Fertility and Sterility

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Fertility and Sterility Extract of Azadirachta indica (neem) leaf induces apoptosis in rat oocytes cultured in vitro Shail K. Chaube, Ph.D., Pramod V. Prasad, Ph.D., Beena Khillare, Ph.D., Tulsidas G. Shrivastav, Ph.D. Fertility and Sterility Volume 85, Pages 1223-1231 (April 2006) DOI: 10.1016/j.fertnstert.2005.11.034 Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 The 10.0 mg/mL neem leaf extract (NLE)–induced morphologic apoptotic changes in oocytes cultured in vitro. (A) Control oocyte showing first polar body after 3.0 h of incubation (arrow). (B) NLE-treated oocyte undergoing shrinkage after 2.0 h (arrow). (C–F) NLE-treated oocyte showing progression of membrane leakage every after 15 min of interval up to 3.0 h (arrow). (G) NLE-treated oocyte showing half of oocyte underwent shrinkage and other half is filled with leaked cytoplasmic fluid after 3.0 h and 15 min (arrow)., (H) NLE-treated oocyte showing cytoplasmic fragmentation after 3 h and 30 min (arrow). (I) Control oocyte showing nigrosin-negative staining after 4.0 h (arrow). (J) NLE-treated oocyte showing nigrosin-positive staining after 4.0 h. (400× magnification). Chaube. Neem leaf extract-induced oocyte apoptosis in rat. Fertil Steril 2006. Fertility and Sterility 2006 85, 1223-1231DOI: (10.1016/j.fertnstert.2005.11.034) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Effects of 10.0 mg/mL neem leaf extract (NLE) with or without 1.0 μmol/L caspase-3 inhibitor (CI) on morphologic apoptotic changes in oocytes cultured in vitro. Chaube. Neem leaf extract-induced oocyte apoptosis in rat. Fertil Steril 2006. Fertility and Sterility 2006 85, 1223-1231DOI: (10.1016/j.fertnstert.2005.11.034) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Western blot analysis of bax protein (corresponding to 21 KDa) in oocytes. C = control oocyte lysate; neem leaf extract (NLE) = 10.0 mg/mL NLE–treated oocyte lysate; NLE+CI = 10.0 mg/mL NLE + 1.0 μmol/L caspase-3–treated oocyte lysate. 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. Neem leaf extract-induced oocyte apoptosis in rat. Fertil Steril 2006. Fertility and Sterility 2006 85, 1223-1231DOI: (10.1016/j.fertnstert.2005.11.034) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 DNA fragmentation in ovulated cumulus oocyte complexes (COCs). (A) TUNEL-negative staining in control oocyte (arrow). (B) TUNEL-positive staining in 10.0 mg/mL NLE-treated oocyte (arrow). Associated cumulus cells did not show TUNEL-positive staining. (400× magnification). Chaube. Neem leaf extract-induced oocyte apoptosis in rat. Fertil Steril 2006. Fertility and Sterility 2006 85, 1223-1231DOI: (10.1016/j.fertnstert.2005.11.034) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 5 The 10 mg/mL neem leaf extract (NLE)-induced DNA fragmentation in oocytes culture in vitro. C = Control oocyte DNA showing intact genomic DNA; T = 10 mg/mL NLE–treated oocytes showing distinct multiples of 180–200-base-pair ladder pattern (arrowheads). Chaube. Neem leaf extract-induced oocyte apoptosis in rat. Fertil Steril 2006. Fertility and Sterility 2006 85, 1223-1231DOI: (10.1016/j.fertnstert.2005.11.034) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions