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Extract of Azadirachta indica (neem) leaf induces apoptosis in rat oocytes cultured in vitro
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., and Tulsidas G. Shrivastav, Ph.D. Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, New Delhi, India
Objective: To determine whether aqueous neem leaf extract (NLE) could induce degeneration of rat oocytes and, if so, whether apoptosis is involved during NLE-induced degeneration of oocytes cultured in vitro. Design: A controlled prospective study. Setting: Laboratory research setting at Department of Reproductive Biomedicine of the Institute. Animal(s): Fifty-four sexually immature female rats that were 24 –25 days of age. Intervention(s): The immature female rats were injected with 10 IU pregnant mare serum gonadotropin for 48 h followed by 10 IU human chorionic gonadotropin (hCG) for 16 h. After 16 h, the rats were killed and ovulated cumulus oocyte complexes were collected from the oviduct. Cumulus-enclosed as well as denuded oocytes were used in the present study. Main Outcome Measure(s): Rates of shrinkage, membrane leakage, degeneration, assessment of morphological apoptotic changes, bax protein expression, and DNA fragmentation. Result(s): The NLE induced morphologic apoptotic changes such as shrinkage, membrane leakage, and cytoplasmic fragmentation prior to degeneration of oocytes. The NLE-treated oocytes that had morphologic apoptotic features showed overexpression of bax protein and DNA fragmentation as evidenced by terminal deoxynucleotidyl transferase nick-end labeling–positive staining and DNA ladder pattern. Conclusion(s): Neem leaf extract induced apoptosis in rat oocytes prior to degeneration in vitro. (Fertil Steril威 2006;85(Suppl 1):1223–31. ©2006 by American Society for Reproductive Medicine.) Key Words: Neem leaf extract, shrinkage, membrane leakage, cytoplasmic fragmentation, degeneration, bax protein expression, DNA fragmentation, apoptosis
Neem plant (Azadirachta indica) has been extensively used in the Ayurvedic system of medicine for a long time. Various parts of this plant are used for the treatment of various diseases. The neem bark aqueous extract is reported to have therapeutic potential for controlling gastric hypersecretion and gastroduodenal ulcer (1). A dental gel formulation containing neem extract has been reported to reduce oral infections, plaque index, and bacterial count (2, 3). The medicinal utilities have been described especially for neem leaf (4). Neem leaf and its constituents are reported to exhibit immunomodulatory, antiinflammatory, antihyperglycemic, antiulcer, antifungal, antibacterial, antimutagenic, anticarcinogenic, nematicidal, antimalarial, antiviral, insecticidal, and antioxidant properties (4 –9). The role of neem products in male fertility regulation has been well studied. The ethereal extract of neem stem bark induced reversible reproductive endocrine malfunction in male rats (10). The ethanolic neem leaf extract (NLE) has been reported to induce abnormal head morphology and reduce mean sperm count in murine (11). Neem leaf extract is also reported to inhibit motility and viability of human Received July 31, 2005; revised and accepted November 8, 2005. Reprint requests: Tulsidas G. Shrivastav, Ph.D., Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, New Mehrauli Road, Munirka, New Delhi–110067, India (FAX: 91-112610 1623; E-mail: [email protected]).
0015-0282/06/$32.00 doi:10.1016/j.fertnstert.2005.11.034
spermatozoa treated in vitro (12). The chloroform extract of neem oil inhibited spermatogenesis and sperm motility in mice (13). Neem oil is used as a vaginal contraceptive (14) and its reversible antifertility effects have been reported in rats and bonnet monkeys (15, 16). The biologically active fraction of neem oil extract that has reversible antifertility effect was identified and characterized (17). The role of neem products in female fertility regulation is not well understood. One recent study has indicated that neem oil inhibits total number of follicles and number of developing follicles in the rat ovary (18). They hypothesized that the treatment of neem oil might have inhibited gondotropin-dependent oocyte growth and maturation pathway, thereby reducing the number of developing follicles (18). Neem oil induced degeneration of eggs and inhibited sperm-egg interaction in vitro (19). A preliminary study carried out by Juncia and Williams (19) indicated that neem oil induced degeneration of mouse egg in vitro. However, the morphologic and biochemical changes prior to neem oil–induced degeneration of mouse eggs were not studied in detail. Thus, it remains unclear whether or not neem products could induce apoptosis in the mammalian egg. Apoptosis is induced by a variety of physiologic and pathologic conditions (20). It is characterized by a series of distinct morphologic, biochemical, and molecular changes in dying cells that include cell shrinkage, membrane blebbing, nu-
Fertility and Sterility姞 Vol. 85, Suppl 1, April 2006 Copyright ©2006 American Society for Reproductive Medicine, Published by Elsevier Inc.
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clear condensation, cytoplasmic fragmentation, overexpression of bax protein, and DNA fragmentation into multiples of 180 –200 base pairs (20 –24). Therefore, the present study was designed to investigate whether NLE could induce degeneration of oocytes and. if so, whether NLE-induced degeneration is mediated through apoptosis in rat oocytes cultured in vitro. MATERIALS AND METHODS Chemicals and Preparation of Culture and Washing Media All chemicals used in the present study were purchased from Sigma Chemical Co., St. Louis, MO, unless stated otherwise. The culture medium (M-199) was prepared per company manual protocol. The pH of culture medium was adjusted to 7.2 ⫾ 0.1 and osmolarity was found to be 290 ⫾ 5 mosmol and supplemented with sodium bicarbonate (0.035% w/v), penicillin (100 IU/mL), and streptomycin (100 g/mL). Washing media was prepared by adding 10% (v/v) fetal bovine serum (FBS) into culture media. Preparation of NLE and Caspase-3 Inhibitor Working Solutions Green leaves of neem were ground in the presence of distilled water and then filtered. The filtrate was then centrifuged at 5000 rpm for 10 min at 4°C and supernatant was collected. The supernatant was mixed with highpressure liquid chromatography– grade chloroform (1:1; v/v) to remove fat-soluble ingredients. The aqueous phase (upper phase) was collected, lyophilized, and kept at ⫺30°C until use. Working concentrations of NLE (2.5, 5.0, 10.0, and 20.0 mg/mL) were freshly prepared before use. In brief, NLE powder was dissolved in distilled water to make stock solution (100 mg/mL) and then further diluted to get the desired concentrations of NLE in culture media. The addition of NLE at final concentrations did not alter the osmolarity (290 ⫾ 5 mosmol) or pH (7.2 ⫾ 0.05) of the culture medium used in the present study. The 1.0 L stock solution (10.0 mmol/L) of caspase-3 inhibitor (Ac-DEVD-CHO; Alexis, San Diego, CA) was diluted in 10.0 mL of culture media to get its final concentration 1.0 mol/L. The various concentrations of NLE were prepared in culture media containing 1.0 mol/L of caspase-3 inhibitor for in vitro studies. Animals and Collection of Oocytes Immature female rats of the Holtzman strain were separated from an existing colony in the departmental animal facility and maintained in normal husbandry conditions with food and water ad libitum. To obtain mature oocytes, the rats (24 –25 days old; 50 ⫾ 5 gm body weight) were given a single subcutaneous injection of 10 IU pregnant mare serum gonadotropin (PMS) in 100 L sterile normal saline to promote growth of a cohort of healthy antral follicles. Fortyeight hours after PMS injection, 10 IU human choronic gonadotropin (hCG) was given to induce superovulation in 1224
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animals. Sixteen hours after hCG injection, the rats were killed. Ovary along with oviduct was removed and transfered to a 35-mm Petri dish containing 2 mL sterile washing media. Superovulated cumulus oocyte complexes (COCs) were collected from the oviduct by manual puncturing with the help of a 26-gauge needle attached to 1-mL insulin syringe and washed three times with washing media. Ovulated cumulus-enclosed oocytes were made free of their cumulus cells by transfering them to culture medium containing 0.1% hyaluronidase for 5 min followed by repeated pipetting through a narrow-bore pipette. The denuded oocytes were used for most of the experiments unless stated otherwise. Groups of 15–16 oocytes were used for one replicate and experiments were repeated at least three times. Effects of Various Doses of NLE on Morphologic Apoptotic Changes in Oocytes Groups of 15 to 20 oocytes were cultured separately in 35-mm Petri dishes containing 2.0 mL culture medium (control group) or medium containing various concentrations (2.5, 5.0, 10.0, and 20.0 mg/mL) of NLE. All Petri dishes were maintained at 37°C for 3.0 h in a CO2 incubator (Thermo Forma, Marietta, OH). At the end of incubation period, oocytes were removed, washed three times with washing media, transfered to grooved slide with 100 L culture medium, and then examined for morphologic apoptotic changes using a phase-contrast microscope (Eclipse E600; Nikon, Tokyo, Japan) at 400⫻ magnification. Time-Course Effects of 10.0 mg/mL NLE on Morphologic Apoptotic Changes in Oocytes Groups of 15–20 oocytes were incubated separately in culture medium with or without 10.0 mg/mL of NLE for various times (1.0, 2.0, and 3.0 h). At the end of each incubation period, oocytes were removed, washed three times with washing medium, and transfered to grooved slide for the analysis of morphologic apoptotic changes using phasecontrast microscope at 400⫻ magnification. Effect of Caspase-3 Inhibitor on 10.0 mg/mL NLE–Induced Morphologic Apoptotic Changes in Oocytes Groups of 15–20 oocytes were incubated separately in plain medium or medium containing 10.0 mg/mL NLE with or without caspase-3 inhibitor (1.0 mol/L) for 3.0 h. At the end of incubation period, oocytes were removed, washed three times with washing medium, and transfered to grooved slide for the analysis of morphological apoptotic changes using phase-contrast microscope at 400⫻ magnification. Eosin/Nigrosin Staining of Oocytes The live and dead status of control and NLE-treated oocytes that underwent degeneration were examined by means of eosin/nigrosin dye-exclusion test per published protocol (25). In
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brief, control oocytes and 10.0 mg/mL NLE–treated oocytes that underwent degeneration were removed, washed three times with washing medium, and then stained with 10 L 1% eosin (w/v) followed by 10 L nigrosin (10% w/v) for 1 min at room temperature. The stained oocytes were observed for live and dead status using a phase-contrast microscope at 400⫻ magnification. Detection of bax Protein by Western Blotting A large number of oocytes (⬃600) are required for the analysis of bax protein expression using Western blotting technique. To collect large number of oocytes, a group of 10 –12 animals were subjected to superovulation induction protocol. The ovulated COCs were first denuded, washed twice with washing medium, and then used for in vitro study. The denuded oocytes were incubated in control medium or medium containing NLE (10 mg/mL) with or without 1.0 mol/L caspase-3 inhibitor. After 3 h of incubation, oocytes were washed three times with washing medium, and 200 oocytes from each group were transfered to 0.6 mL microfuge tube containing 100 L STKM buffer (0.25 mol/L sucrose, 50 mmol/L Tris-HCl, pH 7.5, 25 mmol/L KCl, 5 mmol/L MgCl2, and 0.25% (v/v) Triton X-100) for lysis at 4°C for 15 min. Cell lysates were centrifuged at 4,000g for 20 min at 4°C. The supernatants from all samples were frozen at ⫺30°C until assay. All samples were thawed and total protein content was measured per published protocol (26). Lysates having equal amount of protein (75 g total protein) were then separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing condition and electroblotted following the procedure described earlier (27). In brief, after electroblotting the blot was probed with antibax antibody generated in rabbit (Polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA) diluted (1: 2500) in 1.5% (w/v) BSA-Tris buffered saline supplemented with 0.05% Tween-20 (TBST; 0.05 M Tris-Cl, pH 7.6, 0.15 M NaCl, 0.05% Tween-20) buffer for 1.0 h at room temperature. Nitrocellulose membrane was then washed twice in TBST buffer (5 min each) and further incubated with second antibody (goat-antirabbit IgG conjugated with alkaline phosphatase (Polyclonal; Santa Cruz Biotechnology) diluted (1:10,000) in 1.5% BSA-TBST buffer for 1.0 h at room temperature. The bound antibody was then detected with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; Bio Rad Laboratories, Hercules, CA) color system. In parallel, another set of the same samples was run and probed with anti–-actin antibody generated in rabbit (1:2,500 dilutions; Polyclonal; Santa Cruz Biotechnology) and localized using goat-antirabbit IgG conjugated with alkaline phosphatase (Polyclonal; Santa Cruz Biotechnology) to confirm that equivalent amounts of protein were analyzed. Detection of DNA Fragmentation by TUNEL Assay The DNA fragmentation was detected using a terminal deoxynucleotidyl transferase (TDT) nick-end labeling (TUNEL) kit from R&D Systems (Minneapolis, MN) per company manual protocol. The control and 10 mg/mL NLE–treated cuFertility and Sterility姞
mulus-enclosed oocytes (15–16 oocytes in each group) were fixed in 3.7% (v/v) formaldehyde in PBS for 15 min at 18 –20°C. After washing, oocytes were transfered separately on to poly-L-lysine– coated slides and then air-dried. Slides were treated with 50 L proteinase K solution for 30 min and then immersed in quenching solution for 3– 4 min. Thereafter, slides were immersed in 1⫻ TDT labeling buffer for 5 min and then incubated with 50 L labeling reaction at 37°C for 1.0 h. The slides were immersed in 1⫻ TDT stop buffer to stop the reaction. Washed slides were incubated with 50 L diluted (1:500) biotin-labeled anti-BrdU at 37°C for 1.0 h. Slides were washed with PBS containing 0.05% Tween-20 and then treated with 50 L streptavidin-horseradish peroxidase solution for 10 min. Washed slides were immersed in diaminobenzidine (DAB) solution for 5 min and then in methyl green solution for 2 min after washing. The slides were again washed sequentially by dipping at least ten times in distilled water, 50%, 70%, 95%, and 100% ethanol, and finally in xylene for two changes each. Slides were mounted in distyrene plasticizer xylene (DPX) and then analyzed for TUNEL-positive staining under phase-contrast microscope at 100⫻ and 400⫻ magnifications. The TUNEL analysis was repeated three times and representative photographs are shown in the result section. Isolation and Electrophoresis of Total DNA A large number of oocytes (⬃600) are required for DNA fragmentation analysis using DNA-gel electrophoresis. To collect a large number of oocytes, a group of 10 –12 animals were subjected to superovulation induction protocol. Ovulated cumulus-enclosed oocytes were denuded, washed twice with washing medium. and then used for in vitro study. These denuded oocytes were incubated in control media or media containing 10 mg/mL NLE. After 3 h of incubation, oocytes were removed from control and NLE-treated groups and used for DNA isolation of low-molecular-weight DNA along with genomic DNA (total DNA) following the protocol reported elsewhere (28) with some modifications. In brief, 300 oocytes from control and NLE-treated groups were separately lysed in 100 L lysis buffer (5 mmol/L Tris, 20 mmol/L EDTA, 0.5% Triton X-100, pH 8.0) for 1.0 h on ice. The samples were treated with 5 L RNase A (10 mg/mL) for 1.0 h at 37°C followed by 5 L proteinase K (10 mg/mL) at 50°C for 1.0 h. The DNA samples were kept at ⫺30°C until the collection of DNA samples. All samples were thawed quickly, pooled in respective groups, and then centrifuged at 10000g at 4°C for 20 min. The supernatants were collected and extracted twice with saturated phenol– chloroform–isopropyl alcohol (25:24:1 v/v/v). Then 200 L aqueous phase was transfered to a microcentrifuge tube and kept at ⫺30°C until use. To the 20 L sample, 20 L TE buffer (10 mmol/L Tris, 1 mmol/L EDTA, pH 8.8) was added and mixed gently. Then the 20-L sample was transfered to a 0.5-mL microcentrifuge tube and 4 L 6⫻ gel loading buffer (50% glycerol, 1% bromophenol blue, and 1% xylene cynol in Trisborate EDTA (0.045 M Tris-borate and 0.001 M EDTA, pH 8.0 buffer) was mixed in each sample. Samples were electropho1225
TABLE 1 Effects of various doses of neem leaf extract (NLE) on morphologic apoptotic changes in oocytes (15–20 oocytes in each group) cultured in vitro for 3.0 h. Oocytes
NLE treatment (mg/mL)
Shrinkage (%)
Membrane leakage (%)
Degeneration (%)
Control 2.5 5.0 10.0 20.0
Nil 71.88 ⫾ 1.46ⴱ 90.90 ⫾ 4.55ⴱ 57.21 ⫾ 3.36 Not observed
Nil Nil Nil 43.07 ⫾ 3.41ⴱ 20.0 ⫾ 4.0
Nil Nil Nil Nil 80.0 ⫾ 5.0ⴱ
Note: Values are mean ⫾ SE. ⴱ P⬍.001 (control vs. NLE-treated groups). Chaube. Neem leaf extract–induced oocyte apoptosis in rat. Fertil Steril 2006.
resed on 1% agarose gel (Bio Rad) containing 0.5 g/mL ethidiumbromide for 3 h at 25 V. Gel was visualized and photographed on a UV transilluminator (UVP BioImaging Systems, Upland, CA). The DNA gel electrophoresis was repeated three times. Statistical Analysis Data are expressed as mean ⫾ standard error of the mean (SEM) of triplicate samples. All percentage values were subjected to arc-sine square root transformation before statistical analysis. Data are analyzed by Student t test. A probability of P⬍.001 was considered to be statistically significant. RESULTS Effects of Various Doses of NLE on Morphologic Apoptotic Changes in Oocytes As shown in Table 1, lower doses (2.5 and 5.0 mg/mL) of NLE significantly induced shrinkage of oocytes after 3.0 h of incubation. A higher dose of NLE (10.0 mg/mL) induced
shrinkage (in 57.21% ⫾ 3.36% of oocytes) and membrane leakage (in 43.07% ⫾ 3.41%). If the dose of NLE was further increased to 20.0 mg/mL, only 20.0% ⫾ 4.0% oocytes showed membrane leakage, whereas a majority of oocytes (80.0% ⫾ 5.0%) underwent degeneration (Table 1). Time-Course Effect of 10.0 mg/mL NLE on Morphologic Apoptotic Changes in Oocytes Time-course study revealed that 10.0 mg/mL NLE significantly induced shrinkage of oocytes (in 49.27% ⫾ 4.78%) after 1.0 h of treatment. The rate of oocyte shrinkage was further increased (80.37% ⫾ 1.40%), if the treatment period was increased up to 2.0 h. A shift in the percentage of oocytes undergoing morphologic apoptotic changes from shrinkage to membrane leakage was observed after 2.0 h of NLE treatment, and all treated oocytes underwent either shrinkage (63.13% ⫾ 0.94%) or membrane leakage (37.42% ⫾ 2.20%) after 3.0 h of NLE treatment. Although shrinkage was not observed after 4.0 h of NLE treatment, around 22%
TABLE 2 Time-course effects of 10.0 mg/mL neem leaf extract (NLE) on morphologic apoptotic changes in oocytes (15–20 oocytes in each group) cultured in vitro for 4 h. Oocytes
NLE treatment (in h)
Shrinkage (%)
Membrane leakage (%)
Degeneration (%)
Control 1.0 2.0 3.0 4.0
Nil 49.27 ⫾ 4.78ⴱ 80.37 ⫾ 1.40ⴱ 63.13 ⫾ 0.94 Not observed
Nil Nil Nil 37.42 ⫾ 2.20ⴱ 21.79 ⫾ 1.45
Nil Nil Nil Nil 79.21 ⫾ 2.85ⴱ
Note: Values are mean ⫾ SE. ⴱ P⬍.001 (control vs. NLE-treated groups). Chaube. Neem leaf extract–induced oocyte apoptosis in rat. Fertil Steril 2006.
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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.
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a higher dose of NLE (20.0 mg/mL) induced degeneration of oocytes after 3.0 h of treatment.
FIGURE 2 Effects of 10.0 mg/mL neem leaf extract (NLE) with or without 1.0 mol/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.
of oocytes showed membrane leakage, and 79.21% ⫾ 2.85% underwent degeneration (Table 2).
Assessment of Progression of Morphologic Apoptotic Changes in NLE-Treated Oocytes Oocytes collected after superovulation induction protocol from the oviduct of immature rats were at metaphase I and did not show first polar body. These mature oocytes (absence of GV and nucleolus) extrude first polar body and did not show any morphologic apoptotic changes after 3.0 h of in vitro culture (Fig. 1A). When these oocytes were treated with 10.0 mg/mL NLE for various times, shrinkage of oocyte volume was observed (Fig. 1B) compared to control (Fig. 1A). Though the shrinkage was a first visual change observed in NLE-treated oocytes, a leakage of transparent fluid (membrane leakage) was observed after 2.0 h of incubation and progressed along with shrinkage simultaneously (Fig. 1C–F). Half of the oocyte was shriunk and the other half was filled with leaked transparent fluid after 3.0 h of NLE treatment (Fig. 1G) as compared to control oocytes that had first polar body with normal morphology (Fig. 1A). Some of NLE⫺treated oocytes showed cytoplasmic fragmentation (Fig. 1H) compared to control oocytes (Fig.1A) prior to degeneration only at a higher dose of NLE (20.0 mg/mL) after 3.0 h of treatment. Live and Dead Status of Control and NLE-Treated Oocytes The control oocytes did not take nigrosin stain (Fig. 1I), and NLE-treated oocytes that underwent degeneration showed nigrosin staining (Fig. 1J), further supporting our results that 1228
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Effect of NLE (10 mg/mL) With or Without Caspase-3 Inhibitor (1.0 mol/L) on the Rate of Morphologic Apoptotic Changes in Oocytes When oocytes were treated with 10.0 mg/mL NLE alone, almost all oocytes (98%) showed either shrinkage or membrane leakage after 3.0 h of culture. Addition of caspase-3 inhibitor (1.0 mol/L) prevented these morphologic apoptotic changes (i.e., shrinkage and membrane leakage) in NLEtreated oocytes, with only 14% oocytes showing morphologic apoptotic change, i.e., shrinkage (Fig. 2). Effect of NLE on bax Protein Expression in Oocytes Control and 10.0 mg/mL NLE–treated oocytes with or without caspase-3 inhibitor (1.0 mol/L) were used for the analysis of bax protein expression. As evident from the Western blot analysis (Fig. 3), NLE induced overexpression of bax protein in treated oocytes compared to control oocytes. Further, a densitometry analysis revealed that a treated lane had 3.0 times higher bax protein expression compared to control lane. Addition of caspase-3 inhibitor inhibited NLE-induced overexpression of bax protein, and the result was similar to the control lane. DNA Fragmentation Analysis in Control and NLE-Treated Oocytes Using TUNEL Assay To confirm the occurrence of DNA fragmentation, control and 10 mg/mL NLE–treated cumulus-enclosed oocytes (15–16 oocytes in each group) were subjected to TUNEL assay. Treat-
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 mol/L caspase-3–treated oocyte lysate. Lower portion shows a control assay for -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.
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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.
ment of NLE induced DNA fragmentation (TUNEL-positive staining) selectively in oocytes but not in associated cumulus cells (Fig. 4B) compared to control oocytes that did not show TUNEL staining (Fig. 4A). DNA Fragmentation Analysis in Control and NLE-Treated Oocytes Using DNA Gel Electrophoresis As shown in Figure 5, DNA isolated from control and NLE-treated oocytes were run on 1% agarose gel. The control oocytes that had normal morphology did not show DNA ladder, and intact genomic DNA was seen after 3.0 h of culture (Fig. 5, lane C). On the other hand, the extent of DNA cleavage into multiples of 180 –200 bp was significantly increased and detectable as a ladder pattern in 10.0 mg/mL NLE–treated oocytes that had morphologic apoptotic features (Fig. 5, lane T). DISCUSSION Apoptosis is a genetically controlled, highly conserved, intricate mechanism of cellular suicide and disposal of cells in the absence of inflammatory reactions that can be triggered by a variety of physiologic and pathologic conditions. Apoptosis plays a central role in the development and maintenance of homeostasis in multicellular organisms (20, 24). It is characterized by a series of distinct morphologic, biochemical, and molecular changes in dying cells (21–23). The morphologic changes in dying cells include cell shrinkage, nuclear condensation, cytoplasmic granulation, membrane blebbing, and cytoplasmic fragmentation (22, 29, 30). Data of Fertility and Sterility姞
the present study clearly suggest that NLE induced morphologic apoptotic changes such as shrinkage and membrane leakage in treated oocytes. Shrinkage of oocyte volume was a first visual change that occurred after 1.0 h of NLE treatment. These shrinking oocytes showed a leakage of transparent cytoplasmic fluid out of corona that progressed as the treatment time of NLE was increased. Almost half of the oocyte volume was filled with leaked cytoplasmic fluid after 3.0 h of NLE treatment. Some oocytes showed cytoplasmic fragmentation just prior to degeneration. The NLE-induced degeneration of oocytes was confirmed by live and dead staining using eosin/nigrosin double staining. The NLEinduced morphologic apoptotic changes were inhibited if the oocytes were cotreated with NLE (10.0 mg/mL) along with caspase-3 inhibitor (1.0 mol/L) for 3.0 h. These data clearly suggest that NLE-induced morphologic changes characteristic of apoptosis in oocytes and caspase-3 activation is involved during NLE-induced morphologic apoptotic changes in the present study. The apoptosis was initiated with shrinkage of oocyte volume followed by membrane leakage and then cytoplasmic fragmentation into unequal bodies that is an end point in the process of apoptosis prior to degeneration. The similar sequence of morphologic apoptotic changes has been reported in mouse and rat oocytes cultured in vitro (22, 23, 30, 31). It is generally accepted that a ratio of apoptotic promoter (such as bax expression) to suppressors (such as bcl-2 expression) within a cell determine whether a cell will under go apoptosis or survive (20). Although a ratio of bax/bcl-2 gene products was not analyzed in the present study, we carried 1229
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.
out a Western blot analysis to find out whether NLE induced proapoptotic signals, i.e., overexpression of bax protein in control and NLE-treated oocytes with or without caspase-3 inhibitor. Western blot analysis revealed that NLE-treated oocytes showed overexpression of bax protein. Densitometry analysis further revealed that NLE-treated oocytes had 3.0 times more bax protein than control oocytes. Addition of caspase-3 inhibitor prevented NLE-induced overexpression of bax protein in the present study; the amount of bax protein was similar to control oocytes. These results clearly suggest that overexpression of bax gene might have modulated the ratio of apoptotic promoters to suppressors (bax/bcl-2) within a cell, thereby setting NLE-treated oocytes toward apoptosis. Because caspase-3 inhibitor has prevented NLEinduced overexpression of bax gene, it is quite possible that NLE-induced morphologic apoptotic features might be mediated through bax/bcl-2– caspase-3 pathway in oocytes. Similarly, the involvement of overexpression of bax protein and caspase-3 activation during oocytes apoptosis has been reported in mouse and rat oocytes (22, 24, 32, 33). A unique biochemical event in apoptosis that precedes morphologic changes such as shrinkage, membrane blebbing, and fragmentation of cytoplasm into unequal bodies is due to the activation of Ca⫹⫹/Mg⫹⫹-dependent endonuclease (24). This enzyme cleaves genomic DNA at the internucleosomal region resulting in 180 –200-base-pair DNA oligonucleosomal fragments. These fragmented DNA can be 1230
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detected in a single cell using an in situ technique such as TUNEL or DNA gel electrophoresis, where fragmented DNA appears in a ladder pattern (22, 30, 34 –37). Further, TUNEL-positive staining has been reported in oocytes that had morphologic apoptotic features (22, 30, 31, 34). To analyze NLE-induced DNA fragmentation in the female germ cells, we used cumulus-enclosed oocytes, because denuded oocytes were getting washed off owing to repeated washing during the TUNEL assay. In the present study, TUNEL-positive staining was observed in NLE-treated oocytes and not in the associated cumulus cells, suggesting that NLE might have selective access at the level of oocytes and not in associated cumulus cells. When total DNA was isolated from control and NLE-treated oocytes and electrophoresed on agarose gel, a distinctive DNA ladder (a hallmark feature of apoptosis) was detected in the NLE-treated lane, whereas the control lane had intact genomic DNA. Taken together, these data strongly suggest that NLE induced DNA fragmentation selectively in germ cells (oocytes) and not in associated somatic cell (cumulus cells) in vitro. In conclusion, the present study for the first time clearly demonstrates that NLE induced morphologic apoptotic changes such as shrinkage, membrane leakage, and cytoplasmic fragmentation just before degeneration. These morphologic changes were associated with the overexpression of bax protein and caspase-3 activation, DNA fragmentation as evidenced by TUNEL-positive staining, and a detectable ladder pattern on DNA gel electrophoresis in NLE-treated oocytes. Acknowledgment: We are grateful to Profs. Neeraj K. Sethi, M.D. (Director), Krishnamurthy Kalaivani, M.D. (H.O.D., R.B.M., Dean of Studies), and S. Roy, all from the National Institute of Health and Family Welfare, New Delhi, India, for their keen interest and encouragement throughout the study.
REFERENCES 1. Bandyopadhyay U, Biswas K, Sengupta A, Moitra P, Dutta P, Sarkar D, et al. Clinical studies on the effect of neem (Azadirachta indica) bark extract on gastric secretion and gastroduodenal ulcer. Life Sci 2004; 75:2867–78. 2. Pai MR, Acharya LD, Udupa N. The effect of two different dental gels and a mouthwash on plaque and gingival scores: a 6-week clinical study. Int Dent J 2004;54:219 –23. 3. Pai MR, Acharya LD, Udupa N. Evaluation of antiplaque activity of Azadirachta indica leaf extract gel—a six-week clinical study. J Ethanopharmacol 2004;90:99 –103. 4. Subapriya R, Nagini S. Medicinal properties of neem leaves: a review. Curr Med Chem Anti-Cancer Agents 2005;5:149 –56. 5. Wandscheer CB, Duque JE, da Silva MA, Fukuyama Y, Wohlke JL, Adelmann J, Fontana JD. Larvicidal action of ethanolic extracts from fruit endocarps of Melia azedarach and Azadirachta indica against the dengue mosquito Aedes aegypti. Toxicon 2004;44:829 –35. 6. Sharma V, Walia S, Kumar J, Nair MG, Parmar BS. An efficient method for the purification and characterization of nematicidal azadirectins A, B, and H, using MPLC and ESIMS. J Agric Food Chem 2003;51:3966 –72. 7. Udeinya IJ, Mbah AU, Chijioke CP, Shu EN. An antimalarial extract from neem leaves is antiretroviral. Trans. R Soc Trop Med Hyg 2004; 98:435–7.
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8. Siddiqui BS, Rasheed M, Ilyas F, Gulzar T, Tariq RM, Naqvi SN. Analysis of insecticidal Azadirachta indica. A. Juss. fractions. Z Naturforsch 2004;59:104 –12. 9. Sithisaran P, Supabphol R, Gritsanapan W. Antioxidant activity of Siamese neem tree (VP1209). J Ethanopharmacol 2005;99:109 –12. 10. Raji Y, Udoh US, Mewoyeka OO, Ononye FC, Bolarinwa AF. Implication of reproductive endocrine malfunction in male infertility efficacy of Azadirachta indica extract in rats. Afr J Med Sci 2003;32:159 – 65. 11. Khan PK, Awasthi KS. Cytogenetic toxicity of neem. Food Chem Toxicol 2003;41:1325–1328. 12. Khillare B, Shrivastv TG. Spermicidal activity of Azadirachta indica (neem) leaf extract. Contraception 2003;68:225–9. 13. Yin Z, Jia R, Gao P, Gao R, Jiang D, Liu K, Liu S. Preparation of contraceptive pill microcapsule and its antifertility effect. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi 2004;21:979 – 82. 14. Sharma SK, SaiRam M, Ilavazhagan G, Devendra K, Shiavaji SS, Selvamurthi W. Mechanism of action of NIM-76: a novel vaginal contraceptive from neem oil. Contraception 1996;54:373– 8. 15. Upadhyay SN, Kaushic C, Talwar GP. Antifertility effects of neem (Azadirachta indica) oil by single intrauterine administration: a novel method for contraception. Proc Biol Sci 1990;242;175–9. 16. Upadhyay SN, Dhawan S, Sharma MG, Talwar GP. Long-term contraceptive effects of intrauterine neem treatment (IUNT) in bonnet monkeys: an alternate to intrauterine contraceptive devices (IUCD). Contraception 1994;49:161–9. 17. Garg S, Taluja V, Talwar GP, Upadhyay SN. Immunocontraceptive activity guided fractionation and characterization of active constituents of neem (Azadirachta indica) seed extracts. J Ethnopharmacol 1998; 60:235– 46. 18. Roop JK, Dhaliwal PK, Guraya SS. Extracts of Azadirachta indica and Melia azedarach seeds inhibit follicullogenesis in albino rats. Braz J Med Biol Res 2005;38:943–7. 19. Juncia SC, Williams RS. Mouse sperm-egg interaction in vitro in the presence of neem oil. Life Sci 1993;53:PL279 – 84. 20. Terranova PF, Tayler CC. Apoptosis (cell death). In: Knobil E, Neill J, eds. Encyclopedia of reproduction. New York: Academic Press, 1999. p. 261–73. 21. Anjum R, Khar A. Apoptosis: an overview. Ind J Biotechnol 2002;1: 58 –72. 22. Chaube SK, Prasad PV, Thakur SC, Shrivastav TG. Hydrogen peroxide modulates meiotic cell cycle and induces morphological features characteristic of apoptosis in rat oocytes in vitro. Apoptosis 2005;10:863–74. 23. Chaube SK, Prasad PV, Thakur SC, Shrivastav TG. Estradiol protects
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clomiphine citrate induced apoptosis in rat ovarian follicles and superovulated cumulus oocytes complexes. Fertil Steril 2005;84(Suppl 2):1163–72. Jurisicova A, Acton BM. Deadly decisions: the role of gene regulation programmed cell death in human preimplantation embryo development. Reproduction 2004;128:281–9. Chaube SK, Misro MM. Effect of styrene maleic anhydride (SMA) on viability and integrity of isolated rat oocytes in vitro. Contraception 2002;66:469 –72. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of dye-binding. Analyst Biochem 1976;72:242–54. Towbin H, Staehelin T, Fordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979;76:4350 – 4. Hughes FM, Gorospe WC. Biochemical identification of apoptosis (programmed cell death) in granulosa cells: evidence for a potential mechanism underlying follicular atresia. Endocrinology 1991;129: 2415–22. Schwartz LM, Osborn BA. Programmed cell death, apoptosis and killer genes. Immunol Today 1993;14:582–90. Wu Ji, Zhang L, Wang X. Maturation and apoptosis of human oocytes in vitro are age-related. Fertil Steril 2000;74:1137– 41. Fuzino Y, Ozaki K, Yamamastu S, Ito F, Matsuoka I, Hayashi E, et al. DNA fragmentation of oocytes in aged mice. Hum Reprod 1996;11: 1480 –3. Perez GI, Tao X-Z, Tilly JL. Fragmentation and death (a.k.a. apoptosis) of ovulated oocytes. Mol Hum Reprod 1999;5:414 –20. Thakur SC, Thakur SS, Chaube SK, Singh SP. An ethereal extract of kamala (Mallotus philipinensis (Moll. Arg) Lam.) seed induce adverse effects on reproductive parameters of female rats. Reprod Toxicol 2005;20:149 –56. Yang HW, Hwang KJ, Kwan HC, Kim HS, Choi KW, Oh KS. Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryo. Hum Reprod 1998;13:998 –1002. Roth Z, Hansen PJ. Involvement of apoptosis in disruption of developmental competency of bovine oocytes by heat shock during maturation. Biol Reprod 2004;71:1898 –1906. Hao Y, Lai L, Mao J, Im GS, Bonk A, Prather RS. Apoptosis in parthenogenetic preimplantation porcine embryos. Biol Reprod 2004; 70:1644 –9. Henkel R, Hajimohammad M, Stalf T, Hoogendijk C, Mehnert C, Menkveld R, et al. Influence of deoxyribonucleic acid damage on fertilization and pregnancy. Fertil Steril 2004;81:965–72.
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Objective: To determine whether aqueous neem leaf extract (NLE) could induce degeneration of rat oocytes and, if so, whether apoptosis is involved during NLE-induced degeneration of oocytes cultured in vitro. Design: A controlled prospective study. Setting: Laboratory research setting at Department of Reproductive Biomedicine of the Institute. Animal(s): Fifty-four sexually immature female rats that were 24 –25 days of age. Intervention(s): The immature female rats were injected with 10 IU pregnant mare serum gonadotropin for 48 h followed by 10 IU human chorionic gonadotropin (hCG) for 16 h. After 16 h, the rats were killed and ovulated cumulus oocyte complexes were collected from the oviduct. Cumulus-enclosed as well as denuded oocytes were used in the present study. Main Outcome Measure(s): Rates of shrinkage, membrane leakage, degeneration, assessment of morphological apoptotic changes, bax protein expression, and DNA fragmentation. Result(s): The NLE induced morphologic apoptotic changes such as shrinkage, membrane leakage, and cytoplasmic fragmentation prior to degeneration of oocytes. The NLE-treated oocytes that had morphologic apoptotic features showed overexpression of bax protein and DNA fragmentation as evidenced by terminal deoxynucleotidyl transferase nick-end labeling–positive staining and DNA ladder pattern. Conclusion(s): Neem leaf extract induced apoptosis in rat oocytes prior to degeneration in vitro. (Fertil Steril威 2006;85(Suppl 1):1223–31. ©2006 by American Society for Reproductive Medicine.) Key Words: Neem leaf extract, shrinkage, membrane leakage, cytoplasmic fragmentation, degeneration, bax protein expression, DNA fragmentation, apoptosis
Neem plant (Azadirachta indica) has been extensively used in the Ayurvedic system of medicine for a long time. Various parts of this plant are used for the treatment of various diseases. The neem bark aqueous extract is reported to have therapeutic potential for controlling gastric hypersecretion and gastroduodenal ulcer (1). A dental gel formulation containing neem extract has been reported to reduce oral infections, plaque index, and bacterial count (2, 3). The medicinal utilities have been described especially for neem leaf (4). Neem leaf and its constituents are reported to exhibit immunomodulatory, antiinflammatory, antihyperglycemic, antiulcer, antifungal, antibacterial, antimutagenic, anticarcinogenic, nematicidal, antimalarial, antiviral, insecticidal, and antioxidant properties (4 –9). The role of neem products in male fertility regulation has been well studied. The ethereal extract of neem stem bark induced reversible reproductive endocrine malfunction in male rats (10). The ethanolic neem leaf extract (NLE) has been reported to induce abnormal head morphology and reduce mean sperm count in murine (11). Neem leaf extract is also reported to inhibit motility and viability of human Received July 31, 2005; revised and accepted November 8, 2005. Reprint requests: Tulsidas G. Shrivastav, Ph.D., Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, New Mehrauli Road, Munirka, New Delhi–110067, India (FAX: 91-112610 1623; E-mail: [email protected]).
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spermatozoa treated in vitro (12). The chloroform extract of neem oil inhibited spermatogenesis and sperm motility in mice (13). Neem oil is used as a vaginal contraceptive (14) and its reversible antifertility effects have been reported in rats and bonnet monkeys (15, 16). The biologically active fraction of neem oil extract that has reversible antifertility effect was identified and characterized (17). The role of neem products in female fertility regulation is not well understood. One recent study has indicated that neem oil inhibits total number of follicles and number of developing follicles in the rat ovary (18). They hypothesized that the treatment of neem oil might have inhibited gondotropin-dependent oocyte growth and maturation pathway, thereby reducing the number of developing follicles (18). Neem oil induced degeneration of eggs and inhibited sperm-egg interaction in vitro (19). A preliminary study carried out by Juncia and Williams (19) indicated that neem oil induced degeneration of mouse egg in vitro. However, the morphologic and biochemical changes prior to neem oil–induced degeneration of mouse eggs were not studied in detail. Thus, it remains unclear whether or not neem products could induce apoptosis in the mammalian egg. Apoptosis is induced by a variety of physiologic and pathologic conditions (20). It is characterized by a series of distinct morphologic, biochemical, and molecular changes in dying cells that include cell shrinkage, membrane blebbing, nu-
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clear condensation, cytoplasmic fragmentation, overexpression of bax protein, and DNA fragmentation into multiples of 180 –200 base pairs (20 –24). Therefore, the present study was designed to investigate whether NLE could induce degeneration of oocytes and. if so, whether NLE-induced degeneration is mediated through apoptosis in rat oocytes cultured in vitro. MATERIALS AND METHODS Chemicals and Preparation of Culture and Washing Media All chemicals used in the present study were purchased from Sigma Chemical Co., St. Louis, MO, unless stated otherwise. The culture medium (M-199) was prepared per company manual protocol. The pH of culture medium was adjusted to 7.2 ⫾ 0.1 and osmolarity was found to be 290 ⫾ 5 mosmol and supplemented with sodium bicarbonate (0.035% w/v), penicillin (100 IU/mL), and streptomycin (100 g/mL). Washing media was prepared by adding 10% (v/v) fetal bovine serum (FBS) into culture media. Preparation of NLE and Caspase-3 Inhibitor Working Solutions Green leaves of neem were ground in the presence of distilled water and then filtered. The filtrate was then centrifuged at 5000 rpm for 10 min at 4°C and supernatant was collected. The supernatant was mixed with highpressure liquid chromatography– grade chloroform (1:1; v/v) to remove fat-soluble ingredients. The aqueous phase (upper phase) was collected, lyophilized, and kept at ⫺30°C until use. Working concentrations of NLE (2.5, 5.0, 10.0, and 20.0 mg/mL) were freshly prepared before use. In brief, NLE powder was dissolved in distilled water to make stock solution (100 mg/mL) and then further diluted to get the desired concentrations of NLE in culture media. The addition of NLE at final concentrations did not alter the osmolarity (290 ⫾ 5 mosmol) or pH (7.2 ⫾ 0.05) of the culture medium used in the present study. The 1.0 L stock solution (10.0 mmol/L) of caspase-3 inhibitor (Ac-DEVD-CHO; Alexis, San Diego, CA) was diluted in 10.0 mL of culture media to get its final concentration 1.0 mol/L. The various concentrations of NLE were prepared in culture media containing 1.0 mol/L of caspase-3 inhibitor for in vitro studies. Animals and Collection of Oocytes Immature female rats of the Holtzman strain were separated from an existing colony in the departmental animal facility and maintained in normal husbandry conditions with food and water ad libitum. To obtain mature oocytes, the rats (24 –25 days old; 50 ⫾ 5 gm body weight) were given a single subcutaneous injection of 10 IU pregnant mare serum gonadotropin (PMS) in 100 L sterile normal saline to promote growth of a cohort of healthy antral follicles. Fortyeight hours after PMS injection, 10 IU human choronic gonadotropin (hCG) was given to induce superovulation in 1224
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animals. Sixteen hours after hCG injection, the rats were killed. Ovary along with oviduct was removed and transfered to a 35-mm Petri dish containing 2 mL sterile washing media. Superovulated cumulus oocyte complexes (COCs) were collected from the oviduct by manual puncturing with the help of a 26-gauge needle attached to 1-mL insulin syringe and washed three times with washing media. Ovulated cumulus-enclosed oocytes were made free of their cumulus cells by transfering them to culture medium containing 0.1% hyaluronidase for 5 min followed by repeated pipetting through a narrow-bore pipette. The denuded oocytes were used for most of the experiments unless stated otherwise. Groups of 15–16 oocytes were used for one replicate and experiments were repeated at least three times. Effects of Various Doses of NLE on Morphologic Apoptotic Changes in Oocytes Groups of 15 to 20 oocytes were cultured separately in 35-mm Petri dishes containing 2.0 mL culture medium (control group) or medium containing various concentrations (2.5, 5.0, 10.0, and 20.0 mg/mL) of NLE. All Petri dishes were maintained at 37°C for 3.0 h in a CO2 incubator (Thermo Forma, Marietta, OH). At the end of incubation period, oocytes were removed, washed three times with washing media, transfered to grooved slide with 100 L culture medium, and then examined for morphologic apoptotic changes using a phase-contrast microscope (Eclipse E600; Nikon, Tokyo, Japan) at 400⫻ magnification. Time-Course Effects of 10.0 mg/mL NLE on Morphologic Apoptotic Changes in Oocytes Groups of 15–20 oocytes were incubated separately in culture medium with or without 10.0 mg/mL of NLE for various times (1.0, 2.0, and 3.0 h). At the end of each incubation period, oocytes were removed, washed three times with washing medium, and transfered to grooved slide for the analysis of morphologic apoptotic changes using phasecontrast microscope at 400⫻ magnification. Effect of Caspase-3 Inhibitor on 10.0 mg/mL NLE–Induced Morphologic Apoptotic Changes in Oocytes Groups of 15–20 oocytes were incubated separately in plain medium or medium containing 10.0 mg/mL NLE with or without caspase-3 inhibitor (1.0 mol/L) for 3.0 h. At the end of incubation period, oocytes were removed, washed three times with washing medium, and transfered to grooved slide for the analysis of morphological apoptotic changes using phase-contrast microscope at 400⫻ magnification. Eosin/Nigrosin Staining of Oocytes The live and dead status of control and NLE-treated oocytes that underwent degeneration were examined by means of eosin/nigrosin dye-exclusion test per published protocol (25). In
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brief, control oocytes and 10.0 mg/mL NLE–treated oocytes that underwent degeneration were removed, washed three times with washing medium, and then stained with 10 L 1% eosin (w/v) followed by 10 L nigrosin (10% w/v) for 1 min at room temperature. The stained oocytes were observed for live and dead status using a phase-contrast microscope at 400⫻ magnification. Detection of bax Protein by Western Blotting A large number of oocytes (⬃600) are required for the analysis of bax protein expression using Western blotting technique. To collect large number of oocytes, a group of 10 –12 animals were subjected to superovulation induction protocol. The ovulated COCs were first denuded, washed twice with washing medium, and then used for in vitro study. The denuded oocytes were incubated in control medium or medium containing NLE (10 mg/mL) with or without 1.0 mol/L caspase-3 inhibitor. After 3 h of incubation, oocytes were washed three times with washing medium, and 200 oocytes from each group were transfered to 0.6 mL microfuge tube containing 100 L STKM buffer (0.25 mol/L sucrose, 50 mmol/L Tris-HCl, pH 7.5, 25 mmol/L KCl, 5 mmol/L MgCl2, and 0.25% (v/v) Triton X-100) for lysis at 4°C for 15 min. Cell lysates were centrifuged at 4,000g for 20 min at 4°C. The supernatants from all samples were frozen at ⫺30°C until assay. All samples were thawed and total protein content was measured per published protocol (26). Lysates having equal amount of protein (75 g total protein) were then separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing condition and electroblotted following the procedure described earlier (27). In brief, after electroblotting the blot was probed with antibax antibody generated in rabbit (Polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA) diluted (1: 2500) in 1.5% (w/v) BSA-Tris buffered saline supplemented with 0.05% Tween-20 (TBST; 0.05 M Tris-Cl, pH 7.6, 0.15 M NaCl, 0.05% Tween-20) buffer for 1.0 h at room temperature. Nitrocellulose membrane was then washed twice in TBST buffer (5 min each) and further incubated with second antibody (goat-antirabbit IgG conjugated with alkaline phosphatase (Polyclonal; Santa Cruz Biotechnology) diluted (1:10,000) in 1.5% BSA-TBST buffer for 1.0 h at room temperature. The bound antibody was then detected with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; Bio Rad Laboratories, Hercules, CA) color system. In parallel, another set of the same samples was run and probed with anti–-actin antibody generated in rabbit (1:2,500 dilutions; Polyclonal; Santa Cruz Biotechnology) and localized using goat-antirabbit IgG conjugated with alkaline phosphatase (Polyclonal; Santa Cruz Biotechnology) to confirm that equivalent amounts of protein were analyzed. Detection of DNA Fragmentation by TUNEL Assay The DNA fragmentation was detected using a terminal deoxynucleotidyl transferase (TDT) nick-end labeling (TUNEL) kit from R&D Systems (Minneapolis, MN) per company manual protocol. The control and 10 mg/mL NLE–treated cuFertility and Sterility姞
mulus-enclosed oocytes (15–16 oocytes in each group) were fixed in 3.7% (v/v) formaldehyde in PBS for 15 min at 18 –20°C. After washing, oocytes were transfered separately on to poly-L-lysine– coated slides and then air-dried. Slides were treated with 50 L proteinase K solution for 30 min and then immersed in quenching solution for 3– 4 min. Thereafter, slides were immersed in 1⫻ TDT labeling buffer for 5 min and then incubated with 50 L labeling reaction at 37°C for 1.0 h. The slides were immersed in 1⫻ TDT stop buffer to stop the reaction. Washed slides were incubated with 50 L diluted (1:500) biotin-labeled anti-BrdU at 37°C for 1.0 h. Slides were washed with PBS containing 0.05% Tween-20 and then treated with 50 L streptavidin-horseradish peroxidase solution for 10 min. Washed slides were immersed in diaminobenzidine (DAB) solution for 5 min and then in methyl green solution for 2 min after washing. The slides were again washed sequentially by dipping at least ten times in distilled water, 50%, 70%, 95%, and 100% ethanol, and finally in xylene for two changes each. Slides were mounted in distyrene plasticizer xylene (DPX) and then analyzed for TUNEL-positive staining under phase-contrast microscope at 100⫻ and 400⫻ magnifications. The TUNEL analysis was repeated three times and representative photographs are shown in the result section. Isolation and Electrophoresis of Total DNA A large number of oocytes (⬃600) are required for DNA fragmentation analysis using DNA-gel electrophoresis. To collect a large number of oocytes, a group of 10 –12 animals were subjected to superovulation induction protocol. Ovulated cumulus-enclosed oocytes were denuded, washed twice with washing medium. and then used for in vitro study. These denuded oocytes were incubated in control media or media containing 10 mg/mL NLE. After 3 h of incubation, oocytes were removed from control and NLE-treated groups and used for DNA isolation of low-molecular-weight DNA along with genomic DNA (total DNA) following the protocol reported elsewhere (28) with some modifications. In brief, 300 oocytes from control and NLE-treated groups were separately lysed in 100 L lysis buffer (5 mmol/L Tris, 20 mmol/L EDTA, 0.5% Triton X-100, pH 8.0) for 1.0 h on ice. The samples were treated with 5 L RNase A (10 mg/mL) for 1.0 h at 37°C followed by 5 L proteinase K (10 mg/mL) at 50°C for 1.0 h. The DNA samples were kept at ⫺30°C until the collection of DNA samples. All samples were thawed quickly, pooled in respective groups, and then centrifuged at 10000g at 4°C for 20 min. The supernatants were collected and extracted twice with saturated phenol– chloroform–isopropyl alcohol (25:24:1 v/v/v). Then 200 L aqueous phase was transfered to a microcentrifuge tube and kept at ⫺30°C until use. To the 20 L sample, 20 L TE buffer (10 mmol/L Tris, 1 mmol/L EDTA, pH 8.8) was added and mixed gently. Then the 20-L sample was transfered to a 0.5-mL microcentrifuge tube and 4 L 6⫻ gel loading buffer (50% glycerol, 1% bromophenol blue, and 1% xylene cynol in Trisborate EDTA (0.045 M Tris-borate and 0.001 M EDTA, pH 8.0 buffer) was mixed in each sample. Samples were electropho1225
TABLE 1 Effects of various doses of neem leaf extract (NLE) on morphologic apoptotic changes in oocytes (15–20 oocytes in each group) cultured in vitro for 3.0 h. Oocytes
NLE treatment (mg/mL)
Shrinkage (%)
Membrane leakage (%)
Degeneration (%)
Control 2.5 5.0 10.0 20.0
Nil 71.88 ⫾ 1.46ⴱ 90.90 ⫾ 4.55ⴱ 57.21 ⫾ 3.36 Not observed
Nil Nil Nil 43.07 ⫾ 3.41ⴱ 20.0 ⫾ 4.0
Nil Nil Nil Nil 80.0 ⫾ 5.0ⴱ
Note: Values are mean ⫾ SE. ⴱ P⬍.001 (control vs. NLE-treated groups). Chaube. Neem leaf extract–induced oocyte apoptosis in rat. Fertil Steril 2006.
resed on 1% agarose gel (Bio Rad) containing 0.5 g/mL ethidiumbromide for 3 h at 25 V. Gel was visualized and photographed on a UV transilluminator (UVP BioImaging Systems, Upland, CA). The DNA gel electrophoresis was repeated three times. Statistical Analysis Data are expressed as mean ⫾ standard error of the mean (SEM) of triplicate samples. All percentage values were subjected to arc-sine square root transformation before statistical analysis. Data are analyzed by Student t test. A probability of P⬍.001 was considered to be statistically significant. RESULTS Effects of Various Doses of NLE on Morphologic Apoptotic Changes in Oocytes As shown in Table 1, lower doses (2.5 and 5.0 mg/mL) of NLE significantly induced shrinkage of oocytes after 3.0 h of incubation. A higher dose of NLE (10.0 mg/mL) induced
shrinkage (in 57.21% ⫾ 3.36% of oocytes) and membrane leakage (in 43.07% ⫾ 3.41%). If the dose of NLE was further increased to 20.0 mg/mL, only 20.0% ⫾ 4.0% oocytes showed membrane leakage, whereas a majority of oocytes (80.0% ⫾ 5.0%) underwent degeneration (Table 1). Time-Course Effect of 10.0 mg/mL NLE on Morphologic Apoptotic Changes in Oocytes Time-course study revealed that 10.0 mg/mL NLE significantly induced shrinkage of oocytes (in 49.27% ⫾ 4.78%) after 1.0 h of treatment. The rate of oocyte shrinkage was further increased (80.37% ⫾ 1.40%), if the treatment period was increased up to 2.0 h. A shift in the percentage of oocytes undergoing morphologic apoptotic changes from shrinkage to membrane leakage was observed after 2.0 h of NLE treatment, and all treated oocytes underwent either shrinkage (63.13% ⫾ 0.94%) or membrane leakage (37.42% ⫾ 2.20%) after 3.0 h of NLE treatment. Although shrinkage was not observed after 4.0 h of NLE treatment, around 22%
TABLE 2 Time-course effects of 10.0 mg/mL neem leaf extract (NLE) on morphologic apoptotic changes in oocytes (15–20 oocytes in each group) cultured in vitro for 4 h. Oocytes
NLE treatment (in h)
Shrinkage (%)
Membrane leakage (%)
Degeneration (%)
Control 1.0 2.0 3.0 4.0
Nil 49.27 ⫾ 4.78ⴱ 80.37 ⫾ 1.40ⴱ 63.13 ⫾ 0.94 Not observed
Nil Nil Nil 37.42 ⫾ 2.20ⴱ 21.79 ⫾ 1.45
Nil Nil Nil Nil 79.21 ⫾ 2.85ⴱ
Note: Values are mean ⫾ SE. ⴱ P⬍.001 (control vs. NLE-treated groups). Chaube. Neem leaf extract–induced oocyte apoptosis in rat. Fertil Steril 2006.
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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.
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a higher dose of NLE (20.0 mg/mL) induced degeneration of oocytes after 3.0 h of treatment.
FIGURE 2 Effects of 10.0 mg/mL neem leaf extract (NLE) with or without 1.0 mol/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.
of oocytes showed membrane leakage, and 79.21% ⫾ 2.85% underwent degeneration (Table 2).
Assessment of Progression of Morphologic Apoptotic Changes in NLE-Treated Oocytes Oocytes collected after superovulation induction protocol from the oviduct of immature rats were at metaphase I and did not show first polar body. These mature oocytes (absence of GV and nucleolus) extrude first polar body and did not show any morphologic apoptotic changes after 3.0 h of in vitro culture (Fig. 1A). When these oocytes were treated with 10.0 mg/mL NLE for various times, shrinkage of oocyte volume was observed (Fig. 1B) compared to control (Fig. 1A). Though the shrinkage was a first visual change observed in NLE-treated oocytes, a leakage of transparent fluid (membrane leakage) was observed after 2.0 h of incubation and progressed along with shrinkage simultaneously (Fig. 1C–F). Half of the oocyte was shriunk and the other half was filled with leaked transparent fluid after 3.0 h of NLE treatment (Fig. 1G) as compared to control oocytes that had first polar body with normal morphology (Fig. 1A). Some of NLE⫺treated oocytes showed cytoplasmic fragmentation (Fig. 1H) compared to control oocytes (Fig.1A) prior to degeneration only at a higher dose of NLE (20.0 mg/mL) after 3.0 h of treatment. Live and Dead Status of Control and NLE-Treated Oocytes The control oocytes did not take nigrosin stain (Fig. 1I), and NLE-treated oocytes that underwent degeneration showed nigrosin staining (Fig. 1J), further supporting our results that 1228
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Effect of NLE (10 mg/mL) With or Without Caspase-3 Inhibitor (1.0 mol/L) on the Rate of Morphologic Apoptotic Changes in Oocytes When oocytes were treated with 10.0 mg/mL NLE alone, almost all oocytes (98%) showed either shrinkage or membrane leakage after 3.0 h of culture. Addition of caspase-3 inhibitor (1.0 mol/L) prevented these morphologic apoptotic changes (i.e., shrinkage and membrane leakage) in NLEtreated oocytes, with only 14% oocytes showing morphologic apoptotic change, i.e., shrinkage (Fig. 2). Effect of NLE on bax Protein Expression in Oocytes Control and 10.0 mg/mL NLE–treated oocytes with or without caspase-3 inhibitor (1.0 mol/L) were used for the analysis of bax protein expression. As evident from the Western blot analysis (Fig. 3), NLE induced overexpression of bax protein in treated oocytes compared to control oocytes. Further, a densitometry analysis revealed that a treated lane had 3.0 times higher bax protein expression compared to control lane. Addition of caspase-3 inhibitor inhibited NLE-induced overexpression of bax protein, and the result was similar to the control lane. DNA Fragmentation Analysis in Control and NLE-Treated Oocytes Using TUNEL Assay To confirm the occurrence of DNA fragmentation, control and 10 mg/mL NLE–treated cumulus-enclosed oocytes (15–16 oocytes in each group) were subjected to TUNEL assay. Treat-
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 mol/L caspase-3–treated oocyte lysate. Lower portion shows a control assay for -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.
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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.
ment of NLE induced DNA fragmentation (TUNEL-positive staining) selectively in oocytes but not in associated cumulus cells (Fig. 4B) compared to control oocytes that did not show TUNEL staining (Fig. 4A). DNA Fragmentation Analysis in Control and NLE-Treated Oocytes Using DNA Gel Electrophoresis As shown in Figure 5, DNA isolated from control and NLE-treated oocytes were run on 1% agarose gel. The control oocytes that had normal morphology did not show DNA ladder, and intact genomic DNA was seen after 3.0 h of culture (Fig. 5, lane C). On the other hand, the extent of DNA cleavage into multiples of 180 –200 bp was significantly increased and detectable as a ladder pattern in 10.0 mg/mL NLE–treated oocytes that had morphologic apoptotic features (Fig. 5, lane T). DISCUSSION Apoptosis is a genetically controlled, highly conserved, intricate mechanism of cellular suicide and disposal of cells in the absence of inflammatory reactions that can be triggered by a variety of physiologic and pathologic conditions. Apoptosis plays a central role in the development and maintenance of homeostasis in multicellular organisms (20, 24). It is characterized by a series of distinct morphologic, biochemical, and molecular changes in dying cells (21–23). The morphologic changes in dying cells include cell shrinkage, nuclear condensation, cytoplasmic granulation, membrane blebbing, and cytoplasmic fragmentation (22, 29, 30). Data of Fertility and Sterility姞
the present study clearly suggest that NLE induced morphologic apoptotic changes such as shrinkage and membrane leakage in treated oocytes. Shrinkage of oocyte volume was a first visual change that occurred after 1.0 h of NLE treatment. These shrinking oocytes showed a leakage of transparent cytoplasmic fluid out of corona that progressed as the treatment time of NLE was increased. Almost half of the oocyte volume was filled with leaked cytoplasmic fluid after 3.0 h of NLE treatment. Some oocytes showed cytoplasmic fragmentation just prior to degeneration. The NLE-induced degeneration of oocytes was confirmed by live and dead staining using eosin/nigrosin double staining. The NLEinduced morphologic apoptotic changes were inhibited if the oocytes were cotreated with NLE (10.0 mg/mL) along with caspase-3 inhibitor (1.0 mol/L) for 3.0 h. These data clearly suggest that NLE-induced morphologic changes characteristic of apoptosis in oocytes and caspase-3 activation is involved during NLE-induced morphologic apoptotic changes in the present study. The apoptosis was initiated with shrinkage of oocyte volume followed by membrane leakage and then cytoplasmic fragmentation into unequal bodies that is an end point in the process of apoptosis prior to degeneration. The similar sequence of morphologic apoptotic changes has been reported in mouse and rat oocytes cultured in vitro (22, 23, 30, 31). It is generally accepted that a ratio of apoptotic promoter (such as bax expression) to suppressors (such as bcl-2 expression) within a cell determine whether a cell will under go apoptosis or survive (20). Although a ratio of bax/bcl-2 gene products was not analyzed in the present study, we carried 1229
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.
out a Western blot analysis to find out whether NLE induced proapoptotic signals, i.e., overexpression of bax protein in control and NLE-treated oocytes with or without caspase-3 inhibitor. Western blot analysis revealed that NLE-treated oocytes showed overexpression of bax protein. Densitometry analysis further revealed that NLE-treated oocytes had 3.0 times more bax protein than control oocytes. Addition of caspase-3 inhibitor prevented NLE-induced overexpression of bax protein in the present study; the amount of bax protein was similar to control oocytes. These results clearly suggest that overexpression of bax gene might have modulated the ratio of apoptotic promoters to suppressors (bax/bcl-2) within a cell, thereby setting NLE-treated oocytes toward apoptosis. Because caspase-3 inhibitor has prevented NLEinduced overexpression of bax gene, it is quite possible that NLE-induced morphologic apoptotic features might be mediated through bax/bcl-2– caspase-3 pathway in oocytes. Similarly, the involvement of overexpression of bax protein and caspase-3 activation during oocytes apoptosis has been reported in mouse and rat oocytes (22, 24, 32, 33). A unique biochemical event in apoptosis that precedes morphologic changes such as shrinkage, membrane blebbing, and fragmentation of cytoplasm into unequal bodies is due to the activation of Ca⫹⫹/Mg⫹⫹-dependent endonuclease (24). This enzyme cleaves genomic DNA at the internucleosomal region resulting in 180 –200-base-pair DNA oligonucleosomal fragments. These fragmented DNA can be 1230
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detected in a single cell using an in situ technique such as TUNEL or DNA gel electrophoresis, where fragmented DNA appears in a ladder pattern (22, 30, 34 –37). Further, TUNEL-positive staining has been reported in oocytes that had morphologic apoptotic features (22, 30, 31, 34). To analyze NLE-induced DNA fragmentation in the female germ cells, we used cumulus-enclosed oocytes, because denuded oocytes were getting washed off owing to repeated washing during the TUNEL assay. In the present study, TUNEL-positive staining was observed in NLE-treated oocytes and not in the associated cumulus cells, suggesting that NLE might have selective access at the level of oocytes and not in associated cumulus cells. When total DNA was isolated from control and NLE-treated oocytes and electrophoresed on agarose gel, a distinctive DNA ladder (a hallmark feature of apoptosis) was detected in the NLE-treated lane, whereas the control lane had intact genomic DNA. Taken together, these data strongly suggest that NLE induced DNA fragmentation selectively in germ cells (oocytes) and not in associated somatic cell (cumulus cells) in vitro. In conclusion, the present study for the first time clearly demonstrates that NLE induced morphologic apoptotic changes such as shrinkage, membrane leakage, and cytoplasmic fragmentation just before degeneration. These morphologic changes were associated with the overexpression of bax protein and caspase-3 activation, DNA fragmentation as evidenced by TUNEL-positive staining, and a detectable ladder pattern on DNA gel electrophoresis in NLE-treated oocytes. Acknowledgment: We are grateful to Profs. Neeraj K. Sethi, M.D. (Director), Krishnamurthy Kalaivani, M.D. (H.O.D., R.B.M., Dean of Studies), and S. Roy, all from the National Institute of Health and Family Welfare, New Delhi, India, for their keen interest and encouragement throughout the study.
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