1. Exogenous androstenedione induces formation of follicular cysts and premature luteinization of granulosa cells in the ovary 
Yuki Okutsu, M.D., Masanori T. Itoh, Ph.D., Noriyuki Takahashi, Ph.D., Bunpei Ishizuka, M.D. 
Fertility and Sterility 
Volume 93, Issue 3, Pages (February 2010)
DOI: /j.fertnstert
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Ovarian morphology in control (A–C) and androstenedione-treated (D–G) rats. (F and G) Ovaries without and with corpus luteum (CL), respectively. In E–G, a follicular cyst (FC) was seen. Prepubertal (21 days of age) rats were injected daily with androstenedione or vehicle only. Ovaries were obtained on days 7, 15, or 20 of treatments, and ovarian sections were stained with hematoxylin-polychrome EA50. Representative sections are shown. Scale bars: 500 μm.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
(A–C) Ovarian follicular cyst induced by androstenedione-treatments. (A) FC = follicular cyst; AF = antral follicle, which was probably in the course of cyst formation. (B and C) Higher magnification views of the areas boxed in A. Arrows in C indicate macrophages. (D–G) TUNEL staining in androstenedione-treated rat ovaries. (E and G) Higher magnification views of the areas boxed in D and F. (D and E) Large antral follicle in androstenedione-treated rat ovary. Apoptotic cells (arrows in E) were detected in the inner part of granulosa cell layers. (F and G) Follicular cyst obtained from androstenedione-treated rat ovary, in which few apoptotic cells were detected. Ovaries were obtained from rats on day 20 of androstenedione-treatments. The A–C sections were stained with hematoxylin-polychrome EA50, and the D–G sections were counterstained with Mayer's hematoxylin. Representative sections are shown. Scale bars: 150 μm for A, D, and F; 75 μm for B and C; 60 μm for E and G.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
(A and B) Immunohistochemistry for P450arom in control (A) and androstenedione-treated (B) rat ovaries. P450arom immunoreactivity was localized to the granulosa cells of antral follicles in control and androstenedione-treated rat ovaries, with levels reduced in androstenedione-treated rats, as compared with controls. FC = follicular cyst; AF = antral follicle. (C–F) Immunohistochemistry for P450scc in control (C and D) and androstenedione-treated (E and F) rat ovaries. (D and F) Higher magnification views of the areas boxed in C and E. P450scc immunoreactivity was detected in the granulosa cells of antral follicles in androstenedione-treated rats. (G–J) Immunohistochemistry for p27Kip1 in control (G and H) and androstenedione-treated (I and J) rat ovaries. (H and J) Higher magnification views of the areas boxed in G and I. The p27Kip1 immunoreactivity was detected in the granulosa cells of antral follicles in androstenedione-treated rats. Ovaries were obtained on day 20 of treatments. Sections were counterstained with Mayer's hematoxylin. Representative sections are shown. Scale bars: 150 μm for A, B, C, and E; 300 μm for G and I; 50 μm for D, F, H, and J. (K) Serum levels of T, E2, and P. All values are expressed as mean ± SEM. The number of animals assessed is shown in the parentheses that are located above each bar. Statistically significant differences between the androstenedione-treated rats and matched controls are indicated by the asterisks (P<.05).
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

5. Figure 4
Transmission electron microscopy analysis for granulosa cells in control (A and C) and androstenedione-treated (B and D) mouse follicles, which were cultured for 10 days in the absence or presence of androstenedione. In B, a number of lipid droplets were seen throughout the cytoplasm. In D, numerous mitochondria with complex tubular cristae were also seen. Representative micrographs are shown with original magnifications of ×4000 for A and B and ×20,000 for C and D.
Fertility and Sterility  , DOI: ( /j.fertnstert )
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6. Figure 5
Ovarian follicular cyst formation hypothesis. In the normal ovary, the antral follicle matures, and ovulation subsequently occurs. After ovulation, corpus luteum is formed (luteinized cells are shown in light red). In the androstenedione-treated ovary, the antral follicle is also formed. However, in this case, granulosa cells in the antral follicle are luteinized and apoptosis occurs from the inner part of the granulosa cell layers, which results in the formation of follicular cyst. Furthermore, we demonstrate that the oocyte is degenerated and lost during the course of the formation of the follicular cyst. It has been suggested that the loss of oocyte-granulosa cell communication stimulates premature luteinization in granulosa cells. The follicular cyst has flattered epithelioid cells facing the antrum. In addition, the follicular cyst is not atretic.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions