Effects of testosterone, dihydrotestosterone, and 17β-estradiol on human ovarian tissue survival in culture  Marjut Otala, M.Sc., Sirpa Mäkinen, M.Sc., Timo Tuuri, Ph.D., Jari Sjöberg, M.D., Ph.D., Virve Pentikäinen, M.D., Ph.D., Tiina Matikainen, M.D., Ph.D., Leo Dunkel, M.D., Ph.D.  Fertility and Sterility  Volume 82, Pages 1077-1085 (October 2004) DOI: 10.1016/j.fertnstert.2004.06.032 Copyright © 2004 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Immunohistochemical identification of human ovarian cells containing the androgen receptor (AR). Fresh human ovarian cortical tissue was fixed in 4% formalin and cut into 5μm serial sections. Immunohistochemical analysis was performed with an antibody raised against the N-20 end of the AR as described in Materials and Methods, and the sections were counterstained with hematoxylin. A, Some interstitial cells in the stroma contained nuclear ARs (double arrow). A few of the granulosa cells in some primordial follicles also had ARs in their nuclei (arrows). B, More granulosa cells with AR were detected in primary follicles (arrows). C, Stromal cells containing ARs (double arrow) were usually observed individually, but occasionally small clusters of AR-expressing cells could be found. D, Negative control in which the primary antibody was replaced with nonspecific rabbit IgG. Original magnification, ×400 (A, B, and D) and ×1,000 (C). Fertility and Sterility 2004 82, 1077-1085DOI: (10.1016/j.fertnstert.2004.06.032) Copyright © 2004 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Ovarian apoptosis in tissue cultured for 24 hours. A, Ovarian cortical tissue slices were cultured in serum-free medium, after which DNA was extracted, 3′-end-labeled with Dig-dd-UTP, and subjected to electrophoresis on 2% agarose gels. Immediately after excision (0 hours), the DNA samples showed no apoptotic ladder pattern, whereas after incubation for 24 hours a clear apoptotic ladder pattern was seen in Southern blot analysis. B, Low molecular weight DNA (<1.3 kb) quantification (Scion Image Analyzing System) showed clear induction of apoptosis in the 24-hour cultures. This induction of apoptosis was approximately threefold. Each value represents the mean of a number of independent experiments ± SEM. ***P<.001. The 24-hour time point values were compared with those at the 0-hour time point, and the mean value of the 24-hour cultures was set at 1.0 to depict 100% apoptosis. C, The upper panels show formalin-fixed, hematoxylin-eosin–stained ovarian stromal tissue. Uncultured tissue (0 hours) with normal morphology and cultured tissue (24 hours) with normal and pycnotic (double arrows) cells. The lower panels show in situ 3′-end labeling (ISEL) of apoptotic DNA in ovarian tissue (lower panels). For ISEL analysis, ovarian tissue was fixed and cut into serial sections. Apoptotic cells were detected by ISEL of apoptotic DNA. No apoptotic cells could be identified in the uncultured tissue (0 hours), whereas several apoptotic interstitial cells (double arrow) were often observed in the cultured tissue (24 hours). Sometimes, although quite rarely, apoptotic granulosa cells were also found (arrow). Original magnifications, ×1,000 (histology) and ×400 (ISEL). Fertility and Sterility 2004 82, 1077-1085DOI: (10.1016/j.fertnstert.2004.06.032) Copyright © 2004 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 The effects of T on ovarian tissue apoptosis. Thin slices of ovarian tissue were incubated in the absence or presence of T. The degree of apoptotic laddering in cultures containing various amounts of T was examined by Southern blot analysis and quantified by using the Scion Image Analyzing System. Low molecular weight DNA (<1.3 kb) fragmentation in ovarian tissue cultures in the presence of T was suppressed by 26% at a T concentration of 10−9 mol/L and by 25% at 10−8 mol/L compared with tissue cultured without T. The lowest and highest concentrations of T had very little effect on the tissue. Values are mean ± SEM. NS = not significant, *P<.05. Fertility and Sterility 2004 82, 1077-1085DOI: (10.1016/j.fertnstert.2004.06.032) Copyright © 2004 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Inhibition of in vitro–induced human ovarian tissue apoptosis by DHT. Ovarian tissue slices were incubated under serum-free conditions in the presence or absence of DHT. Southern blot analysis, quantification of low molecular weight DNA fragmentation, and ISEL of apoptotic DNA were performed as described in Figure 2. Immunolocalization of Ki-67 was performed by using a rabbit polyclonal antibody to human Ki-67. A, DHT inhibited apoptotic DNA laddering by 37% at 10−7 mol/L, by 31% at 10−8 mol/L, by 25% at 10−9 mol/L, and by 30% at 10−10 mol/L. B, Numerous apoptotic cells (arrows) were detected in the untreated tissue, and individual apoptotic cells were also found in the tissue treated with DHT (upper panels). The overall morphology of the control and the treated tissue was similar. Little or no stromal cell proliferation could be detected in the control tissue or tissue treated with DHT (lower panels), although occasional granulosa cells in both groups seemed to be in the process of cell division (arrow). Original magnification, ×400. C, The AR antagonist casodex (CX) effectively blocked the anti-apoptotic effect of DHT. NS = not significant, *P<.05; **P<.01. Fertility and Sterility 2004 82, 1077-1085DOI: (10.1016/j.fertnstert.2004.06.032) Copyright © 2004 American Society for Reproductive Medicine Terms and Conditions

FIGURE 5 The effects of 17β-estradiol on ovarian tissue apoptosis. Tissue culture and low molecular weight DNA detection and quantification were performed as in Figure 2. 17β-estradiol was not able to suppress ovarian tissue apoptosis in the cultures. Fertility and Sterility 2004 82, 1077-1085DOI: (10.1016/j.fertnstert.2004.06.032) Copyright © 2004 American Society for Reproductive Medicine Terms and Conditions

FIGURE 6 Localization of the AR in cultured granulosa cells obtained from immature antral follicles and the effect of DHT on the apoptosis of these cells. (A) Individual granulosa cells were analyzed immunohistochemically using rabbit polyclonal antibody to human AR (N-20) as described in Materials and Methods. The left-hand side shows the negative control in which the primary antibody was replaced with PBS. The right-hand side shows strong staining in the granulosa cells. Original magnification, ×1,000. (B) Granulosa cells were cultured in serum-free conditions for 24 hours with or without DHT. DNA was extracted from the granulosa cells and subjected to Southern blotting. (C) Quantification of the low molecular weight DNA was performed as described in the legend of Figure 2. NS = not significant. Fertility and Sterility 2004 82, 1077-1085DOI: (10.1016/j.fertnstert.2004.06.032) Copyright © 2004 American Society for Reproductive Medicine Terms and Conditions