodies worked well. For unknown reasons, xylene treatment helped expose epitopes in the immune-fluorescence assays. We could not exclude the possibility that SPAG11E might interact through its receptors or nonprotein molecules, which might block the accession of antibodies to their specific epitopes. As a member of cationic β-defensin family, SPAG11E may interact with the lipid matrix of cell membranes like the other defensin family members such as β-defensin 3, which can interact with the lipid matrix of a cell membrane.[34] Binding of SPAG11E to the xylene-soluble molecules on the sperm membrane might block the recognition of the epitope by mcAbs, which implied a direct interaction of SPAG11E with the sperm membrane. However, in the lumen fluids, unbound SPAG11E should be accessible for antibody, and this may explain why antibodies had antifertility activity. However, unbound SPAG11E could be washed out in the process of sperm immunofluorescence assay, which might explain the absence of detectable SPAG11E signal on sperm with common treatments. These observations also suggest that free SPAG11E in the epididymal fluids rather than sperm-bound SPAG11E might be the effective targets of antibodies.

Although SPAG11E in the rat sperm head could be recognized with mcAbs, no signal was observed in the flagellum in immunofluorescence staining assays, which is inconsistent with the SPAG11E signals in the flagellum disclosed by EM. The reason might be that the tightly condensed fibers prevent SPAG11E recognition under normal conditions, whereas in the ultrathin sections, SPAG11E in the flagellum were exposed and accessible to mcAbs.

To our knowledge, the localization of SPAG11E in the sperm nucleus and flagellum has not been reported. It is interesting that cationic AMPs LL-37 and human β-defensin-1 (HBD1) have been reported to be localized in cell nucleus.[35],[36] HBD1, another AMP without consensus nuclear localization signal, was also observed in the nuclei of human keratinocytes and malignant salivary gland tumors.[37],[38] The possible transmission mechanisms for AMPs include a cationic sequence-based mechanism and transport through the nuclear pore complex.[38] The nucleus-localized LL-37, HBD1, and other AMPs were hypothesized to be involved in the modulation of gene expression and/or protein synthesis,[38] but the postulated mechanism seems not convincing for sperm because sperm have rare gene transcription and protein synthesis. Considering that SPAG11E was involved in sperm motility in vitro[4] and in vivo,[16],[18] the localization of SPAG11E in the sperm flagellum and nucleus possibly contributes to sperm motility, but the detailed mechanisms remain to be clarified.

It is reported that almost half of all pregnancies are still unwanted or unplanned.[39] Clearly, there is a need for expanded and reversible contraceptive options. There are more than a dozen existing methods of contraception, but all except two were designed for women.[40] Because SPAG11E was considered as a novel epididymal target for male contraception,[14] the analysis of predominant epitope motifs might help accelerate the validation of its male contraception potential.

With our specific mcAbs, the fertility of male mice could be reduced to 50%. Although different animal models were utilized, mcAbs targeting to specific epitope of SPAG11E showed higher efficacy compared with the previous report immunized with synthetic antigen peptides in the rat.[18] The synthetic SPAG11E peptide fragment (MCRSGERKGDICSDP conjugated with KLH)[18] happens to contain one of the epitope motifs, “RSGERKGD.” Lack of the other two epitope motifs in their antigen peptide might explain the limited decrease in motility and fertility after immunization with their synthetic antigen. Our complete epitope mapping makes it possible to combine all the effective epitope motifs in a human-made antigen with a hope to improve antifertility effects of SPAG11E vaccine since all the motifs showed antifertility effects.

The epitope “SDPWNRCC” is a consensus peptide sequence of mammalianSPAG11E [Supplementary Figure 1] including human beings. The recognition of human SPAG11E by the mcAbs in both Western blotting and IHC staining indicates a potential application of these antibodies in human semen and sperm analysis. Because the alteration in SPAG11 function might contribute to the reduced sperm motility seen in asthenozoospermic patients, our present findings provide powerful tools for further investigation of sperm motility-related male factor infertility and subfertility.

Financial support and sponsorship

This work was supported by grants from the Natural Science Foundation of China (No. 81671508) and the Innovation-oriented Science and Technology Grant from NPFPC Key Laboratory of Reproduction Regulation (No. CX2017-01).

Conflicts of interest

There are no conflicts of interest.


  Supplementary Materials Top



  List of Oligo DNAs Synthesized for Epitope Peptide Expression and Epitope Mapping Top


Part A – The oligo DNA synthesized for the first-round mapping of 16-mer epitope peptides. The corresponding peptide sequences are displayed as follows.

S: Segment; s: Sense strand; r: Reverse complementary strand.

S1s 5′-gatcc gac att cca cct gga atc cgc aac acc gtg tgc ttc atg cag cgg ggc taag-3′

S1r 5′-tcgactta gcc ccg ctg cat gaa gca cac ggt gtt gcg gat tcc agg tgg aat gtc g-3′

S2s 5′-gatcc aac acc gtg tgc ttc atg cag cgg ggc cac tgt cgc ctc ttc atg tgc cgt taag-3′

S2r 5′-tcgactta acg gca cat gaa gag gcg aca gtg gcc ccg ctg cat gaa gca cac ggt gtt g-3′

S3s 5′-gatcc cac tgt cgc ctc ttc atg tgc cgt tct ggg gag cgc aag ggg gat att taag-3′

S3r 5′-tcgactta aat atc ccc ctt gcg ctc ccc aga acg gca cat gaa gag gcg aca gtg g-3′

S4s 5′-gatcc tct ggg gag cgc aag ggg gat att tgc tct gac ccg tgg aac cgc tgc taag-3′

S4r 5′-tcgactta gca gcg gtt cca cgg gtc aga gca aat atc ccc ctt gcg ctc ccc aga g-3′

S5s 5′-gatcc tgc tct gac ccg tgg aac cgc tgc tgc gta tcc agt tcc att aaa aac taag-3′

S5r 5′-tcgactta gtt ttt aat gga act gga tac gca gca gcg gtt cca cgg gtc aga gca g-3′

S6s 5′-gatcc gta tcc agt tcc att aaa aac cgc taag-3′

S6r 5′-tcgactta gcg gtt ttt aat gga act gga tac g-3′

Part B – The oligo DNA synthesized for the second round of (8-mer) epitope mapping.

S: Segment; s: Sense strand; r: Reverse complementary strand.

S2-1s 5′-gatcc gtg tgc ttc atg cag cgg ggc aac acc gtg taag-3′

S2-1r 5′-tcgactta cac ggt gtt gcc ccg ctg cat gaa gca cac g-3′

S2-2s 5′-gatcc atg cag cgg ggc aac acc gtg tgc taag-3′

S2-2r 5′-tcgactta gca cac ggt gtt gcc ccg ctg cat g-3′

S2-3s 5′-gatcc cag cgg ggc aac acc gtg tgc ttc taag-3′

S2-3r 5′-tcgactta gaa gca cac ggt gtt gcc ccg ctg g-3′

S2-4s 5′-gatcc cgg ggc aac acc gtg tgc ttc atg taag-3′

S2-4r 5′-tcgactta cat gaa gca cac ggt gtt gcc ccg g-3′

S2-5s 5′-gatcc ggc aac acc gtg tgc ttc atg cag taag-3′

S2-5r 5′-tcgactta ctg cat gaa gca cac ggt gtt gcc g-3′

S2-6s 5′-gatcc aac acc gtg tgc ttc atg cag cgg taag-3′

S2-6r 5′-tcgactta ccg ctg cat gaa gca cac ggt gtt g-3′

S2-7s 5′-gatcc acc gtg tgc ttc atg cag cgg ggc taag-3′

S2-7r 5′-tcgactta gcc ccg ctg cat gaa gca cac ggt g-3′

S2-8s 5′-gatcc gtg tgc ttc atg cag cgg ggc cac taag-3′

S2-8r 5′-tcgactta gtg gcc ccg ctg cat gaa gca cac g-3′

S2-9s 5′-gatcc tgc ttc atg cag cgg ggc cac tgt taag-3′

S2-9r 5′-tcgactta aca gtg gcc ccg ctg cat gaa gca g-3′

S2-10s 5′-gatcc ttc atg cag cgg ggc cac tgt cgc taag-3′

S2-10r 5′-tcgactta gcg aca gtg gcc ccg ctg cat gaa g-3′

S2-11s 5′-gatcc atg cag cgg ggc cac tgt cgc ctc taag-3′

S2-11r 5′-tcgactta gag gcg aca gtg gcc ccg ctg cat g-3′

S2-12s 5′-gatcc cag cgg ggc cac tgt cgc ctc ttc taag-3′

S2-12r 5′-tcgactta gaa gag gcg aca gtg gcc ccg ctg g-3′

S2-13s 5′-gatcc cgg ggc cac tgt cgc ctc ttc atg taag-3′

S2-13r 5′-tcgactta cat gaa gag gcg aca gtg gcc ccg g-3′

S2-14s 5′-gatcc ggc cac tgt cgc ctc ttc atg tgc taag-3′

S2-14r 5′-tcgactta gca cat gaa gag gcg aca gtg gcc g-3′

S2-15s 5′-gatcc cac tgt cgc ctc ttc atg tgc cgt taag-3′

S2-15r 5′-tcgactta acg gca cat gaa gag gcg aca gtg g-3′

S3-1s 5′-gatcc tgt cgc ctc ttc atg tgc cgt tct taag-3′

S3-1r 5′-tcgactta aga acg gca cat gaa gag gcg aca g-3′

S3-2s 5′-gatcc cgc ctc ttc atg tgc cgt tct ggg taag-3′

S3-2r 5′-tcgactta ccc aga acg gca cat gaa gag gcg g-3′

S3-3s 5′-gatcc ctc ttc atg tgc cgt tct ggg gag taag-3′

S3-3r 5′-tcgactta ctc ccc aga acg gca cat gaa gag g-3′

S3-4s 5′-gatcc ttc atg tgc cgt tct ggg gag cgc taag-3′

S3-4r 5′-tcgactta gcg ctc ccc aga acg gca cat gaa g-3′

S3-5s 5′-gatcc atg tgc cgt tct ggg gag cgc aag taag-3′

S3-5r 5′-tcgactta ctt gcg ctc ccc aga acg gca cat g-3′

S3-6s 5′-gatcc tgc cgt tct ggg gag cgc aag ggg taag-3′

S3-6r 5′-tcgactta ccc ctt gcg ctc ccc aga acg gca g-3′

S3-7s 5′-gatcc cgt tct ggg gag cgc aag ggg gat taag-3′

S3-7r 5′-tcgactta atc ccc ctt gcg ctc ccc aga acg g-3′

S4-1s 5′-gatcc ggg gag cgc aag ggg gat att tgc taag-3′

S4-1r 5′-tcgactta gca aat atc ccc ctt gcg ctc ccc g-3′

S4-2s 5′-gatcc gag cgc aag ggg gat att tgc tct taag-3′

S4-2r 5′-tcgactta aga gca aat atc ccc ctt gcg ctc g-3′

S4-3s 5′-gatcc cgc aag ggg gat att tgc tct gac taag-3′

S4-3r 5′-tcgactta gtc aga gca aat atc ccc ctt gcg g-3′

S4-4s 5′-gatcc aag ggg gat att tgc tct gac ccg taag-3′

S4-4r 5′-tcgactta cgg gtc aga gca aat atc ccc ctt g-3′

S4-5s 5′-gatcc ggg gat att tgc tct gac ccg tgg taag-3′

S4-5r 5′-tcgactta cca cgg gtc aga gca aat atc ccc g-3′

S4-6s 5′-gatcc gat att tgc tct gac ccg tgg aac taag-3′

S4-6r 5′-tcgactta gtt cca cgg gtc aga gca aat atc g-3′

S4-7s 5′-gatcc att tgc tct gac ccg tgg aac cgc taag-3′

S4-7r 5′-tcgactta gcg gtt cca cgg gtc aga gca aat g-3′

S4-8s 5′-gatcc tgc tct gac ccg tgg aac cgc tgc taag-3′

S4-8r 5′-tcgactta gca gcg gtt cca cgg gtc aga gca g-3′

S5-1s 5′-gatcc tct gac ccg tgg aac cgc tgc tgc taag-3′

S5-1r 5′-tcgactta gca gca gcg gtt cca cgg gtc aga g-3′

S5-2s 5′-gatcc gac ccg tgg aac cgc tgc tgc gta taag-3′

S5-2r 5′-tcgactta tac gca gca gcg gtt cca cgg gtc g-3′

S5-3s 5′-gatcc ccg tgg aac cgc tgc tgc gta tcc taag-3′

S5-3r 5′-tcgactta gga tac gca gca gcg gtt cca cgg g-3′

S5-4s 5′-gatcc tgg aac cgc tgc tgc gta tcc agt taag-3′

S5-4r 5′-tcgactta act gga tac gca gca gcg gtt cca g-3′

S5-5s 5′-gatcc aac cgc tgc tgc gta tcc agt tcc taag-3′

S5-5r 5′-tcgactta gga act gga tac gca gca gcg gtt g-3′

S5-6s 5′-gatcc cgc tgc tgc gta tcc agt tcc att taag-3′

S5-6r 5′-tcgactta aat gga act gga tac gca gca gcg g-3′

S5-7s 5′-gatcc tgc tgc gta tcc agt tcc att aaa taag-3′

S5-7r 5′-tcgactta ttt aat gga act gga tac gca gca g-3′

S5-8s 5′-gatcc tgc gta tcc agt tcc att aaa aac taag-3′

S5-8r 5′-tcgactta gtt ttt aat gga act gga tac gca g-3′


  Methods of Epitope Mapping Based on Recombinant Fusion Expression of Epitope Peptides and Western Blotting Analysis Top


The DNA fragments encoding each 16- or 8-mer peptide had a “gattc” sequence (BamHI cohesive end) on the 5′ end (the sense strand) and a “taag” sequence (termination codon TAA-SalI cohesive end) on the 3′ end. The recombinant plasmids pXXGST-S1 to pXXGST-S6 were transformed into the E. coli strain BL21 (DE3; Novagen, Inc.). Each clone was first cultured in 3 mL of Luria broth (LB) containing 100-μg/mL ampicillin at 30°C, until the cell density reached an OD600 of 0.6–0.8, and then incubated at 42°C for 5 h to induce expression. Finally, cell pellets containing 16- or 8-mer peptide fusion proteins were collected and stored at −20°C. Cell culture after induction was added to 6× loading buffer and boiled for 5 min before analysis on 15% SDS-PAGE. Samples separated by SDS-PAGE were either stained with Coomassie blue R-250 or transferred onto polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia Biotech) in a continuous buffer system at 20 V for 30 min, using a semidry blotter (Bio-Rad). The blotted PVDF membrane was blocked with 1× NET (50-mM Tris-HCl [pH 7.5], 150-mM NaCl, 0.1% NP-40, 1-mM EDTA [pH 8.0], and 0.25% gelatin), and immunodetection was carried out with the ECL Plus western blotting detection system (GE Healthcare, UK), with 1:5000 dilution of primary antibody and 1:1000 dilution of secondary antibody (HRP-conjugated goat anti-IgG). The stained bands were visualized on x-ray films.

Epitope sequences were determined by alignment of the overlapping sequences recognized by antisera and monoclonal antibodies. All the recombinant plasmids with epitope inserts were sequenced (Invitrogen Co., Shanghai). On the basis of the 16-mer epitope motif analysis, the 8-mer peptides overlapping each other by 7 residues were also expressed as fusion proteins with GST188 protein and used for epitope mapping in the same manner.



 
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