A block in the road to fertility: autoantibodies to heat-shock protein 90-β in human ovarian autoimmunity  Eusebio S. Pires, Ph.D., Vrinda V. Khole, Ph.D.  Fertility and Sterility  Volume 92, Issue 4, Pages 1395-1409 (October 2009) DOI: 10.1016/j.fertnstert.2008.08.068 Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A) Pie chart showing percentage of predominance of patient sera from both the study groups identifying an immunoreactive band at a 90-kd locus where 46.66% (7 out of 15) of patients for antiovarian antibodies (AOA) in the premature ovarian failure (POF) group and 47% (8 out of 17) of patients positive for AOA in the IVF group made antibodies to this protein. (B) Representative Western blot using sera from control and AOA-negative (lanes C1 to C5) patients showing no reactivity to any other ovarian antigen apart from the 66-kd albumin locus and specific reactivity to 90-kd protein by AOA 90-kd–positive sample (lanes P1 to P7). A “no primary” control (lane NC) showed no immunoreaction to any of the ovarian proteins. (C) Representative immunohistochemistry showing immunoreactivity of 90-kd sera toward the ooplasm of the (A) primordial follicles, (B) secondary follicle, (C) antral follicle, (D) Graafian follicle, (E) interstitium of the corpora lutea, and (F) to the thecal cells. None of the controls or AOA-negative patients showed any reactivity to any other cell type of the ovary. (Magnification: A, E ×100; B, C, F ×40; D ×20). Fertility and Sterility 2009 92, 1395-1409DOI: (10.1016/j.fertnstert.2008.08.068) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 2 The 90-kd protein was located on a 1D Coomassie-stained SDS-PAGE. The corresponding band was cut from multiple gels. (A) Lane 1: A closely associated 97-kd band cut to differentiate between the two. Lane 2: PFK84 phosphofructokinase 84-kd prestained marker used as a landmark. (B) The protein from the gel was then electroeluted, and the homogeneity of the protein was checked by rerunning the protein on a silver-stained SDS-PAGE. Lanes 3, 4: Homogeneity of 97-kd and 90-kd eluted proteins, respectively. Lane 5: Aligned lane with molecular-weight marker. (C) Lanes E1–E5: Reactivity of the 90-kd–positive sera and the eluted protein EP90. Lanes RO, ELU: Coomassie-stained gel profile of total rat ovarian protein and eluted 90-kd protein, respectively. Lane E6: Control sera showing no immunoreactivity. Fertility and Sterility 2009 92, 1395-1409DOI: (10.1016/j.fertnstert.2008.08.068) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (A) Panel showing the glycosylation status of EP90 protein using a commercially available kit to detect proteins that are glycosylated. A1: No obvious pink stain at the desired locus, indicating no glycosylation (lane EP90). A2: Respective silver stained gel of the same experimental set. (B) Western blot analysis where eluted EP90 was probed with phosphoserine/threonine and tyrosine monoclonal antibodies. EP90 is phosphorylated only at the serine and threonine. Fertility and Sterility 2009 92, 1395-1409DOI: (10.1016/j.fertnstert.2008.08.068) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 4 (A) Immunohistochemistry showing the expression of EP90 in development right from (1) day-0 ovary, (3) day-10 ovary, (5) day-20 ovary to (7) matured rat ovary of day 45. Note that EP90-positive sera clearly show strong immunoreactivity to the oocyte ooplasm and theca (red arrows). (2, 4, 6, 8) Control sera showing no reactivity to any cell types in any of the ovarian sections in development. (B) Confocal images of the expression of EP90 in embryogenesis showing immunostaining in the (1) cytoplasm of unfertilized oocyte, (2) germinal vesicle breakdown oocyte (GVBD), (3) two-cell embryo, (4) morula, and (5) blastocyst. Nucleus was counterstained with propidium iodide. Using secondary alone control, there was no immunoreactivity to (6) unfertilized oocyte or (7) morula. (Magnification: ×640). Fertility and Sterility 2009 92, 1395-1409DOI: (10.1016/j.fertnstert.2008.08.068) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 5 Expression of EP90 across the species. (A) Western blot analysis using (P) one patient's sera (P) shows reactivity in rat ovary (RO), commercially available human ovary (HO), and pig ovary (PO) at the 90-kd locus. The control sera (C) showed no reactivity apart from the 66-kd locus in any of the ovarian preparations. (B) Immunohistochemistry of expression of EP90 in the oocyte ooplasm in (1.2) monkey ovarian (MO) section and (2.2) rabbit ovarian (RbO) section with reactivity to theca and some stromal reactivity as indicated by red arrows. The control sera to (1.1) monkey and (2.1) rabbit ovarian sections showed no reactivity. Fertility and Sterility 2009 92, 1395-1409DOI: (10.1016/j.fertnstert.2008.08.068) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 6 (A) Eluted rat ovarian EP90 protein sequence from 1D Coomassie-stained gel processed for liquid chromatography mass spectrophotometry (LC/MS). The bold-underline peptide sequences in indicate fragments obtained after tryptic digestion. The peptide fragments obtained matched to human heat-shock protein 90-beta (HSP90β). (B, C) Specificity of the 90-kd sera in picking up three spots in a 2D SDS-PAGE Western blot and its location on a silver-stained gel, respectively. (D) Cored and microsequenced spots by MALDI TOF-TOF. The bold-underline peptide sequences indicate fragments obtained after tryptic digestion. The peptide fragments obtained matched to human heat-shock protein 90-beta (HSP90β). Fertility and Sterility 2009 92, 1395-1409DOI: (10.1016/j.fertnstert.2008.08.068) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 7 (A) Western blot using eluted EP90 probed with (1) antibody to HSP90, (2) SP2/0 culture supernatant negative control, and (3) antibody to β-catenin (unrelated protein). Only lane 1 shows immunoreactivity. (B) (1) Immunoreactivity of the mAb to HSP90 to the ooplasm of the oocyte. No staining in the cumulus granulosa cells was seen. (2) A SP2/0 culture supernatant serving as negative control showed no immunostaining. (C) (1) Western blot analysis using recombinant HSP90 transferred on nitrocellulose membrane and probed with randomly selected 90-kd positive sera (P1–P8). (2) Representative lane indicating no immunoreactivity with control or AOA-negative sera. A “no primary control” (NPC) showed no immunoreactivity. A monoclonal antibody to human HSP90 was used as positive control (+ve). (D) Status of immunoreactivity of all 90-kd–positive sera in ELISA coated with recombinant HSP90 protein. Q1 to Q4 represents quartiles where the 90-kd patients were segregated into groups of approximately four patients in order of increasing absorbance values. C represents absorbance values obtained from controls. The 4/15 EP90-positive sera show absorbance readings in the range observed with sera from controls, therefore now termed as true HSP90-negative sera (circles). Fertility and Sterility 2009 92, 1395-1409DOI: (10.1016/j.fertnstert.2008.08.068) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions