Aberrant expression of apoptosis proteins and ultrastructural aberrations in uterine leiomyomas from patients with hereditary leiomyomatosis and renal cell carcinoma  Noel C. Wortham, M.Biochem., N. Afrina Alam, M.D., Ella Barclay, M.D., Patrick J. Pollard, Ph.D., Bart E. Wagner, B.Sc., Sanjiv Manek, M.D., George Elia, B.Sc., Ian P.M. Tomlinson, M.D.  Fertility and Sterility  Volume 86, Issue 4, Pages 961-971 (October 2006) DOI: 10.1016/j.fertnstert.2006.02.106 Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Immunohistochemical staining of sporadic and HLRCC leiomyomas, and normal myometrium. Representative images (original magnification: all 400×) for staining with antibodies where differences in staining were observed related to H&E-stained sections (A). Increases in expression of (B) Bcl-2 and (C) PCNA were observed in both sporadic and HLRCC leiomyomas compared with myometrium. Increased expression of Bcl-x (D) was observed in HLRCC leiomyomas, but not in sporadic leiomyomas. Wortham. Apoptosis in HLRCC uterine leiomyomas. Fertil Steril 2006. Fertility and Sterility 2006 86, 961-971DOI: (10.1016/j.fertnstert.2006.02.106) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Immunohistochemical staining for Bak. Representative images (original magnification: all 400×) for staining of Bak in HLRCC and non-HLRCC myometrium, and HLRCC and sporadic uterine leiomyomas. Decreased expression of Bak is observed in HLRCC myometrium compared with non-HLRCC myometrium. Decreased Bak expression is also observed in HLRCC leiomyomas compared with HLRCC myometrium. No changes are observed between sporadic leiomyomas and non-HLRCC myometrium. Wortham. Apoptosis in HLRCC uterine leiomyomas. Fertil Steril 2006. Fertility and Sterility 2006 86, 961-971DOI: (10.1016/j.fertnstert.2006.02.106) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Electron micrographs of myometrium, HLRCC, and sporadic leiomyomas. Low magnification of myometrium (A) indicates ordered structure, light nuclei, and a common alignment of cells. The HLRCC leiomyomas (B) display some breakdown of this order, and an apparent increased cell density. Sporadic leiomyomas (C) display islands of cells, with an extensive collagen matrix and no apparent order. Collagen structure also differs among the three tissue types: In myometrium (D), it is ordered with tight bundles, whereas in HLRCC leiomyomas (E) and sporadic leiomyomas (F), there is increasing evidence of breakdown of structure. Collagen fibers are more loosely packed, with little common orientation. Filament structures within the cells also demonstrate aberrations. Myofilaments and intermediate filaments in myometrium (G) are ordered and aligned to allow the cell to function efficiently. In the leiomyomas (H–K), however, the structure breaks down. Bundles of 8–12-nm filaments are scattered in the cell, forcing other organelles and cytoskeletal structures to the cell periphery. In HLRCC leiomyomas (H, I), these filaments form distinct bundles in the cytoplasm, whereas sporadic leiomyomas (J, K) have more diffuse spreading of the disordered filaments. Other observations included the presence of autophagosomes in some cells (L) and some alterations in mitochondrial morphology, including swelling (M). Scale bars are provided on all micrographs. Wortham. Apoptosis in HLRCC uterine leiomyomas. Fertil Steril 2006. Fertility and Sterility 2006 86, 961-971DOI: (10.1016/j.fertnstert.2006.02.106) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Electron micrographs of myometrium, HLRCC, and sporadic leiomyomas. Low magnification of myometrium (A) indicates ordered structure, light nuclei, and a common alignment of cells. The HLRCC leiomyomas (B) display some breakdown of this order, and an apparent increased cell density. Sporadic leiomyomas (C) display islands of cells, with an extensive collagen matrix and no apparent order. Collagen structure also differs among the three tissue types: In myometrium (D), it is ordered with tight bundles, whereas in HLRCC leiomyomas (E) and sporadic leiomyomas (F), there is increasing evidence of breakdown of structure. Collagen fibers are more loosely packed, with little common orientation. Filament structures within the cells also demonstrate aberrations. Myofilaments and intermediate filaments in myometrium (G) are ordered and aligned to allow the cell to function efficiently. In the leiomyomas (H–K), however, the structure breaks down. Bundles of 8–12-nm filaments are scattered in the cell, forcing other organelles and cytoskeletal structures to the cell periphery. In HLRCC leiomyomas (H, I), these filaments form distinct bundles in the cytoplasm, whereas sporadic leiomyomas (J, K) have more diffuse spreading of the disordered filaments. Other observations included the presence of autophagosomes in some cells (L) and some alterations in mitochondrial morphology, including swelling (M). Scale bars are provided on all micrographs. Wortham. Apoptosis in HLRCC uterine leiomyomas. Fertil Steril 2006. Fertility and Sterility 2006 86, 961-971DOI: (10.1016/j.fertnstert.2006.02.106) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Immunohistochemical staining of sporadic and HLRCC leiomyomas, and normal myometrium for the muscle intermediate filament protein desmin. Representative images (original magnification: all 400×) are related to H&E-stained sections. Desmin staining in myometrium (A) is diffuse and ordered, with apparent polarity aligned with the cell. Staining in HLRCC leiomyomas (B) displays distinct foci identical to those observed by electron microscopy (Fig. 3H, 3I) and less staining elsewhere in the cell. Staining in sporadic leiomyomas (C) is more diffuse, displaying some evidence of aggregation, but with no apparent polarity. Wortham. Apoptosis in HLRCC uterine leiomyomas. Fertil Steril 2006. Fertility and Sterility 2006 86, 961-971DOI: (10.1016/j.fertnstert.2006.02.106) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions