Androstenedione induces abnormalities in morphology and function of developing oocytes, which impairs oocyte meiotic competence  Wataru Tarumi, M.Sc., Sanae Tsukamoto, Yuki Okutsu, M.D., Noriyuki Takahashi, Ph.D., Toshitaka Horiuchi, Ph.D., Masanori T. Itoh, Ph.D., Bunpei Ishizuka, M.D.  Fertility and Sterility  Volume 97, Issue 2, Pages 469-476 (February 2012) DOI: 10.1016/j.fertnstert.2011.11.040 Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Early secondary follicle develops to the preovulatory stage by the single follicle culture system, and thereafter hCG/EGF treatment induces the expansion of cumulus cells. The follicles were cultured without androstenedione. (A) An early secondary follicle (diameter, 100 μm) immediately after isolation from a 14-day-old mouse ovary. White and black arrows indicate a GV-stage oocyte and granulosa cell layers, respectively. (B) A follicle (diameter, 350 μm) cultured for 6 days. The granulosa cells proliferated. (C) A follicle (diameter, 600 μm) cultured for 12 days. Antrum (black arrow) was expanded, and cumulus cells (white arrow) were visible. (D) A cumulus-oocyte complex isolated at 16 hours after hCG/EGF treatment. The expansion of cumulus cells (black arrow) was seen. Scale bars, 100 μm. Fertility and Sterility 2012 97, 469-476DOI: (10.1016/j.fertnstert.2011.11.040) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Androstenedione reduces survival rates of follicles, prompts the formation of follicles with abnormal morphology, and increases E2 and P secretion in follicles. (A) Changes in diameters of follicles and oocytes and in survival rates of follicles. All values are expressed as mean ± SEM (n = 12–20 follicles/group). ∗P<.05 and ∗∗P<.01 vs. controls. (B–D) More than 20% of androstenedione-treated follicles exhibited morphologic abnormalities as follows: after 8 days of culture (B and C), a follicle in which cumulus and mural granulosa cells were lacking and (D) an oocyte with abnormal morphology, which was probably in the course of degeneration. Panel C is a higher magnification view of the area boxed in panel B. Scale bars, 50 μm. (E) Changes in E2 and P concentrations in media of individually cultured follicles. All values are expressed as mean ± SEM (n = 4 or 5/group). ∗P<.05 and ∗∗P<.01 vs. controls. Fertility and Sterility 2012 97, 469-476DOI: (10.1016/j.fertnstert.2011.11.040) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Androstenedione prevents the change of chromatin configuration in oocytes of in vitro–grown follicles and reduces oocyte GDF9 expression. (A–C) When chromatin was stained with Hoechst 33342, chromatin configuration in oocytes of individually cultured follicles was divided into two types: (A) chromatin distributed diffusely around the nucleolus (NSN) and (B) chromatin compacted in a ring around the nucleolus (SN). Arrows indicate the nucleolus of oocytes. Scale bars, 10 μm. (C) Percentages of oocytes (n = 10–24/group) in NSN and SN types at days 4, 8, and 12 of culture with or without androstenedione (10−5 M). (D) Relative GDF9 expression (normalized to β-actin) in the oocytes of follicles cultured with or without androstenedione (10−5 M), which were determined by real-time RT-PCR. All values are expressed as mean ± SEM (n = 7–11/group). Data are normalized to controls at day 4. ∗P<.05 and ∗∗P<.01. Fertility and Sterility 2012 97, 469-476DOI: (10.1016/j.fertnstert.2011.11.040) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Androstenedione inhibits oocyte maturation and prevents spindle assembly and chromosome alignment in oocytes. Follicles were individually cultured for 12 days and thereafter were treated with hCG/EGF for 16 hours. As shown in panels A–C, during this period, the normal oocyte changed from GV stage via GVBD stage to the metaphase II (MII) stage. The arrow in the image of panel C shows a first polar body. Scale bars, 20 μm. The percentages of each stage were estimated in the follicles cultured with or without androstenedione (10−11, 10−9, and 10−5 M). All values are expressed as mean ± SEM (n = 12–20/group). ∗P<.05 and ∗∗P<.01 vs. controls. (D–J) Spindles and chromosomes in oocytes. At 0, 8, and 16 hours after hCG/EGF treatment, spindles were detected using immunohistochemistry for α-tubulin (green), and chromatin and chromosomes were stained with Hoechst 33342 (dark blue). (D–F) Oocytes in control follicles and (G–J) oocytes in 10−5 M androstenedione-exposed follicles. (J) Higher magnification view of the area boxed in panel I. Scale bars, 10 μm. Fertility and Sterility 2012 97, 469-476DOI: (10.1016/j.fertnstert.2011.11.040) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions