| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
a Center for Recombinant Gamete Contraceptive Vaccinogens, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
b Department of Obstetrics and Gynecology, Hyogo Medical College, Hyogo, Japan
c Department of Obstetrics and Gynecology and Reproductive Medicine, Health Science Center, Stony Brook, New York 11794-8091
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A complete understanding of the mechanism behind immunologic infertility, as well as improved diagnosis and treatment, depends on knowledge of the identities of specific sperm antigens capable of eliciting the production of functionally relevant sperm antibodies. For example, were a cocktail of recombinant sperm surface antigens available, it might serve as the target for an immunoassay of patient serum to detect the presence of specific antibodies mediating infertility, thus improving diagnosis of immunological infertility. Further, ASA and their cognate antigens may provide the basis for immunologic control of fertility in the form of a birth control vaccine.
Several groups have reported on the identification of sperm antigens recognized by systemic and/or local auto- and iso-antibodies from infertile individuals using immunoblotting techniques [17, 18–32]. In most cases, however, the reactions of sperm antigens with control sera from fertile individuals were not described, so the functional relevance of these antigens in fertility remains unclear. Furthermore, most of the earlier studies used unidimensional gel electrophoresis for the separation of sperm proteins [22, 25, 31–35]. In the present study, we adopted high-resolution two-dimensional (2D) electrophoresis with separation of sperm antigens in the first dimension by either isoelectric focusing (IEF) or nonequilibrium pH gradient electrophoresis (NEPHGE) to screen a range of acidic and basic proteins, followed by PAGE and a sensitive Western blotting method. In addition, the serum samples were initially screened for the presence of ASA by the immunobead binding test (IBT) [36], and only those samples showing significant reactivity for the presence of ASA were utilized to identify the immunodominant antigens. Moreover, sperm antigens unique to infertile patients were identified by excluding those antigens recognized by serum samples from clinically fertile subjects using computerized comparison of 2D immunoblots. A database of 2D gel images of silver-stained proteins (sperm proteome) and a database of vectorially labeled sperm surface proteins (sperm surface index) [37] allowed the definition of a subset of sperm surface antigens relevant to antibody-mediated infertility.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
All reagents used for electrophoresis were of the highest purity obtainable.
Human Serum
Serum samples were obtained from infertile men and women with unexplained infertility. The infertile subjects did not have any previously diagnosed hormonal, infective, or physical causes for their infertility. The male subjects had not undergone vasectomy.
Immunobead Binding
ASA in the serum samples from infertile females were detected by indirect immunobead binding as previously described [8]. The ASA that bound to the spermatozoa of male patients were detected by direct immunobead binding [8]. All the serum and sperm samples were tested for the presence of IgG-, IgM-, and IgA-specific ASA and included samples from 18 infertile male subjects, 9 infertile female subjects, 5 fertile male subjects, and 5 fertile female subjects.
Criteria for Choosing Sera for Western Blot Analysis
A serum sample was chosen for further study based on a high IBT score; i.e., more than 60% of the spermatozoa were observed to bind beads indicative of IgG- and/or IgM-specific ASA. All the selected samples contained antibodies directed against the sperm head or the entire spermatozoa. A total of 15 serum samples were chosen from infertile men, and 6 serum samples were chosen from infertile women for Western blot analysis.
Preparation of Sperm Antigens
Semen specimens were obtained from fertile donors with normal sperm quality. Only ejaculates with normal semen characteristics [38] were used in this study. After liquefaction of the semen, the mature sperm were separated from the seminal plasma, immature germ cells, and non-sperm cells (mainly white blood cells and epithelial cells) by Percoll (Pharmacia Biotech, Uppsala, Sweden) density centrifugation as described by Naaby-Hansen et al. [37]. Prior to the last centrifugation, the cells were counted, and all samples showed > 90% motility. The spermatozoa obtained from 8–12 individuals were pooled and frozen immediately until further use. All samples were obtained under informed consent using forms approved by the University of Virginia Human Investigation Committee.
Radioiodination
Radioiodination was performed according to the procedure adopted and standardized by Naaby-Hansen et al. [37]. Percoll-purified spermatozoa were suspended in Ham's F-10 medium to a final concentration of 20 x 106/ml. Washed Iodo-beads (Pierce Chemical Co., Rockford, IL) (one bead per 8 x 106 spermatozoa) and carrier-free 125I-Na (10 µCi/106 spermatozoa) were added to the sample. Radiolabeling was performed by incubating the sample for 10 min at 20°C on a rocking table. The cells were removed from the Iodo-beads by pipetting and immediately were subjected to a second Percoll density gradient centrifugation before being washed three times in Ham's F-10 medium. "Surface" proteins studied here are predominantly plasma membrane proteins accessible to external labeling on ejaculated sperm, which, with the preparation methods employed, consist almost entirely of cells that have not undergone the acrosome reaction. The cells were counted before the final wash, and the resulting pellet was used for extraction of sperm proteins. Autoradiography was performed using a sandwich of components in the following order: intensifying screen, blot, two layers of film, and intensifying screen [39]; x-ray films were routinely exposed for 3 wk.
Solubilization Procedures
Spermatozoa were solubilized in a lysis buffer containing 2% octyl-ß-glucopyranoside (OBG) containing 100 mM dithiothreitol (DTT), urea (9.8 M), and the protease inhibitors: 2 mM PMSF, 5 mM iodoacetamide, 5 mM EDTA, 3 mg/ml L-1-chlor-3-(4-tosylamido)-7-amino-2-heptanon-hydrochloride, 1.46 mM pepstatin A, and 2.1 mM leupeptin. Cells (5 x 108/ml) were solubilized by constant shaking at 4°C for 45 min. Insoluble material was removed by centrifugation at 10 000 x g for 2 min, and the supernatant was applied to the first electrophoretic dimension.
Electrophoresis
IEF was performed using the gel composition as described earlier [37]. Carrier ampholine compositions were 20% pH 5–7, 20% pH 7–9, and 60% pH 3.5–10. Sixty-five microliters of sperm extract (~0.15 mg of protein) was applied per rod. The tubes were filled by gently overlaying the sample with a buffer containing 5% Nonidet P-40, 1% ampholines (pH 3.5–10), 8 M urea, and 100 mM DTT. Focusing was conducted for a total of 17 700 volt-hours using voltage stepping: 2 h at 200 V, 5 h at 500 V, 11 h at 800 V, and 3 h at 2000 V.
NEPHGE was performed in 14 x 0.15-cm acrylamide rods using the gel composition described by Celis et al. [40] and Naaby-Hansen et al. [37]. The carrier ampholine composition was 13% pH 3.5–10, 37% pH 5–8, 13% pH 6.5–9, and 37% pH 8–10.5. Fifty-five microliters (~0.13 mg of protein) of spermatozoa extract was applied per rod. Electrophoresis was conducted for a total of 8600 volt-hours using 400 V for 5 h and 550 V for 3 h.
Two-dimensional SDS-PAGE was carried out in 0.15-cm-thick, 16 x 16-cm slab gels using linear gradient gels (T = 9–15%; T = concentration of acrylamide in the gel) in a Protean II xi Multi-cell apparatus (Bio-Rad, Richmond, CA) [37]. Electrotransfer to nitrocellulose membranes was carried out as described previously [37].
Immunoblotting Analysis
After electrophoretic transfer of 2D-separated polypeptides, the membranes were rinsed twice for 5 min in PBS (pH 7.4), and excess binding sites were blocked by incubation for 1 h at room temperature in PBS (pH 7.4) containing 5% dry milk and 0.05% Tween 20. The blots were incubated with the serum at 1:2000 dilution at 4°C overnight under constant slow rocking. In some cases a given blot was incubated with more than one serum. In these instances, the blots were washed in PBS (pH 7.4) containing 0.05% Tween 20 after incubation with the first serum at 4°C overnight and incubated with another serum under the same conditions as described above. Serum samples from 15 infertile men and 6 infertile women were analyzed that contained ASA as shown by the IBT. Secondary enzyme-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA) (goat antihuman IgG+IgM) were diluted 1:5000 in PBS containing 0.05% Tween 20, and the blots were incubated for 1 h at 20°C. The secondary antibody alone did not bind to blotted human sperm proteins (data not shown). Horseradish peroxidase conjugates were visualized by enhanced chemiluminescence (ECL) using the manufacturer's protocol (Amersham, Buckinghamshire, UK).
Scanning and Computer Analysis
X-ray films from chemiluminescence preparations and the autoradiograms from the radioiodination experiments were scanned with a Kodak camera (Eastman Kodak, Rochester, NY). The resulting 2D images were analyzed using the Bio Image "2D Analyzer" version 6.1 (Bio Image, Ann Arbor, MI). This software automates the identification, quantification, and comparison of 2D-gel-separated protein spots. A composite image representing the aggregate protein spots recognized by serum samples of fertile subjects of both sexes was obtained after matching the computer-scanned images of the ECL blots corresponding to fertile male and female individuals by using protein spots common to each image as reference points or "anchors." The anchors were chosen based on the electrophoretic mobility, the constellation of the protein spots, and the shape of the spots. The resulting composite image of the separated protein spots was compared and matched with the images corresponding to infertile male and female groups to subtract the protein spots recognized by serum samples of fertile subjects from the protein spots recognized by samples of infertile subjects, thus identifying the major auto- and iso-antigens that are uniquely recognized by sera of infertile subjects.
A composite image of the sperm surface proteins was created following matching of autoradiograms obtained after surface radioiodination. Care was taken to include only those spots that were consistently labeled as described earlier [37]. The computer-generated images containing the auto- and iso-antigens were matched with the sperm surface information to identify the surface auto- and iso-antigens.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Silver-stained 2D gels (16 x 15 cm) of nonionic detergent/urea-solubilized human sperm proteins resolved more than 1300 protein spots by IEF/PAGE (Fig. 1A) and NEPHGE/PAGE (Fig. 1B). These gels represent the repertoire of protein spots obtained from a pool of 10 individuals' sperm. The protein pattern was highly reproducible, although minor variations between donors were observed with regard to the relative abundance and charge of some proteins that were found to vary when individual samples were compared by computer analysis (data not shown). Figure 1 presents the identities of some of the major protein spots as identified by microsequencing of individual spots or immunoblotting with antisera to specific proteins. For example, different heat shock protein family members were resolved. These proteins are abundant on the sperm membrane (unpublished results). PH-20, a sperm hyaluronidase [41], is a membrane-bound protein represented by three 53-kDa isoforms (unpublished results). The major cytosolic proteins, 
-tubulin and ß-tubulin, are also noted, as is human sperm calreticulin (unpublished results). In the NEPHGE gel the position of gastrin-binding protein is indicated.
|
Figure 2, A and B, illustrates representative 2D Western blots of sperm proteins resolved by IEF or NEPHGE and probed with serum from a fertile male subject (Fig. 2A) or from an infertile male subject (Fig. 2B). The serum from the infertile subject showed stronger immunoreactivity to some spots than that of the fertile subject, although the importance of such differences in intensity is not presently understood. In addition, a number of antigenic spots among both acidic and basic proteins were reactive with sera from infertile but not fertile subjects (arrows). Similarly, differences in immunological reactivity were noted in comparing Western blots probed with an infertile female serum (Fig. 3B) to those reactive with fertile female serum (Fig. 3A); the infertile subject's serum recognized more antigenic spots (arrows) and also showed stronger reactivity to some spots than did the serum from the fertile female.
|
|
Analysis of the Acidic Antigens
Analysis of individual serum samples from infertile subjects showed marked variability in the range of specific proteins recognized but also demonstrated that some antigens were common to different individuals. The variation in the immunoreactivity of 4 such infertile male samples is shown in Figure 4, A–D (the major differences in protein spots recognized by sera from these individuals are highlighted with arrows). This observation of a range of recognized sperm antigens implies considerable diversity in the ASA repertoire in the human male. On the other hand, similarity in the patterns of immunoreactivity for certain sperm antigens was frequently noted as demonstrated by the serum samples from 2 infertile females in Figure 5, A and B (arrows indicate identical spots between Fig. 5, A and B). Computer analysis and comparison of blots reacted with sera from infertile and fertile subjects indicated that the 4 protein spots at ~60 kDa (note arrows at 60 kDa) recognized by the sera of both infertile individuals belong to a subset of proteins not recognized by the fertile subjects' sera.
|
|
A comprehensive overview of the repertoire of immunoreactive sperm proteins recognized by serum samples from fertile and infertile subjects of both sexes was obtained by serially incubating blots with 5 serum samples from each group of subjects (Fig. 6, A–D). Serum samples from the infertile subjects were selected based on their high immunoreactivity, heterogeneity in the immunoreactivity, and unique recognition of certain protein spots following individual Western blot analysis of the sera. Figure 6, A and B, presents the repertoire of sperm proteins bound by sera from 5 fertile males (Fig. 6A) and 5 fertile females (Fig. 6B). Comparison of the sperm antigens recognized by sera from fertile males (Fig. 6A) and fertile females (Fig. 6B) reveals a remarkable overall similarity in the repertoire between the two sexes, although the samples from fertile males recognized a greater number of antigens than did that from fertile females. Figure 6, C and D, shows the Western blots serially probed with sera from 5 infertile male (Fig. 6C) and 5 infertile female subjects (Fig. 6D), respectively. Comparison of the Western blots probed with sera from fertile and infertile patients (Fig. 6, A+B to C+D) demonstrates that several discrete sperm antigens were recognized by sera from infertile patients (arrows). The molecular masses of prominent protein spots uniquely recognized by sera from male patients were 34 kDa (pI 4.2), 42 kDa (pI 4.3), 88.5 kDa (pI 4.0), 106 kDa (pI 4.2), 41.8 kDa (pI 5.3, pI 5.4, pI 5.5), 61.5 kDa (pI 5.62), 60.4 kDa (pI 5.75), and 60 kDa (pI 6.6 and 6.7). Major iso-antigens recognized by sera from infertile females were at the molecular masses of 60 kDa (pI 6.2, pI 6.4, pI 5.82, pI 5.88), 32.3 kDa (pI 6.7, pI 6.8, pI 6.85), and 41.2 kDa (pI 5.1).
|
Variation in Immunoreactivity between Sera from Male and Female Patients
Computer image analysis of the immunoblots showed that a total of 310 antigens were recognized by the sera from infertile male patients whereas 205 antigens were recognized by the sera from infertile female patients. The results suggest that the immunoreactivity to sperm antigens shown by infertile males is probably higher than that of infertile females, although more serum samples from female subjects require analysis before a definitive conclusion can be reached. Further, a distinct variation in the pattern of immunoreactivity was discernible between the two sexes, each of these groups showing specificity for different sets of proteins (Fig. 6, C and D) even though proteins common to both subject groups were noted.
Computer-aided subtraction of the antigens recognized by sera of fertile subjects, both male and female, from the total repertoire of antigens recognized by the infertile male groups revealed 86 protein antigens that were exclusively recognized by samples from infertile males (Fig. 7A) compared to samples from fertile subjects. Of these 86 antigens recognized by the infertile male patients' sera, 72 were specific to only infertile male patients and were not recognized by sera from infertile females. Similar computer-aided analysis of the 2D blots yielded 26 protein spots unique to the infertile female subjects (Fig. 7B). Twelve of these antigens were specific to only infertile females and were not recognized by infertile males. Among the sperm antigens exclusively recognized by the sera from infertile patients (both males and females), 14 antigens were commonly recognized by samples from both sexes (arrows).
|
Sperm Surface Antigens Exclusively Recognized by the ASA-Positive Patients
Figure 8 presents a computer-generated composite image of human sperm surface proteins obtained after computer scanning of autoradiograms obtained from 2D IEF-PAGE of vectorially iodinated sperm proteins [37]: 103 proteins were identified on the sperm surface by this method. Computer-aided matching of the sperm auto- and iso-antigens with the composite image of the sperm surface proteins allowed identification of a subset of sperm surface auto- and iso-antigens. The locations of six sperm surface auto-antigens and iso-antigens are depicted in Figure 8 (arrows). Prominent are two 60-kDa proteins with pIs of 6.2 and 6.4 that were recognized by samples from 2 infertile women. (Fig. 5, A and B and Fig. 6D). Among the sperm antigens recognized by sera from infertile males, a 88.2-kDa sperm surface protein with a pI of 4.0 was recognized by samples from 2 subjects (Fig. 6C). Sera from 2 subjects also recognized two sperm surface proteins at molecular masses of 34 kDa and 38 kDa, both at a pI of 4.2 (Fig. 6C). Table 1 lists the masses and pI of the sperm surface iso- and auto-antigens.
|
|
Correlation of the Western Blot Results with the IBT Results
It was interesting to note that serum samples with high scores in the IBT for binding to various surface domains contained antibodies shown in the 2D analysis to recognize sperm surface proteins on the Western blots. Table 1 also includes the immunobead pattern of samples from these subjects. Each of these samples showed IBT reactivity toward IgG- and/or IgM-specific antibody. The antibodies were directed toward the head or the entire surface of the sperm.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The results obtained here by immunoblot analysis of individual serum samples confirm earlier reports on the heterogeneity of antigens recognized by human sperm auto- and iso-antibodies and imply that there is considerable diversity in the antisperm immunoglobulin repertoire [25, 44]. In one of these earlier studies [44], a limited number of antigens identified with individual serum samples also showed variable intensity of staining on one-dimensional gels. The finding in the present study that many of the patients' sera did not recognize the same set of antigens may be due in part to the complexity of sperm architecture and the diversity of antigens involved in the autoimmune response. It perhaps also suggests that the mechanism and pattern of infertility in these patients might differ. Antibodies recognizing different sperm surface proteins may influence the ability of sperm to fertilize by acting at different stages of the fertilization process. Differences in the antibody repertoire might also relate to differences in the mode of immunization. Previous studies have shown that the subclasses of antisperm IgA antibodies vary in men who have autoimmunity to sperm [8].
Our Western blot data confirm the presence of sperm antibodies in a high proportion of both fertile and infertile individuals, although the fertile individuals' serum samples recognized many fewer sperm antigens and the relative intensity of the reactivity was weaker compared to that of the infertile individuals. These data imply that most of the sperm antigens recognized by serum samples from fertile subjects are internal, because each of these samples was negative for ASA in the IBT. Tung et al. [45] have noted in immunofluorescent studies that sera from some fertile subjects are directed to internal antigens and that molecular mimicry with bacterial antigens may be occurring. Several immunoblotting assays earlier demonstrated the presence of ASA in a high proportion of fertile subjects [21, 28, 31]. ASA in a fertile population may therefore reflect a natural physiological phenomenon [21, 28, 46], and the occurrence of the antibodies to intracellular components seems to be relatively ubiquitous [7]. Several of these intracellular antigens have been microsequenced and identified as components of sperm specific structures such as the fibrous sheath and outer dense fibers (unpublished data). In the present study, systematic identification of a subset of these antigens has aided in the identification of sperm antigens eliciting production of functionally relevant and/or irrelevant antibodies that may have a role in the impairment of reproductive function.
The serial incubation of serum samples for immunoblotting combined with computer analysis succeeded in providing information on the major auto- and iso-antigens unique to the infertile patients. Male serum samples recognized many more antigens than did samples from females (310 vs. 205), although a comprehensive evaluation with more samples from female subjects is required to statistically validate the conclusion. A longer constant exposure of sperm proteins to the immune system among the male patients may explain the higher reactivity among males. In addition, a distinct difference in the pattern of immunoreactivity between male and female patients was discernible. The male patients' sera were reactive to several acidic proteins (pI 4.0–5.0) that were not recognized by any of the samples from females (see Fig. 6, C and D). Conversely, major iso-antigens recognized by the female patients in the pI range of 6.0–7.5 were poorly reactive to sera from the male patients (Fig. 6, C and D).
A rational basis for identifying fertility-related sperm antigens relies on the identification of antigens on the cell surface. Hence it is especially critical to distinguish between immunity to sperm surface antigens and the internal antigens of spermatozoa. Naaby-Hansen et al. [37] have identified 181 sperm surface proteins utilizing radiolabeling with 125I followed by IEF/PAGE as well as NEPHGE/PAGE separation. Employing the same method, followed by computer analysis, we have obtained a composite image consisting of 103 surface-labeled proteins in IEF/PAGE gels. By utilizing this image we were able to categorize a subset of 6 sperm surface antigens that may have a role in immunoinfertility. One of these antigens is a 88.2-kDa protein at a pI of 4.0, recognized by two male infertile patients. Naaby-Hansen et al. [37] have shown this protein to be a sperm surface protein, phosphorylated on tyrosine residues. Interestingly, serum samples from two women showed striking similarity in immunoreactivity, recognizing the same protein spots (Fig. 4, A and B) at 60 kDa and pI of 6.2 and 6.4. Both these sperm proteins were found to be on the surface of the sperm as demonstrated by surface labeling of the sperm with radioactive 125I. Further, both samples contained IgG antibodies directed toward the head of the sperm as shown by IBT, confirming that the antigens are exposed on the sperm surface, probably located on the sperm head. Two male patients' sera strongly recognized 2 protein spots at 34 kDa and 38 kDa that were identified on the sperm surface.
Although the current analysis of sera, based on both IBT and Western blotting to diagnose immunity to sperm, is able to detect the presence of ASA and identify the cognate antigens on the sperm surface, identification of the epitopes against which these antibodies are directed should improve the reliability of the analysis. Recognition of multiple antigens that appear to have a potential role in fertility could occur due to common epitopes. Earlier reports have shown that monoclonal antibodies of the same isotype but directed against different antigens present on the sperm head could block sperm attachment to the zona pellucida [47] or sperm-egg membrane fusion [48, 49]. Further, antibodies directed against different epitopes of the same sperm surface antigen may also affect sperm fertilizing ability in different ways [47, 49]. As knowledge of the specific sperm proteins against which ASA are directed increases, their locus of action in impairing fertilization will enhance their clinical value.
The computer digitization of information on the 2D gel patterns of surface-labeled sperm proteins opens the possibility for a new tool in the diagnosis of ASA and possible treatment of antibody-mediated infertility. Based on the sperm proteome database and the index of sperm surface proteins, 2D immunoblotting with patients' sera can now be employed to identify those patients with antibodies to protein antigens accessible at the cell surface and hence obtain more specific information on a patient's repertoire of auto- or iso-antibodies. As these databases expand with the identification of additional iso- and auto-antigens, and as microsequence information and microsequence-based cloning provides characterization of the relevant proteins, the possibility may emerge for a rational basis for immunotherapy of antibody-mediated infertility using defined native or recombinant sperm antigens. Further, diagnosis of antibody-mediated infertility may be simplified by using 2D immunoblots with a patient's serum and referencing the result to the database of surface antigens.
|
FOOTNOTES |
|---|
2 Correspondence: John C. Herr, University of Virginia, Health Sciences Center, Box #439, Charlottesville, VA 22908. FAX: 804 982 3912; jch7k{at}virginia.edu
Accepted: February 8, 1999.
Received: November 20, 1998.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  S. A Khan, A. R Suryawanshi, S. A Ranpura, S. V Jadhav, and V. V Khole Identification of novel immunodominant epididymal sperm proteins using combinatorial approach Reproduction, July 1, 2009; 138(1): 81 - 93. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. H. WEIDLE, V. EVTIMOVA, S. ALBERTI, E. GUERRA, N. FERSIS, and S. KAUL Cell Growth Stimulation by CRASH, an Asparaginase-like Protein Overexpressed in Human Tumors and Metastatic Breast Cancers Anticancer Res, April 1, 2009; 29(4): 951 - 963. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Wolkowicz, L. Digilio, K. Klotz, J. Shetty, C. J. Flickinger, and J. C. Herr Equatorial Segment Protein (ESP) Is a Human Alloantigen Involved in Sperm-Egg Binding and Fusion J Androl, May 1, 2008; 29(3): 272 - 282. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Domagala, S. Pulido, M. Kurpisz, and J.C. Herr Application of proteomic methods for identification of sperm immunogenic antigens Mol. Hum. Reprod., July 1, 2007; 13(7): 437 - 444. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Capkova, F. Elzeinova, and P. Novak Increased expression of secretory actin-binding protein on human spermatozoa is associated with poor semen quality Hum. Reprod., May 1, 2007; 22(5): 1396 - 1404. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Lefievre, K. Bedu-Addo, S. J Conner, G. S M Machado-Oliveira, Y. Chen, J. C Kirkman-Brown, M. A Afnan, S. J Publicover, W C. L Ford, and C. L R Barratt Counting sperm does not add up any more: time for a new equation? Reproduction, April 1, 2007; 133(4): 675 - 684. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. K. Naz Antisperm Immunity for Contraception J Androl, March 1, 2006; 27(2): 153 - 159. [Full Text] [PDF]
|
|
|
|
 |
|
 A. I. Yudin, S. E. Generao, T. L. Tollner, C. A. Treece, J. W. Overstreet, and G. N. Cherr Beta-Defensin 126 on the Cell Surface Protects Sperm from Immunorecognition and Binding of Anti-Sperm Antibodies Biol Reprod, December 1, 2005; 73(6): 1243 - 1252. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. L. Pixton, E. D. Deeks, F. M. Flesch, F. L.C. Moseley, L. Bjorndahl, P. R. Ashton, C. L.R. Barratt, and I. A. Brewis Sperm proteome mapping of a patient who experienced failed fertilization at IVF reveals altered expression of at least 20 proteins compared with fertile donors: Case report Hum. Reprod., June 1, 2004; 19(6): 1438 - 1447. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.K. Rajeev and K.V.R. Reddy Sperm membrane protein profiles of fertile and infertile men: identification and characterization of fertility-associated sperm antigen Hum. Reprod., February 1, 2004; 19(2): 234 - 242. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Wolkowicz, J. Shetty, A. Westbrook, K. Klotz, F. Jayes, A. Mandal, C. J. Flickinger, and J. C. Herr Equatorial Segment Protein Defines a Discrete Acrosomal Subcompartment Persisting Throughout Acrosomal Biogenesis Biol Reprod, September 1, 2003; 69(3): 735 - 745. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Shetty, M. J. Wolkowicz, L. C. Digilio, K. L. Klotz, F. L. Jayes, A. B. Diekman, V. A. Westbrook, E. M. Farris, Z. Hao, S. A. Coonrod, et al. SAMP14, a Novel, Acrosomal Membrane-associated, Glycosylphosphatidylinositol-anchored Member of the Ly-6/Urokinase-type Plasminogen Activator Receptor Superfamily with a Role in Sperm-Egg Interaction J. Biol. Chem., August 15, 2003; 278(33): 30506 - 30515. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Shibahara, Y. Shiraishi, Y. Hirano, T. Suzuki, S. Takamizawa, and M. Suzuki Diversity of the inhibitory effects on fertilization by anti-sperm antibodies bound to the surface of ejaculated human sperm Hum. Reprod., July 1, 2003; 18(7): 1469 - 1473. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Bohring and W. Krause Immune infertility: towards a better understanding of sperm (auto)-immunity: The value of proteomic analysis Hum. Reprod., May 1, 2003; 18(5): 915 - 924. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Rao, J. C. Herr, P. P. Reddi, M. J. Wolkowicz, L. A. Bush, N. E. Sherman, M. Black, and C. J. Flickinger Cloning and Characterization of a Novel Sperm-Associated Isoantigen (E-3) with Defensin- and Lectin-Like Motifs Expressed in Rat Epididymis Biol Reprod, January 1, 2003; 68(1): 290 - 301. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. C. McCauley, B. E. Kurth, E. J. Norton, K. L. Klotz, V. A. Westbrook, A. J. Rao, J. C. Herr, and A. B. Diekman Analysis of a Human Sperm CD52 Glycoform in Primates: Identification of an Animal Model for Immunocontraceptive Vaccine Development Biol Reprod, June 1, 2002; 66(6): 1681 - 1688. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Hao, M. J. Wolkowicz, J. Shetty, K. Klotz, L. Bolling, B. Sen, V. A. Westbrook, S. Coonrod, C. J. Flickinger, and J. C. Herr SAMP32, a Testis-Specific, Isoantigenic Sperm Acrosomal Membrane-Associated Protein Biol Reprod, March 1, 2002; 66(3): 735 - 744. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Wolkowicz, S. Naaby-Hansen, A. R. Gamble, P. P. Reddi, C. J. Flickinger, and J. C. Herr Tektin B1 Demonstrates Flagellar Localization in Human Sperm Biol Reprod, January 1, 2002; 66(1): 241 - 250. [Abstract] [Full Text]
|
|
|
|
|
|
N. P. Haden, J. R. Hickox, C. Scott Whisnant, and D. M. Hardy Systematic Characterization of Sperm-Specific Membrane Proteins in Swine Biol Reprod, December 1, 2000; 63(6): 1839 - 1847. [Abstract] [Full Text]
|
|
|
|
|
|
I. A. Brewis Proteomics in reproductive research: The potential importance of proteomics to research in reproduction Hum. Reprod., December 1, 1999; 14(12): 2927 - 2929. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
