Estradiol protects clomiphene citrate–induced apoptosis in ovarian follicular cells and ovulated cumulus–oocyte complexes Shail K. Chaube, Ph.D., Pramod.

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Estradiol protects clomiphene citrate–induced apoptosis in ovarian follicular cells and ovulated cumulus–oocyte complexes Shail K. Chaube, Ph.D., Pramod V. Prasad, M.Sc., Sonu C. Thakur, Ph.D., Tulsidas G. Shrivastav, Ph.D. Fertility and Sterility Volume 84, Pages 1163-1172 (October 2005) DOI: 10.1016/j.fertnstert.2005.03.073 Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Ovarian histology. (A) Control ovary showing ruptured follicle after superovulation induction (arrows). (B) Ovary treated with 10 mg of clomiphene citrate (CC) per kilogram of body weight showing unruptured follicles in ovary (arrows). (C) Ovary treated with 10 mg of CC per kilogram of body weight and 2.0 mg of E2 per kilogram of body weight showing ruptured follicles in the ovary (arrows; magnification, ×100). Chaube. Estradiol protects clomiphene citrate-induced apoptosis in rats. Fertil Steril 2005. Fertility and Sterility 2005 84, 1163-1172DOI: (10.1016/j.fertnstert.2005.03.073) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Fragmentation of DNA in ovarian follicles. (A) TUNEL-negative staining in granulosa cells of control ovary. (B) TUNEL-positive staining in granulosa cells of animal ovary treated with 10 mg of CC per kilogram of body weight (arrows). (C) TUNEL-negative staining in granulosa cells of animal ovary treated with 10 mg of CC per kilogram of body weight and 2.0 mg of E2 per kilogram of body weight (magnification, ×400). Panel C shows greener color because of overexposure to methyl green (a counterstain). Chaube. Estradiol protects clomiphene citrate-induced apoptosis in rats. Fertil Steril 2005. Fertility and Sterility 2005 84, 1163-1172DOI: (10.1016/j.fertnstert.2005.03.073) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Fragmentation of DNA in ovulated cumulus oocyte complexes (COCs). (A) TUNEL-negative staining in control COCs. (B) TUNEL-positive staining in COCs collected from animals treated with 10 mg of clomiphene citrate per kilogram of body weight. (C) TUNEL-negative staining in COCs collected from animals treated with 10 mg of CC per kilogram of body weight and 2.0 mg of E2 per kilogram of body weight (magnification, ×100). (D–F) Higher magnifications (×400) of panels A–C; respectively. To avoid further loss of COCs from slides from the repeated washing, slides for COCs were not stained with methyl green; however, histological slides were stained with methyl green because of tight attachment. Panel E shows TUNEL-positive staining in cumulus cells and associated oocyte. The faint brown color of the associated oocyte cytoplasm is caused by the large size of the oocyte and its haploid DNA content. The color of the TUNEL stain is dependent on the cell size and its compactness. Chaube. Estradiol protects clomiphene citrate-induced apoptosis in rats. Fertil Steril 2005. Fertility and Sterility 2005 84, 1163-1172DOI: (10.1016/j.fertnstert.2005.03.073) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Representative photograph showing membrane blebbing in oocytes isolated and cultured in vitro for 1 hour. (A) Control oocytes with normal morphology with first polar body. (B) Oocytes collected from animals treated with 10 mg of clomiphen citrate (CC) per kilogram of body weight showing membrane blebbing (arrows). (C) Oocytes collected from animals treated with 10 mg of CC per kilogram of body weight and 2.0 mg of E2 per kilogram of body weight (magnification, ×400). Chaube. Estradiol protects clomiphene citrate-induced apoptosis in rats. Fertil Steril 2005. Fertility and Sterility 2005 84, 1163-1172DOI: (10.1016/j.fertnstert.2005.03.073) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 5 Western blot analysis of bax protein (corresponding to 21 kDa) in ovaries (A) and cumulus oocyte complexes (COCs) (B). C = control; CC = Clomiphene citrate treated; CC+E2 = CC+E2 treated. Lower portion shows a control assay for β-actin protein (corresponding to 45 kDa) to confirm that the equivalent amounts of protein were analyzed. Chaube. Estradiol protects clomiphene citrate-induced apoptosis in rats. Fertil Steril 2005. Fertility and Sterility 2005 84, 1163-1172DOI: (10.1016/j.fertnstert.2005.03.073) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions