Identification and validation of candidate biomarkers involved in human ovarian autoimmunity  Purvi V. Mande, Firuza R. Parikh, Indira Hinduja, Kusum Zaveri, Rama Vaidya, Rahul Gajbhiye, Vrinda V. Khole  Reproductive BioMedicine Online  Volume 23, Issue 4, Pages 471-483 (October 2011) DOI: 10.1016/j.rbmo.2011.06.013 Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 (A) Bar graph showing percentage of ovarian autoantigens targeted by the sera of women of reproductive age undergoing IVF (group I) and women with idiopathic premature ovarian failure (group II), using data obtained from Western blot analysis. (B) Coomassie blue-stained sodium dodecyl sulphate polyacrylamide gel electrophoresis profile of total rat ovarian protein lysate, showing molecular size of autoantigens (1–5: 45, 80, 90, 97 and 120kDa, respectively). (C) Representative Western blot of immunodominant autoantigens in rat whole ovarian extract using sera from anti-ovarian antibody-positive patients, targeting 45, 80, 90, 97 and 120kDa antigens (lanes 1–5). Proven fertile control (lane NC1) and no primary antibody control (lane NC2) did not show any reactivity to any antigenic target. Albumin is seen as a 66-kDa band. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control. Reproductive BioMedicine Online 2011 23, 471-483DOI: (10.1016/j.rbmo.2011.06.013) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 (A) Representative immunohistochemistry showing immunoreactivity of anti-ovarian antibody-positive patient sera to oocyte ooplasm of secondary follicle and to zona pellucida (1), antral follicle (2), Graafian follicle (3), theca (4), corpus luteum (5) and granulosa (6), indicated with black arrowheads. Length of the bar is 1cm. Magnification is same for all panels and a representative bar is inserted in the first panel. Sera from healthy control women (7) and no primary antibody control (8) showed no immunoreactivity to any cell type. Scale bar represents=140μm for all panels. (B) Bar graph showing the frequency of different cell types in rat ovarian tissue targeted by antibodies from the sera of patients from groups I and II. Reproductive BioMedicine Online 2011 23, 471-483DOI: (10.1016/j.rbmo.2011.06.013) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 (A) Representative silver-stained gel of the eluted proteins PM45 (lane 1), PM80 (lane 2), PM97 (lane 3) and PM120 (lane 4). (B) Western immunoblots showing reactivity of patients’ sera with eluted proteins, showing five different 45-kDa-positive patient sera reactive to eluted PM45 protein (panel a, lanes 1–5), 80-kDa-positive patient sera reactive to eluted PM80 protein (panel b, lanes 1–5), 97-kDa-positive patient sera reactive to eluted PM97 protein (panel c, lanes 1–5) and 120-kDa-positive patient sera reactive to eluted PM120 protein (panel d, lanes 1–5). Sera from control women (panels a, b, c and d, lane 6) showed no reactivity to any of the eluted proteins. Reproductive BioMedicine Online 2011 23, 471-483DOI: (10.1016/j.rbmo.2011.06.013) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 (A) Western blot analysis of rat total ovarian lysate (lane 1) and eluted proteins (lane 2) using: anti-β-actin monoclonal antibody (a), anti-heat shock 70 protein 5 polyclonal antibody (b) and anti-α-actinin 4 polyclonal antibody (c). Myeloma culture supernatant (lane 3) served as negative control and showed no reactivity to any of the eluted proteins. (B) Representative Western blots showing reactivity of patients’ sera with: recombinant glutathione-S-transferase (GST)-tagged β-actin (lane a3); recombinant GST-tagged heat shock 70 protein 5 (lane b3); and recombinant GST-tagged α-actinin 4 (lane c3). A no primary antibody control (lane 1) and proven fertile control sera (lane 2) showed no immunoreactivity to any of the recombinant proteins. (C) Representative immunofluorescent and immunohistochemical localization of autoimmune targets in the ovarian section using: anti-β-actin antibody with ooplasm, granulosa and theca, as indicated with white arrowheads (panels a2 and a3); anti-heat shock 70 protein 5 antibody with ooplasm, theca, granulosa and corpus luteum, as indicated with black arrowheads (panels b2 and b3); and anti-α-actinin 4 antibody with ooplasm, theca and corpus luteum, as indicated with white arrowheads (panels c2 and c3). Myeloma culture supernatant serving as negative control did not show reactivity to any of the rat ovarian proteins (panels a1, b1 and c1). Scale bar represents=40μm for all panels. Length of the bar is 1cm. Green=positive stain; blue=counter stain. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Reproductive BioMedicine Online 2011 23, 471-483DOI: (10.1016/j.rbmo.2011.06.013) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Bar diagram showing the reactivity of group I (black box), group II (red box) patients’ sera and control sera (blue box) against recombinant α-actinin 4 (ACTN4), heat shock 70 protein 5 (HSPA5) and β-actin (ACTB). The optical densities (OD) of group I and group II sera were significantly higher (all P<0.0001) than the control sera. Reproductive BioMedicine Online 2011 23, 471-483DOI: (10.1016/j.rbmo.2011.06.013) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 6 Immunohistochemical localization of autoimmune targets in ovary from human (A) and rat (B) using patients’ sera targeting 45kDa (β-actin, panels A2, B2), 80kDa (heat shock 70 protein 5, panels A3, B3) and 97kDa (α-actinin 4, panels A4, B4). Control sera (panels A1, B1) did not show any reactivity to the human and rat ovaries. Scale bar represents=140μm for all panels. Length of the bar is 1cm. Reproductive BioMedicine Online 2011 23, 471-483DOI: (10.1016/j.rbmo.2011.06.013) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 7 Expression pattern of α-actinin 4 in rat somatic tissues: lung (1), thyroid (2), muscle (3), adrenal (4), epididymis (5), testis (6), gastric mucosa (7), brain (8), kidney (9), liver (10), pancreas (11), thymus (12), heart (13), spleen (14) and kidney (15), as indicated with black arrowheads. α-Actinin 4-positive sera only showed reactivity to kidney (15). No reactivity to any of the somatic tissues tested was observed using β-actin-positive and heat shock 70 protein 5-positive sera and control sera (data not shown). Scale bar represents=40μm for all panels. Length of the bar is 1cm. Reproductive BioMedicine Online 2011 23, 471-483DOI: (10.1016/j.rbmo.2011.06.013) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions

Figure 8 Immunohistochemical localization of autoimmune targets in rat ovary during ontogeny using patients’ sera targeting β-actin (a), heat shock 70 protein 5 (b) and α-actinin 4 (c) proteins in day 0 (1), day 10 (2), day 20 (3) and day 30 ovaries (4), as indicated with black arrowheads. Control sera showed no reactivity to any cell types at any stage of development (d). Scale bar represents=140 μm for all panels. Length of the bar is 1cm. All the panels are of the same magnification and a representative bar has been inserted in the panel 1. Reproductive BioMedicine Online 2011 23, 471-483DOI: (10.1016/j.rbmo.2011.06.013) Copyright © 2011 Reproductive Healthcare Ltd. Terms and Conditions