Jong Yeob Choi, Ph. D. , Min Wha Jo, M. S. , Eun Young Lee, M. S

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The role of autophagy in follicular development and atresia in rat granulosa cells Jong Yeob Choi, Ph.D., Min Wha Jo, M.S., Eun Young Lee, M.S., Byung-Koo Yoon, M.D., Ph.D., Doo Seok Choi, M.D., Ph.D. Fertility and Sterility Volume 93, Issue 8, Pages 2532-2537 (May 2010) DOI: 10.1016/j.fertnstert.2009.11.021 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Immunolocalization of LC3 protein in rat ovarian follicles at different stages of development: (A) primordial, (B) primary, (C) preantral, (D) early antral, (E) medium antral, and (F) large antral follicles. GC, granulosa cells; TC, thecal cells; O, oocytes. Magnification ×400. Fertility and Sterility 2010 93, 2532-2537DOI: (10.1016/j.fertnstert.2009.11.021) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 The expression of cleaved caspase-3 and LC3-II proteins in granulosa cells from immature rats at different times (0–5 days) after pregnant mare serum gonadotropin injection (15 IU, intraperitoneally). (A, B) Densitometric quantification (top) and representative immunoblots (bottom) of cleaved caspase-3 and LC3 proteins. Experiments were repeated three times, and data were expressed as mean ± standard error. ∗P<.05, statistically significant. (C) Correlation between LC3 and cleaved caspase-3 protein expression. Fertility and Sterility 2010 93, 2532-2537DOI: (10.1016/j.fertnstert.2009.11.021) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Colocalization of cleaved caspase-3 and LC3 proteins on adjacent sections of (A, B) healthy and/or (C, D) atretic early (I) and medium (II) antral follicles from immature rats 0–5 days after pregnant mare serum gonadotropin injections (15 IU, intraperitoneally). GC, granulosa cells; TC, theca cells. Magnification ×200. Fertility and Sterility 2010 93, 2532-2537DOI: (10.1016/j.fertnstert.2009.11.021) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Hormone dependency of cleaved caspase-3 and LC3 protein expression in cultured rat granulosa cells after incubation under serum-free conditions in the absence or presence of FSH. (A, B) Densitometric quantification (top) and representative immunoblots (bottom) of cleaved caspase-3 and LC3 proteins. Experiments were repeated three times; data are expressed as mean ± standard error. ∗P<.05, statistically significant. (C) Immunofluorescence images of granulosa cells cultured in serum-free conditions. LC3-II was associated with punctate structures in granulosa cells cultured without FSH (top), and LC3-I had a diffuse expression pattern in granulosa cell cultured with FSH (bottom). (D) Transmission electron microscopic images of granulosa cells cultured without FSH (left) and with FSH (right). Arrows indicate representative autophagic vacuoles. N, nucleus. Fertility and Sterility 2010 93, 2532-2537DOI: (10.1016/j.fertnstert.2009.11.021) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions