Vrinda V. Khole

Spermatid-Specific Expression of the Novel X-Linked Gene Product SPAN-X Localized to the Nucleus of Human Spermatozoa

Abstract Formation of mature spermatozoa involves a series of dramatic molecular and morphological changes in the male germ cell lineage. These changes result from the temporally regulated transcription and translation of several testis-specific gene products. Here, we describe a novel, testis-specific protein designated SPAN-X for sperm protein associated with the nucleus on the X chromosome. SPAN-X sequences showed no significant similarity with known cDNA or peptide sequences. The SPAN-X peptide sequences contained three overlapping consensus nuclear localization signals, a high percentage (33%–37%) of charged amino acid residues, and a relatively acidic isoelectric point (pI; 4.88–6.05). Northern analysis of mRNA from multiple human tissues identified a SPAN-X transcript exclusively in the testis. In situ hybridization of human testes sections showed SPAN-X mRNA expression in haploid, round, and elongating spermatids. The SPANX gene was mapped to chromosome Xq27.1 by fluorescence in situ hybridization and by Southern blot analysis of human/mouse somatic cell hybrids. On Western blots of human sperm proteins, antirecombinant SPAN-X antibodies reacted with broad bands migrating between 15–20 kDa. Immunofluorescent labeling of human spermatozoa demonstrated SPAN-X localization to nuclear craters and cytoplasmic droplets. Expression of SPAN-X, an X-linked gene product, exclusively in haploid spermatids leads to interesting questions regarding the transcription of sex-linked genes during spermiogenesis.

A block in the road to fertility: autoantibodies to heat-shock protein 90-β 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 patients 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.

Multiple endometrial antigens are targeted in autoimmune endometriosis

Endometriosis is defined as the growth of endometrial glands and stroma in ectopic locations. Its aetiology is multifactorial, but autoimmunity has been shown to play a role in its onset and development. The present study aimed to investigate the presence of both IgG and IgM anti-endometrial antibodies in sera of endometriosis patients in comparison with age-matched controls, and to also investigate the cognate endometrial proteins involved. Sera from these groups were screened by western blot and immunohistochemistry. Thirteen out of the 40 sera tested were positive for IgG isotype, and 10/27 IgG negative patients were positive for IgM isotype. These findings indicate that endometrial antibodies of IgG and IgM classes could be detected in almost 60% of endometriosis patients. Of the various identified endometrial antigens, 30 and 45 kDa antigens were immunodominant in both IgG and IgM positive endometriosis patients. With immunohistochemistry, positive sera showed reactivity in luminal epithelium, glandular epithelium and stroma. These anti-endometrial antibodies might be partially responsible for failure of implantation leading to infertility. Identification of specific targets would be a help in understanding the pathophysiology of endometriosis, and would also help in setting up a non-invasive test for the diagnosis of endometriosis.

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.

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.

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.

Identification and validation of novel serum markers for early diagnosis of endometriosis

BACKGROUND Non-invasive diagnosis of endometriosis is urgently required to prevent the long delay between the onset of symptoms and diagnosis. A biomarker that possesses both high sensitivity and specificity is greatly required. Here, we describe the use of a proteomic approach to identify potential novel endometrial antigens using sera from endometriosis patients and healthy controls, with evaluation of biomarkers for non-invasive diagnosis of endometriosis. METHODS A cross-sectional study was conducted to identify specific endometrial antigens using 1D and 2D western blots in women with early endometriosis (n = 17), advanced endometriosis (n = 23) and without endometriosis (n = 30). Five immunoreactive spots were analyzed using matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry with MASCOT analysis. ELISAs were established for specific epitopes and autoantibody titres were estimated in an independent cohort comprising women with early endometriosis (n = 18), advanced endometriosis (n = 32) and without endometriosis (n = 27) for validation. RESULTS The 2D western blot analysis resulted in the identification of three endometrial antigens, tropomyosin 3 (TPM3), stomatin-like protein 2 (SLP2) and tropomodulin 3 (TMOD3). Serum levels of antibodies against the epitopes from the immunodominant region of proteins TPM3, SLP2 and TMOD3 were significantly elevated in endometriosis patients when compared with controls. Sensitivity and specificity of serum anti-TPM3a-autoAb (61%, 93%), anti-TPM3c-autoAb (44%, 93%), anti-TPM3d-autoAb (78%, 89%), anti-SLP2a-autoAb (50%, 96%), anti-SLP2c-autoAb (61%, 93%), anti-TMOD3b-autoAb (61%, 96%), serum anti-TMOD3c-autoAb (78%, 93%) and anti-TMOD3d-autoAb (78%, 96%) were better than those of serum CA125 levels (21%, 89%) in the detection of early stages of endometriosis. CONCLUSIONS Serum anti-TPM3a-autoAb, anti-TPM3c-autoAb, anti-TPM3d-autoAb, anti-SLP2a-autoAb, anti-SLP2c-autoAb, anti-TMOD3b-autoAb, anti-TMOD3c-autoAb and anti-TMOD3d-autoAb could be new markers for the early diagnosis of endometriosis.

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.

Spot-ting differences between the ectopic and eutopic endometrium of endometriosis patients

OBJECTIVE To examine proteins aberrantly expressed in the ectopic endometrium compared with eutopic endometrium from the same patient. DESIGN Experimental study. SETTING Research institute and an obstetrics and gynecology clinic (National Institute for Research in Reproductive Health and Sanjeevani Diagnostic Center and Maternity Home, India). PATIENT(S) Twenty participants with (n=11) and without (n=9) endometriosis. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Protein identification by two-dimensional (2D) electrophoresis and mass spectrometry as well as validation of the identified proteins by studying protein expression via Western blot and protein localization via immunohistochemical analysis. RESULT(S) Computer-assisted image analysis detected the presence of 53 protein spots in ectopic 2D gels that were conspicuous by their absence in the 2D maps of eutopic and control endometrium, i.e., these spots were detected only in ectopic gels. Eleven spots were identified by mass spectrometry. The expression of four of these proteins-haptoglobin, Rho-GDIα, SM-22α, and Rab37-have been validated by immunohistochemical and Western blotting analysis. CONCLUSION(S) This study assumes significance, as there are no reports on the comparison of the global protein profiles of paired eutopic and ectopic endometrium. Furthermore, the study demonstrates a definitive difference in the protein repertoire of the ectopic endometrium compared with its uterine counterpart in the same patient. Such studies are relevant in deciphering the complex biology of the endometriotic lesion.

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 β-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.