Immune-mediated destruction of ovarian follicles associated with the presence of HSP90 antibodies.

We previously established that the presence of autoantibodies to heat-shock protein 90 (HSP90) is one common causes of female infertility, and demonstrated that its presence leads to detrimental effects on ovarian and reproductive function in mice. The pathophysiological mechanism and alteration in the immune physiology, however, remain unknown. We therefore carried out detailed analysis of various immune cells in the spleen and ovary following immunization of C57BL/6 female mice to generate antibodies to HSP90 in the general circulation. We observed a significant increase in levels of CD45- cells; CD4+ T cells; Ly6G6C+ cells; and CD11b+ Ly6G+ cells in the spleen of these mice, which correlate with the increased anti-HSP90 antibody production. Ovarian- and granulosa-cell populations also showed increased infiltration of CD45+ leukocytes and neutrophil and monocyte populations, which may have led to the observed ovarian follicular degeneration that predominantly manifested as empty follicles. A decrease in the number of functional ovarian follicles was also associated with a decrease in the level of Gdf9 gene expression. Thus, changes in the immune physiology of the spleen and ovary that leads to the generation of antibodies to HSP90 can also bring about the destruction of ovarian follicles.

Liprin α3: a putative estrogen regulated acrosomal protein.

Liprin α3 was reported for the first time using sperm proteomics. Present study reports its localization on sperm and immunochemical characterization. Liprin α3 is identified as a 133 kDa protein in testis and epididymal protein extracts. In testis, immunohistochemical localization was seen in pachytenes, diplotenes, round spermatids whereas it was localized in the epithelial cells and luminal sperm in all the three regions of epididymis. Protein was localized in acrosome of rat sperm, which was further confirmed by sequential treatment of sperm with hypertonic solution. In the spermatogenic cells the protein was found to be located in developing acrosome as evident by its co-localization with Golgi marker. Protein was found to be developmentally regulated. In silico analysis of Liprin α3 revealed presence of the estrogen responsive elements upstream to initiation site and its regulation by estrogen was experimentally validated using a tamoxifen treated rat model. Western blot analysis of epididymosomes showed the presence of Liprin α3, indicating its involvement in trafficking of vesicle. The protein expression was seen in both mouse and human sperm indicating conserved nature and a probable role in acrosome reaction.

Immunization with ovarian autoantigens leads to reduced fertility in mice following follicular dysfunction.

Immunoproteomics using sera of women with ovarian autoimmune diseases such as primary ovarian insufficiency and IVF embryo transfer recruits led to identification of three proteins namely alpha actinin 4 (α-ACTN4), heat-shock 70 protein 5 (HSPA5), and actin beta (ACTB). This study deals with the establishment of a peptide ELISA for screening sera of antiovarian antibody (AOA)-positive patients and further delves into understanding the role of these three proteins in ovarian autoimmunity in a mouse model. Using in silico approach, antigenic peptides of these proteins were identified and used for peptide ELISA. ELISA results indicated that AOA-positive sera showed reactivity with only specific peptides. The functional significance of the dominant peptides was studied by active immunization of female mice with these peptides. All immunized mice generated high antibody titers and profound effect on ovaries with few primordial (2.4±0.1, 2.4±0.2, and 2±0.1), primary (2.4±0.5, 1.7±0.3, and 2.4±0.3), preantral (2.3±0.5, 3.4±0.3, and 2.9±0.3), antral (0.9±0.2, 1.6±0.8, and 2.3±0.6) follicles, and corpora lutea (2.8±0.8, 2.9±1.7, and 4.6±2.3), and increased number of atretic follicles (5.5±0.4, 4.9±1.8, and 7.5±1.0) in ACTN4-, HSPA5-, and ACTB-immunized mice compared with control animals (3.0±0.2, 3.5±0.6, 3±0.1, 3.6±0.2, 4.7±0.3, and 1.5±0.3) respectively. These mice when mated with fertile male mice showed an overall 25-43% reduction in fertility compared with controls. The data clearly suggest that the dominant antigenic epitopes of the three proteins play critical role in fertility and could possibly be the key autoimmune targets. These epitopes could be used to develop a more specific and sensitive diagnostic test for women with ovarian autoimmune diseases and to design therapy for disease management for reinstatement of ovarian function.

Anti-HSP90 autoantibodies in sera of infertile women identify a dominant, conserved epitope EP6 (380-389) of HSP90 beta protein.

BACKGROUND: We earlier reported a simple specific test for detection of anti-ovarian antibodies in infertile women and identified number of specific molecular and cellular targets of which human heat shock protein 90-beta (HSP90 beta) was found to be the most immunodominant. The present study focuses on prediction and validation of the immunodominant epitope/s of this protein using sera from infertile women having anti-HSP90 autoantibodies. METHODS: Delineation of the immunodominant epitopes of HSP90 beta was done by using epitope prediction algorithms and 10 peptides (EP1-EP10) were custom synthesized. Their immunoreactivity was measured by ELISA using sera from patients and controls. To determine the most immunodominant epitope, the results were subjected to statistical analysis. The immunoreactivity of the immunodominant peptides were confirmed by dot blots using sera from patients. A rabbit polyclonal antibody against the immunodominant epitope was generated and its immunoreactivity to the parent protein in ovarian extracts as well in oocytes and embryos was investigated. RESULTS: Experimentally and statistically, peptide EP6 (380-389) seems to be the major antigenic epitope for the serum antibody binding followed by EP1 (1-12) and EP8 (488-498). Predicted 3D structures of these peptides demonstrated that they exist in the loop conformation which is the most mobile part of the protein. Also, analysis of the sequences of HSP90 beta across several species reveals that EP6 peptide forms a part of a well conserved motif. The polyclonal antibody generated to the immunodominant epitope- EP6 confirms similar biochemical and cellular immunoreactivity as seen with the patients' sera having anti-HSP90 autoantibodies. CONCLUSIONS: The decapeptide EP6 is a major immunogenic epitope of HSP90 followed by EP1 and EP8. Knowledge of binding epitopes on the autoantigen is necessary to understand the subsequent pathologic events. The study might generate new tools for the detection of disease-inducing epitopes and a possible therapeutic intervention.

Identification and validation of candidate biomarkers involved in human ovarian autoimmunity.

Antibodies to multiple ovarian antigens have been proposed as markers of ovarian autoimmunity. The role of ovarian autoantibodies has been widely discussed in the pathophysiology of premature ovarian failure and unexplained infertility, but the autoantigens are yet to be identified. Three immunodominant ovarian autoantigens, α-actinin 4 (αACTN4), heat shock 70 protein 5 (HSPA5) and ß-actin (ACTB), have been identified using anti-ovarian antibody-positive sera from women with idiopathic premature ovarian failure (n=50) and women undergoing IVF (n=695), using mass spectrometry. These autoantigens were subsequently validated using Western blot, immunohistochemistry and enzyme-linked immunosorbent assay. These autoantigens are localized to different components of the ovary such as the ooplasm of the oocyte, theca, granulosa, corpus luteum and zona pellucida. All the above antigens were found to be expressed in the ooplasm throughout follicular development. All the autoantigens are expressed specifically in the oocyte except αACTN4. The three autoantigens could contribute to the array of biomarkers to be used for developing specific and sensitive tests for diagnosis of women at risk of premature ovarian failure and IVF failure due to ovarian autoimmunity and could give an insight into the molecular mechanisms involved in the pathophysiology of these conditions.

Can anti-ovarian antibody testing be useful in an IVF-ET clinic?

OBJECTIVE: To establish importance of anti-ovarian antibodies (AOA) testing in infertile women. DESIGN: A clinical reproductive outcome comparative study between two groups of women undergoing IVF-ET. Group 1 consists of women tested positive for AOA, put on corticosteroid therapy, reverted to AOA negative and then taken up for IVF-ET. Group 2 were seronegative for AOA. SETTING: Major urban infertility reference centre and National research institute. PATIENT(S): Five hundred seventy infertile women enrolled for IVF-ET. INTERVENTION(S): AOA testing, corticosteroid therapy and IVF-ET/ICSI. MAIN OUTCOME MEASURE(S): Comparable clinical outcome and significance of AOA testing established. RESULTS: AOA positive serum samples were sent periodically to re-investigate presence of AOA after corticosteroid therapy and women turned AOA negative were taken up for IVF-ET. Of the 70/138 women in group 1 who were treated with corticosteroids and turned seronegative for AOA, 22/70 were poor responders and needed donor oocyte-recipient cycles. Results demonstrated that fertilization and clinical pregnancy rates between both groups are comparable. Nevertheless, it is also observed that there is poor response to stimulation protocol, smaller number of oocytes retrieved and more spontaneous abortions in group 1 women. Hence not all outcomes following the treatment are comparable between the two groups. Usefulness of the test was established in two case studies. CONCLUSIONS: AOA testing could be included in the battery of tests investigating and treating infertility.

Thiol based mechanism internalises interacting partners to outer dense fibers in sperm.

The sperm tail outer dense fibres (ODFs) contribute passive structural role in sperm motility. The level of disulphide cross-linking of ODFs and their structural thickness determines flagellar bending curvature and motility. During epididymal maturation, proteins are internalized to modify ODF disulphide cross-linking and enable motility. Sperm thiol status is further altered during capacitation in female tract. This suggests that components in female reproductive tract acting on thiol/disulphides could be capable of modulating the tail stiffness to facilitate modulation of the sperm tail rigidity and waveform en route to fertilization. Understanding the biochemical properties and client proteins of ODFs in reproductive tract fluids will help bridge this gap. Using recombinant ODF2 (aka Testis Specific Antigen of 70 kDa) as bait, we identified client proteins in male and female reproductive fluids. A thiol-based interaction and internalization indicates sperm can harness reproductive tract fluids for proteins that interact with ODFs and likely modulate the tail stiffness en route to fertilization.

Identification of novel immunodominant epididymal sperm proteins using combinatorial approach.

Functionally immature spermatozoa leave the testis mature during epididymal transit. This process of maturation involves either addition of new proteins or modification of existing proteins onto the sperm domains that are responsible for domain-specific functions. Epididymal proteins are preferred targets for immunocontraception. In an attempt to identify epididymis-specific sperm proteins, we used a novel combinatorial approach comprising subtractive immunization (SI) followed by proteomics. Following SI, sera of mice were used for immunoproteomics, which led to the identification of 30 proteins, of which four proteins namely sperm head protein 1, sperm flagella protein 2 (SFP2), SFP3, and SFP4 are being reported for the first time on sperm. Another group of four proteins namely collagen alpha-2 (I) chain precursor, homeodomain-interacting protein kinase 1, GTP-binding protein Rab1, and ubiquinol cytochrome c reductase core protein II although reported earlier in testis are being reported for the first time in epididymal sperm. Furthermore, seven out of these eight novel proteins could be validated using peptide ELISA. These data are a useful repository, which could be exploited to develop targets for post-testicular immunocontraception or biomarkers for infertility diagnosis and management.

Specific and sensitive immunoassays detect multiple anti-ovarian antibodies in women with infertility.

Serum anti-ovarian antibodies (AOAs) have been shown in autoimmune premature ovarian failure and in vitro fertilization-embryo transfer (IVF-ET) cases. The specificity of assays detecting these antibodies has been questioned. Researchers have used several techniques (e.g., ELISA and indirect immunofluorescence). Few have reported on the non-specificity and the type of molecular and cellular targets. We reported earlier on the presence of naturally occurring anti-albumin antibodies as the likely factor for non-specificity. Having developed a novel blocking recipe, we show substantial elimination of this non-specificity. With these standardized tests, we hereby report multiple targets at protein and histological levels. In our study group, 15 of 50 (30%) patients with premature ovarian failure and 13 of 50 (26%) IVF-ET patients showed the presence of AOAs. Western blotting showed a large number of patients making AOAs to a 90-kDa protein, followed by 97- and 120-kDa proteins. Histochemically, it was evident that the sera of these patients predominantly react with the oocyte; other somatic cellular targets are also involved. The specific non-invasive test developed by us was found to be useful because it could carry out a reliable diagnosis of an autoimmune etiology that would be very helpful to select patients in whom immune-modulating therapy could be recommended, which in turn may restore ovarian function and fertility.

Naturally occurring anti-albumin antibodies are responsible for false positivity in diagnosis of autoimmune premature ovarian failure.

Autoimmunity is a well-established causative factor of premature ovarian failure (POF), and evidence for the same has been well reported in the literature. Detection of specific autoantibodies remains the most practical clinical research marker of any autoimmune disease. Variation in efficiency and specificity in the detection of ovarian autoantibodies has been reported. However, the frequency of false positivity and a solution to overcome this has not yet been reported. Herein, we report autoantibody to albumin as the likely responsible agent for false positivity. Our data indicate that presence of naturally existing autoalbumin antibodies in the circulation of normal women is responsible for the false signal seen in SDS-PAGE Western blot analysis and in immunohistochemistry (IHC). Having shown the presence of anti-albumin antibody in normal women as well as in the sera of POF patients, we have developed a novel blocking agent to overcome this problem. A high titer polyclonal antibody against human serum albumin was generated. This antibody showed immunoreactivity to albumin obtained from various sources. Preincubation of Western blots and IHC sections with this antibody drastically reduced background signals. The advantage of using this blocking was evident by identification of specific anti-ovarian antibodies in a group of POF patients. This blocking procedure made it possible to obtain a clear indication of the ovarian antibody status in women presenting with autoimmune POF.

Monoclonal antibody from vasectomized mouse identifies a conserved testis-specific antigen TSA70.

Vasectomy results in the occlusion of testicular outflow, leading to autoimmunity characterized by the production of antisperm antibodies (ASA). Reports on the rise in ASA following vasectomy in several species are available; however, not much is known about the specific sperm autoantigens to which postvasectomy antibodies are directed. In the present study, monoclonal antibodies were generated using a vasectomized mouse. One of the monoclonal antibodies, D5E5, identified an approximately 70-kd antigen localized on the principal piece of the tail and also on the tip of the acrosome of mouse sperm. The cognate antigen was expressed postmeiotically in a stage-specific manner during spermiogenesis, starting from step 8 of elongating spermatids during spermiogenesis up to mature spermatozoa. The protein was conserved across the species, as observed by its presence in rat, bull, marmoset, and human sperm. Following capacitation, the antigen on the head was seen to shift to the acrosomal region and was lost after the acrosome reaction. However, the localization on tip of the acrosome still persisted, which indicates that the antigen may play a role post-acrosome reaction in sperm egg interaction. Resistance to Triton X-100 solubilization indicates that TSA70 could be an acrosomal matrix protein. In addition, we observed a significant reduction in forward progressive motility of mouse sperm treated in vitro with D5E5. In view of its testis specificity, acrosome and tail localization, and conserved nature, TSA70 is likely to play an important role in sperm function.

Monoclonal antibodies to epididymis-specific proteins using mice rendered immune tolerant to testicular proteins.

Monoclonal antibodies (mabs) have been used as a powerful tool for identification of newer sperm proteins. However, conventional hybridoma technology rarely provides chance to obtain mabs to epididymal proteins. To increase this chance, we have used an alternate method of neonatal tolerization. In this protocol, animals were tolerized at birth using testicular proteins followed by immunization with cauda epididymal sperm protein (which is a cocktail of proteins both from testicular and epididymal origin). This protocol induced a specific immune response to epididymal sperm proteins. Spleen from one of these animals was then used for preparation of mabs. This fusion resulted in a number of mabs reacting specifically to epididymal proteins. Although mabs identified a protein of approximately similar molecular weight on 1-dimensional Western blot analysis, there were differences in regional localization on rat sperm as seen by indirect immunofluorescence. Immunohistochemical localization of these proteins in rat epididymis showed region specific synthesis. The synthesis of proteins was seen in the distal caput epididymis, and maximum expression was seen in supranuclear region of corpus epithelium. The proteins were localized on sperm from corpus and cauda region. Epididymis specific synthesis of the proteins and agglutinating nature of the mabs to these underlines the functional importance of these proteins in sperm maturation in epididymis. These antibodies could therefore, be used as tools for understanding the physiology of maturation of sperm in epididymis and role of the epididymal protein in fertilization.

HSP90 antibodies: a detrimental factor responsible for ovarian dysfunction.

PROBLEM: Earlier studies from our group have established that about 47% cases of autoimmune ovarian failure are due to presence of autoantibodies to Heat Shock Protein 90 (HSP90). However, there are no reports correlating pathological effects of HSP90 autoantibodies leading to ovarian failure. METHOD OF STUDY: Antibodies to HSP90 in female mouse model were generated by active immunization with an immunodominant peptide of HSP90, followed by detailed analysis of several reproductive parameters. RESULT: Estrous cyclicity remains unchanged; however, there was a significant drop in the fertility index due to an increase in pre- and post-implantation loss, associated with an increased incidence of degenerated eggs and embryos. The ovaries showed an increase in the number of empty and degenerated follicles and extensive granulosa cell deaths, which was reflected by the decrease in the levels of Nobox and Gja1 gene expression. CONCLUSION: This study underlines a critical role played by HSP90 in ovarian folliculogenesis and highlights the implications of the presence of anti-HSP90 antibodies in infertile women.

Epididymosome-mediated acquisition of MMSDH, an androgen-dependent and developmentally regulated epididymal sperm protein.

A differential proteomics approach led to the identification of several novel epididymal sperm proteins. One of the novel proteins was methylmalonate-semialdehyde dehydrogenase (MMSDH). In the present study, we carried out an in-depth characterization to study its regulation by androgen, its appearance during ontogeny, and the mechanism of its interaction with and acquisition by the sperm. Western blotting and immunohistochemical studies suggest that the protein is present in both tissue and sperm from all regions of the epididymis, indicating synthesis as well as acquisition of the protein in these regions. Androgen depletion resulted in reduction of the MMSDH protein level in the epididymis, which completely disappeared 1 week after castration. The protein reappeared after testosterone propionate injection, indicating that the protein is regulated by androgens. Ontogeny studies indicated that the protein appeared from day 10 postnatal with a gradual increase at day 30, which maximized at day 50, indicating that the protein is developmentally regulated and is probably involved in epididymal development. Sequential extraction of sperm proteins indicated that MMSDH exists both as a peripheral and integral form on the plasma membrane. We also found that the protein can be transferred from the epididymosomes to testicular sperm in vitro. The study provides evidence regarding the acquisition of this multidomain androgen and developmentally regulated protein in the epididymis via the epididymosomes. The molecule has generated enough interest and deserves to be investigated further for its physiological relevance.

Differential proteomics leads to identification of domain-specific epididymal sperm proteins.

The alteration in the protein signatures of the testicular sperm during its epididymal sojourn makes it functionally competent for successful fertilization. The present study was undertaken to identify the proteins acquired on its 2 domains, that is, the head and the flagellum, during the epididymal transit using a differential proteomics approach. Testicular sperm proteome was compared with cauda epididymal sperm proteome in rat. The protein spots exclusively present in the cauda epididymal sperm proteome were searched in the cauda sperm head proteome and the cauda sperm flagella proteome, and a total of 335 spots were found by alignment and auto-matching of the gels, of which 140 could be identified by mass spectrometry. Database search revealed that of these 9 proteins were novels. Gene Ontology annotation revealed that the identified proteins were distributed across different cellular components and were primarily involved in metabolic processes. The study also provides information on the localization of these proteins on the sperm domains, which indirectly gives a clue about its putative function. Validation of 3 proteins, namely MMSDH, NDUFS1, and UQCRC2, using antibodies very elegantly demonstrates that the strategy has been very effective. This comprehensive data of domain-specific epididymal sperm proteins will be useful in development of newer targets for posttesticular contraception and diagnostic markers for infertility.

Evaluation of contraceptive potential of a novel epididymal sperm protein SFP2 in a mouse model.

PROBLEM: Sperm flagellar protein 2 (SFP2), which was earlier identified using a novel combinatorial approach, was evaluated for its contraceptive potential in mice. METHOD OF STUDY: Male mice were actively immunized with two synthetic peptides of SFP2. Antipeptide antibody was characterized by Western blot and indirect immunofluorescence. Immune response was monitored, and mating studies were performed 6 and 22 weeks post-immunization. RESULT: Antibodies to the SFP2 peptide 1 recognized a doublet at 220- to 230-kDa region only in the epididymal protein extract. Peptide 1 antibody recognized the cognate protein on spermatozoa from mouse, rat, and human. Histological analysis of testis and epididymis of the immunized mice indicated no deleterious effect. Incubation of sperm with the immune sera of peptide 1 caused significant reduction in motility and viability but did not agglutinate sperm. Only synthetic peptide 1 gave rise to high-level antibodies in all the immunized mice, which on mating resulted in reduced fertility rate (20%) when compared with PBS control animals (100%). The antibody levels in the immunized males declined by 22 weeks post-immunization, resulting in 100% reinstatement of fertility. CONCLUSION: These data provide an experimental basis for the development of effective contraceptive vaccine based on new epididymal target.

A block in the road to fertility: autoantibodies to heat-shock protein 90-beta in human ovarian autoimmunity.

OBJECTIVE: To report autoantibodies to human heat-shock protein 90-beta (HSP90 beta) in sera of women with infertility. DESIGN: Prospective, controlled observations. SETTING: Major urban infertility referral center and research institution. PATIENT(S): Fifty women with premature ovarian failure, 65 infertile women enrolled in the in vitro fertilization-embryo transfer program, and 60 normally menstruating fertile women as controls. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Identification and complete characterization of a 90-kd protein, the most immunodominant autoantigen. RESULT(S): Our previous studies employing a novel blocking demonstrated several cellular and molecular ovarian antigenic targets using patient's serum. Of all these antigens, the 90-kd protein designated as EP90 was found to be conserved across species, was serine-threonine phosphorylated, and was expressed from the primordial stage to the graafian-stage ooplasm of the oocytes during follicular development. Using high-throughput proteomic technologies like liquid chromatography/mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF), and tandem mass spectrometry analysis revealed the identity of this protein to be HSP90 beta. Commercially available recombinant protein immunoreacted with the sera from patients with antiovarian antibodies against the 90-kd antigen. In parallel, using monoclonal antibody to human HSP90, we found that it reacts with the eluted protein from a crude ovarian extract. CONCLUSION(S): This is the first report to show the presence of ovarian autoantibodies to human HSP90 in sera of women with infertility. This protein could be involved in human ovarian autoimmunity and thereby be a causative factor in early ovarian failure.