An increase of granulosa cell apoptosis mediates aqueous neem (Azadirachta indica) leaf extract-induced oocyte apoptosis in rat

Abstract

Objective: Neem plant (Azadirachta indica) has been extensively used in Ayurvedic system of medicine for female fertility regulation for a long time, but its mechanism of action remains poorly understood. Hence, the present study was aimed to determine whether an increase of granulosa cell apoptosis is associated with aqueous neem leaf extract (NLE)-induced oocyte apoptosis.

Materials and methods: Sexually immature female rats of 20 days old were fed NLE (50 mg/day) for 10 days and then subjected to superovulation induction protocol. The morphological changes in cumulus oocyte complexes (COCs), rate of oocyte apoptosis, hydrogen peroxide (H2O2), total nitrite, and cytochrome c concentrations, inducible nitric oxide synthase (iNOS), cytochrome c, p53, Bcl2 and Bax expressions, deoxyribonucleic acid (DNA) fragmentation, and estradiol 17β level in granulosa cells collected from preovulatory COCs were analyzed.

Results: Aqueous NLE increased H2O2 concentration and decreased catalase activity, increased iNOS expression and total nitrite concentration, increased p53, Bax, and p53 expressions but decreased Bcl2 expression, increased cytochrome c concentration and induced DNA fragmentation in granulosa cells. An increased granulosa cell apoptosis resulted in reduced estradiol 17β concentration and induced apoptosis in ovulated oocytes.

Conclusion: We conclude that aqueous NLE-induced granulosa cell apoptosis through the mitochondria-mediated pathway, reduced estradiol 17β concentration and induced apoptosis in ovulated oocytes. Thus, granulosa cell apoptosis mediates NLE-induced oocyte apoptosis during female fertility regulation in rat.

Keywords: Aqueous neem leaf extract; Bax/Bcl2; deoxyribonucleic acid fragmentation; granulosa cell apoptosis; rat oocytes; reactive oxygen species.

Figure 1

Representative photographs showing neem leaf extract (NLE) induced morphological changes in ovulated cumulus oocyte complexes (COCs). (a) Control, cumulus oocytes complex showing greater number and tightly intact cumulus granulosa cells with oocyte (arrows). NLE-treated-A, NLE (50 mg/day for 10 days) reduced number of granulosa cells and induced shrinkage in the majority of COCs (shrinkage; arrow). NLE-treated-N, Remaining COCs showing normal morphology with a reduced number of granulosa cells (arrows). (b) Effect of NLE treatment on the rate of oocytes apoptosis. Data are mean ± SE of three replicates. *Denotes significantly (P < 0.05) higher as compare to control (n = 3/group; Student's t-test). Bar = 40 μm

Figure 2

Neem leaf extract (50 mg/day for 10 days) treatment increased H₂O₂ concentration (a) and reduced catalase activity (b) in granulosa cells of preovulatory cumulus oocyte complexes. Data are mean ± SE of three replicates. *Denotes significantly (P < 0.05) different as compare to control (n = 3/group; Student's t-test)

Figure 3

Representative photograph showing the effect of neem leaf extract (NLE) (50 mg/day for 10 days) on immunocytochemical localization of inducible nitric oxide synthase (iNOS) expression and total nitrite concentration in granulosa cells of preovulatory COCs. Most of the granulosa cells collected from preovulatory COCs of NLE-treated animal show higher iNOS expression as compared to control (a; arrows). NLE treatment increased total nitrite concentration in granulosa cells of preovulatory COCs (b). Data are mean ± SE of three replicates. *Denotes significantly (P < 0.05) higher as compare to control (n = 3/group; Student's t-test). Bar = 40 μm

Figure 4

Representative immunofluorescence photograph showing the effects of neem leaf extract (NLE) (50 mg/day for 10 days) treatment on p53, Bax, and Bcl2 expression in granulosa cells of preovulatory cumulus oocyte complexes. NLE treatment increased p53 (a; arrows), Bax (b; arrows), and decreased Bcl2 (c; arrows) expression in granulosa cells. Bar = 40 μm

Figure 5

Representative photograph showing effects of neem leaf extract (NLE) (50 mg/day for 10 days) treatment on immunocytochemical localization of cytochrome c expression and cytochrome c contration in granulosa cells isolated from cumulus oocyte complexes (COCs) collected from ovary. Most of the granulosa cells collected from preovulatory COCs of NLE-treated animal show higher cytochrome c expression as compared to control (a; arrows) and increased cytochrome c concentration (b) in granulosa cells collected from ovary. Data are mean ± SE of three replicates. *Denotes significantly (P < 0.05) higher as compare to control (n = 3/group; Student's t-test). Bar = 40 μm

Figure 6

Representative photograph showing effects of neem leaf extract (NLE) (50 mg/day for 10 days) treatment on deoxyribonucleic acid (DNA) fragmentation in granulosa cells isolated from preovulatory as well as ovulated cumulus oocyte complexes (COCs). (a) Granulosa cells collected from preovulatory COCs did not show any DNA fragmentation as evidenced by green fluorescence of acridine orange (AO). (b) NLE-induced apoptosis in most of the granulosa cells of preovulatory COCs as evidenced by yellowish orange fluorescence of ethidum bromide (EB) (arrows). (c) The initiation of apoptosis as evidenced by yellow fluorescence of EB was observed in granulosa cells of ovulated COCs (arrows). (d) NLE treatment induced cell death as evidenced by reddish fluorescence of EB and reduced number of granulosa cells of ovulated COCs (arrows) (n = 3/group). Bar = 40 μm

Figure 7

Representative photograph showing neem leaf extract (NLE) (50 mg/day for 10 days) induced deoxyribonucleic acid (DNA) fragmentation in granulosa cells encircling oocytes that showed normal morphology at the time of collection. (a) Granulosa cells encircling preovulatory oocytes did not show DNA fragmentation as evidenced by green fluorescence of acridine orange (AO). (b) NLE-induced apoptosis in most of the granulosa cells encircling preovulatory oocyte as evidenced by yellowish orange fluorescence of ethidium bromide (EB) (arrows). (c) The initiation of apoptosis was also observed in granulosa cells encircling ovulated oocyte as evidenced by yellow fluorescence of EB (arrow). (d) NLE treatment induced cell death thereby reduced number of granulosa cells encircling ovulated oocyte as evidenced by reddish fluorescence of EB (arrows) (n = 3/group). Bar = 40 μm

Figure 8

Neem leaf extract (50 mg/day for 10 days) treatment reduced estradiol 17β concentration in granulosa cells isolated from preovulatory cumulus oocyte complexes. Data are mean ± SE of three replicates. *Denotes significant (P < 0.05) decrease as compare to control granulosa cell lysate (n = 3/group; Student's t-test)