Hum. Reprod. Advance Access originally published online on July 27, 2006
Human Reproduction 2006 21(11):2985-2995; doi:10.1093/humrep/del255
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Ginkgolide B induces apoptosis and developmental injury in mouse embryonic stem cells and blastocysts
Department of Bioscience Technology and Center for Nanotechnology, Chung Yuan Christian University, Chung Li, Taiwan
E-mail: whchan{at}cycu.edu.tw
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Abstract |
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BACKGROUND: Ginkgolide B, the major active component of Ginkgo biloba extracts, can both stimulate and inhibit apoptotic signalling. We previously showed that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards early post-implantation blastocyst development and increases early-stage blastocyst death. Here, we report more detailed examinations of the cytotoxic effects of ginkgolide B on mouse embryonic stem cells (ESCs) and blastocysts and their subsequent development in vitro and in vivo. METHODS AND RESULTS: Using cell culture assay model, we revealed in our results that ginkgolide B treatment of ESCs (ESC-B5) induced apoptosis via reactive oxygen species (ROS) generation, c-Jun N-terminal kinase (JNK) activation, loss of mitochondrial membrane potential (MMP) and the activation of caspase-3. Furthermore, an in vitro assay model showed that ginkgolide B treatment inhibited cell proliferation and growth in mouse blastocysts. Finally, an in vivo model showed that treatment with 10 µM ginkgolide B caused resorption of post-implantation blastocysts and fetal weight loss. CONCLUSIONS: Our results reveal for the first time that ginkgolide B retards the proliferation and development of mouse ESCs and blastocysts in vitro and causes developmental injury in vivo.
Key words: apoptosis/blastocysts/development/ginkgolide B/stem cell
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Introduction |
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Ginkgolide B, the major component of extracts from leaves of Ginkgo biloba L. traditionally used in Chinese herbal medicine, exerts a wide range of biological activities, including antiangiogenesis and antioxidation (Ahlemeyer and Krieglstein, 2003
; DeFeudis et al., 2003; Bate et al., 2004). Ginkgolides are terpenoid compounds and ameliorate tissue irrigation by augmenting cellular energy in the arterial, venous and capillary circulation. In particular, ginkgolide B possesses antioxidant activity and is a potent peroxy radical scavenger (Maitra et al., 1995). Ginkgolides decrease singlet oxygen, hydrogen peroxide and hydroxyl radical production, supporting their role as antioxidants (Pincemail et al., 1987). Recent reports show that the increase in free radicals and associated lipid peroxidation reactions is suppressed in the central nervous system and heart upon treatment with extracts of Ginkgolide biloba leaves (Shen and Zhou, 1995; Pietri et al., 1997). In addition, G. biloba extracts inhibit 
-amyloid aggregation-induced caspase-3 activation and cell apoptosis (Luo et al., 2002). Another study demonstrated that G. biloba extracts induce injury of oral cavity tumours via apoptosis processes (Kim et al., 2005). Thus, extracts of this compound appear to have both anti- and pro-apoptotic effects. However, although multiple biological functions of G. biloba extracts have been identified to date, the precise molecular mechanisms underlying these actions remain to be established.
Apoptosis, which is widely observed in different cells of various organisms, is a unique morphological pattern of cell death characterized by chromatin condensation, membrane blebbing and DNA fragmentation (Ellis et al., 1991). Apoptosis plays an important role in the homeostasis of multicellular organisms. Moreover, abnormal apoptotic function is associated with several human diseases including neurodegenerative disorders and cancers (Thompson, 1995). In normal embryogenesis, the role of apoptosis is to clear abnormal or redundant cells in preimplantation embryos (Hardy, 1997; Hardy et al., 2003). However, apoptosis does not occur before the blastocyst stage in normal mouse embryonic development (Byrne et al., 1999). Apoptosis triggered in the early stages, such as that due to teratogen hazard exposure, causes embryonic development injury (Dong et al., 2002; Little et al., 2003). These results imply that teratogen-induced injury in the early stages of embryonic development occurs via its apoptotic trigger properties. Reactive oxygen species (ROS) generation plays a critical role in apoptotic progression in embryonic stem cells (ESCs) (Hsuuw et al., 2005). However, the precise mechanisms are unclear at present.
ROS are oxygen-containing molecules having either unpaired electrons or the ability to acquire electrons from other molecules. Many chemical and physical treatments capable of inducing apoptosis are known to provoke oxidative stress via ROS generation (Halliwell and Gutteridge, 1990; Hsuuw et al., 2005; Pathak and Khandelwal, 2006; Yan et al., 2006), suggesting a close relationship between oxidative stress and apoptosis. Recent studies demonstrate that UV irradiation, hyperosmotic shock, heat shock, photodynamic therapy and several other factors induce apoptosis via oxidative stress in various mammalian cells (Chan et al., 1999, 2000, 2003; Chan and Wu, 2004; Pathak and Khandelwal, 2006; Yan et al., 2006). In addition, oxidative stress triggered by oxidants such as H2O2 may directly induce apoptosis, whereas the addition of antioxidants may block this effect (Buttke and Sandstrom, 1994). These lines of evidence collectively indicate that ROS are important inducers of apoptotic processes.
Although the precise molecular mechanisms for apoptosis have not been clearly defined, caspase activation, mitochondrial membrane potential (MMP) changes and c-Jun N-terminal kinase (JNK) activation are thought to play critical roles (Chan and Wu, 2004; Hsuuw et al., 2005). Caspase zymogens are activated by proteolysis, leading to apoptosis; this effect may be inhibited in vitro and in vivo by small tetrapeptidic inhibitors (Milligan et al., 1995; Nicholson and Thornberry, 1997). MMP changes and the release of mitochondrial cytochrome C are mediated by Bcl-2 family members, which play important roles in regulating apoptosis (Tsujimoto and Shimizu, 2000). The Bcl-2 proteins mediate changes in the permeability of the outer mitochondrial membrane and may be divided into anti-apoptotic and pro-apoptotic subgroups (Adams and Cory, 1998; Tsujimoto and Shimizu, 2000). Finally, changes in protein kinase activity can be observed during apoptosis in various cell types (Anderson, 1997), indicating that protein phosphorylation is likely to be involved in the regulation of apoptosis. One major protein kinase, JNK, acts as a key component in regulating the apoptotic entry of several cell types (Xia et al., 1995; Verheij et al., 1996; Seimiya et al., 1997).
ESCs are pluripotent and early embryo-derived cells. Upon culture in the presence of anti-differentiation agents, such as embryonic fibroblasts or leukaemia-inhibitory factor (LIF), ESCs proliferate while maintaining the capacity to differentiate into any cell type in the body (Evans and Kaufman, 1981). When the anti-differentiation agent is withdrawn, ESCs spontaneously differentiate and develop in a manner that recapitulates early embryogenesis (Keller, 1995). When ESCs differentiate in suspension culture, they form typical 3D aggregates called embryoid bodies (EBs), which consist of ectodermal, mesodermal and endodermal tissues, thus resembling the egg-cylinder stage of an embryo. These properties have made EB formation a useful in vitro system for studying early embryo development and differentiation processes.
In an effort to examine the effects of components of G. biloba extracts on embryogenesis, we previously exposed mouse blastocysts to ginkgolide A and ginkgolide B, which are the main active ingredients in G. biloba extracts. We found that ginkgolide treatment triggered apoptosis in the inner cell mass (ICM), blastocoele and trophectoderm (TE) and also negatively affected early post-implantation embryonic development on culture dishes in vitro. However, the precise mechanisms governing ginkgolide-induced apoptosis of blastocyst cells were previously unknown.
Earlier studies by our group demonstrated that ginkgolide B has an apoptotic effect on mouse blastocysts in vitro. Here, we investigate whether ginkgolide B has a hazardous effect on embryo development in cell culture and animal assay models and discuss the possible mechanisms of ginkgolide B-induced embryo development injury. Currently, the effects of dietary G. biloba L. extracts on human embryo development are unclear. Our data provide important evidence that dietary extracts of the leaves of G. biloba L. affect embryogenesis in an in vivo pregnancy mouse model. On the basis of the results, we propose that pregnant women should avoid using G. biloba L. leaf extracts for medical therapy. However, the precise mechanisms and physiological effects of ginkgolide B on human embryogenesis remain to be established. We demonstrate that ginkgolide B treatment triggers ROS generation, loss of MMP and the activation of JNK and caspase-3. In addition, treatment of blastocysts with ginkgolide B significantly inhibits embryonic development and induces apoptosis, both in vitro and in vivo.
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Materials and methods |
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Materials
Dulbecco’s modified Eagle’s medium (DMEM), ginkgolide B, sodium pyruvate, 2',7'-dichlorofluorescin diacetate (DCF-DA), dihydrorhodamine 123 (DHR 123) and pregnant mare’s serum gonadotrophin (PMSG) were obtained from Sigma (St Louis, MO, USA). HCG was purchased from Serono (NV Organon Oss, the Netherlands). Z-DEVD-AFC (flourogenic substrate for caspase-3) was obtained from Calbiochem (La Jolla, CA, USA). CDP-StarTM (a chemiluminescent substrate for alkaline phosphatase) was purchased from Boehringer Mannheim (Mannheim, Germany), whereas bicinchoninic acid (BCA) protein assay reagent was obtained from Pierce (Rockford, IL, USA). The anti-JNK1 (C17) and anti-p-JNK (G-7) antibodies as well as alkaline phosphatase-conjugated goat anti-rabbit and anti-mouse immunoglobulin G (IgG) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The terminal deoxynucleotidyl transferase (TdT)-mediated dUDP nick-end labelling (TUNEL) in situ cell death detection kits were obtained from Roche Molecular Biochemicals (Mannheim, Germany), and the CMRL-1066 medium was a product of Gibco Life Technologies (Grand Island, NY, USA).
Cell culture and ginkgolide B treatment
Mouse ESCs (ESC-B5) were cultured in DMEM supplemented with 20% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were plated on 60-mm culture dishes, and ginkgolide B treatments were performed the following day. For ginkgolide B treatment, cells were incubated in medium containing various concentrations of ginkgolide B (5–10 µM) at 37°C in a CO2 incubator for 24 h. Cells were then washed twice with ice-cold phosphate-buffered saline (PBS) and lysed on ice for 10 min in 400 µl of lysis buffer [20 mM Tris–HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM benzamidine, 1 mM phenylmethylsulphonyl fluoride, 50 mM NaF, 20 µM sodium pyrophosphate and 1 mM sodium orthovanadate]. Cell lysates were sonicated on ice for 3 x 10 s followed by centrifugation at 15 000 x g for 20 min at 4°C. The supernatants were used as cell extracts.
MTT assay
The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test is a colorimetric assay that measures the percentage of cell survival. Following treatment of ESC-B5 cells with ginkgolide B, 100 µl of 0.45 g/l MTT solution was added to each well in 96-well culture plate. Cells were incubated at 37°C for 60 min to allow colour development, and 100 µl of 20% sodium dodecyl sulphate (SDS) in DMF:H2O (1:1) solution was added to each well to halt the reaction. The plates were then incubated overnight at 37°C to dissolve the formazan products. The results were analysed by spectrophotometry using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 570 nm.
Apoptosis assay
Cells (1 x 105) were treated with or without various concentrations of ginkgolide B at 37°C for 24 h, and oligonucleosomal DNA fragmentation in apoptotic cells was measured using the Cell Death Detection ELISAplus kit, according to the manufacturer’s protocol (Roche Molecular Biochemicals). Spectrophotometric data were obtained at 405 nm using an ELISA reader.
Caspase-3 activity assay
Caspase-3 activity was measured using the fluorogenic substrate, Z-DEVD-AFC. Cell lysates (100 µg) were incubated in 250 µl of caspase assay buffer [25 mM HEPES (pH 7.5), 0.1% CHAPS, 10 mM dithiothreitol (DTT) and 100 U/ml of aprotinin] containing 0.1 mM Z-DEVD-AFC for 3 h at 37°C. Ice-cold caspase assay buffer (1.25 ml) was added to the mixture, and the relative caspase-3 activity was determined using a fluorescence spectrophotometer (F-2000, Hitachi; excitation 400 nm, emission 505 nm).
ROS assay
ROS were measured in arbitrary units using DCF-DA and DHR 123 dye. Cells (1.0 x 06) were incubated in 50 µl of PBS containing 20 µM DCF-DA for 1 h at 37°C, and relative ROS units were determined using a fluorescence ELISA reader (excitation 485 nm, emission 530 nm). An aliquot of the cell suspension was lysed, the protein concentration was determined and the results were expressed as arbitrary absorbance units per milligram protein.
Detection of MMP
ESC-B5 cells were grown in 96-well plates for 24 h and then incubated with 5–10 µM ginkgolide B. Twenty-four hours after the addition of the ginkgolide B, fluorescence dyes [DiOC6(3) (20 nM) or tetramethylrhodamine ethyl ester, perchlorate (TMRE) (0.1 µM)] were added to the wells, and the plates were incubated for 15 min. Fluorescent emissions were then measured with a plate spectrofluorometer [excitation: 485 nm for DiOC6(3) and 535 nm for TMRE; emission: 535 nm for DiOC6(3) and 590 nm for TMRE].
JNK activity assay
JNK activity, measured by the presence of phosphorylated c-Jun protein, was analysed using the AP-1/c-Jun ELISA kit, according to the manufacturer’s protocol (Active Motif, Carlsbad, CA, USA). Briefly, AP-1 heterodimeric complexes in cellular nuclear extracts were collected by binding to a consensus 5'-TGA(C/G)TCA-3' oligonucleotide coated on a 96-well plate. The phospho-c-Jun was assayed using a phospho-c-Jun primary antibody and a secondary horse-radish peroxidase-conjugated antibody in a colorimetric reaction.
Immunoblots
Immunoblotting was essentially performed according to a previous report by our group (Chan, 2005). Briefly, proteins were resolved by SDS–polyacrylamide gel electrophoresis (PAGE), transferred to polyvinylidene difluoride (PVDF) membranes and immunoblotted with anti-JNK, anti-phospho JNK, anti-caspase-3, anti-p-STAT3 and anti-OCT4 antibodies (0.25 µg/ml). Proteins of interest were detected with alkaline phosphatase-conjugated goat anti-rabbit or anti-mouse IgG antibodies, and visualized using the CDP-StarTM chemiluminescent substrate, according to the manufacturer’s protocol.
EB formation
EBs were formed as previously described (Dang et al., 2002). Briefly, ESC-B5 cells were dissociated by trypsin–EDTA (0.25%) and cultured in LIF-free stem cell culture medium to induce differentiation. Cell suspension liquid cultures (<103 cells/ml) were dispensed to 10-cm Petri dishes at 10 ml per dish. Hanging drop cultures were prepared using 10-µl droplets, each containing an appropriate number of ESC-B5 cells for the initiation of EB formation. The ESC-B5 cells were allowed to aggregate in the hanging drops for 2 days and were then transferred to liquid suspension culture. For cell differentiation study, embryoid body cells were treated with 50 ng/ml of nerve growth factor (NGF) for 14 days to induce differentiation into nerve cells, along with expression of microtubule-associated protein-2 (MAP-2), a major nerve cell biomarker.
Collection of mouse blastocysts
CD-1-(ICR) virgin albino mice (6–8 weeks old) were induced to superovulate by the injection of 5 IU PMSG followed 48 h later by the injection of 5 IU HCG. The mice were then mated overnight with a single fertile male of the same strain. Female mice with vaginal plugs were separated and used for experiments. All mice were maintained on breeder chow and kept under a 12-h day/12-h night regimen, with food and water available ad libitum. All animals received humane animal care, as outlined in the Guidelines for Care and Use of Experimental Animals (Canadian Council on Animal Care, Ottawa, 1984). The day a vaginal plug was found was defined as day 0 of pregnancy. Blastocysts were obtained by flushing the uterine horn on day 4; the flushing solution consisted of CMRL-1066 culture medium containing 1 mM glutamine and 1 mM sodium pyruvate. The blastocysts from different females were expanded, pooled and randomly selected for experiments.
Preparation of G. biloba extract
Dried G. biloba leaves were mixed with 20-fold weight ratio of distilled water and warmed at 60°C for 2 h with stirring. The warm mixture was then sonicated for 3 min. The mixture was then centrifuged at 250 x g for 15 min to remove large particles. The supernatant was further centrifuged at 10 000 x g for 60 min to give a clear supernatant mixture. The aqueous extract was stored at –20°C for cells or blastocysts treatment.
Ginkgolide B treatment and TUNEL assay of blastocysts
Blastocysts were incubated in medium containing various concentrations of ginkgolide B for 24 h. For TUNEL staining, embryos were washed in ginkgolide B-free medium, fixed, permeabilized and subjected to TUNEL labelling using an in situ cell death detection kit (Roche Molecular Biochemicals) according to the manufacturer’s protocol. Photographic images were obtained using fluorescence microscopy under bright light.
Cell proliferation assay of ginkgolide B-treated blastocysts
Blastocysts were incubated with culture medium containing 5 or 10 µM ginkgolide B. Twenty-four hours later, the blastocysts were washed with ginkgolide B-free medium, and dual differential staining (Pampfer et al., 1990) was used to facilitate the counting of cell numbers in the ICM and TE. Briefly, blastocysts were incubated in 0.4% ponase in M2-bovine serum albumin (BSA) medium (M2 medium containing 0.1% BSA) for the removal of the zona pellucida. The denuded blastocysts were exposed to 1 mM trinitrobenzenesulphonic acid in BSA-free M2 medium containing 0.1% polyvinylpyrrolidone at 4°C for 30 min and then washed with M2 medium (Hardy et al., 1989). The blastocysts were further treated with 30 µg/ml anti-dinitrophenol–BSA complex antibody in M2-BSA at 37°C for 30 min and then with M2 medium supplemented with 10% whole guinea-pig serum as a source of complement, 20 µg/ml bisbenzimide and 10 µg/ml propidium iodide (PI) at 37°C for 30 min. The immunolysed blastocysts were gently transferred to slides and protected from light before observation. Under UV light excitation, the ICM cells (which take up bisbenzimidine but exclude PI) were stained blue, whereas the TE cells (which take up both fluorochromes) were stained orange-red. Because multinucleated cells are not common in preimplantation embryos (Gardner and Davies, 1993), the number of nuclei was considered to represent an accurate measure of the cell number.
Morphological development analysis of mouse embryos in vitro
Outgrowing embryo culture
Blastocysts from ICR mice were collected on day 4 of pregnancy and incubated in the presence or absence of 10 µM ginkgolide B, as described in our previous study (Chan, 2005). After 24 h, the treated blastocysts were individually transferred to fibronectin-coated culture wells and cultured in the absence of ginkgolide B for 72 h in CMRL-1066 medium supplemented with 20% FBS (CMRL-FBS) (Armant et al., 1986). Under these culture conditions, each hatched blastocyst attached to the fibronectin and outgrew to form a cluster of ICM cells over the trophoblastic layer, in a process called TE outgrowth.
Morphology of outgrowing embryos
After a total incubation of 96 h, morphological scores for outgrowth were assessed. The outgrowing embryos were classified as either attached or outgrowth, with the latter defined by the presence of a cluster of ICM cells over the trophoblastic layer. The ICM clusters were then scored according to their shapes, ranging from compact-rounded ICM (+++) to a few scattered cells (+) over the trophoblastic layer, as previously described (Pampfer et al., 1994). The surface area of the trophoblastic layer (TE outgrowth) was photographed and measured by first weighing the cut-out images and then by indirectly evaluating the surface images with regard to a standard curve of weight versus area.
Cell proliferation of outgrowths
The cell proliferation of outgrowths was estimated by direct nuclear count, using Giemsa staining (Wuu et al., 1999) and the cell spreading technique previously used for rat implanting embryos (Pampfer et al., 1994). Briefly, the culture medium was carefully removed and replaced with 0.05% hypotonic sodium citrate (30 µl/well) at room temperature for 5 min. This solution was then evaporated under partial vacuum (200 Torr) at 50–55°C for 60–90 min. The outgrowths were fixed with 50 µl per well of FixDenat fixative at room temperature for 30 min, and nuclei were stained with a 4% Giemsa solution at room temperature for 30 min. The proliferation of each individual outgrowth was expressed as the number of Giemsa-stained nuclei. Dead cells in the outgrowths were identified by the presence of fragmented nuclei (karyorrhexis), as detected by bisbenzimide staining, or by the presence of DNA strand breaks (karyolysis), as detected by the TUNEL method, as previously described (Huang et al., 2003).
Development of blastocysts in vivo by embryo transfer
To examine the ability of expanded blastocysts to implant and develop in vivo, we transferred the generated embryos to recipient mice. ICR females were mated with vasectomized males (C57BL/6J) to produce pseudo-pregnant mothers as recipients for the embryo transfer experiments. To assess the impact of ginkgolide B on post-implantation growth in vivo, we exposed the blastocysts to 0 or 10 M ginkgolide B for 24 h and then transferred five to eight embryos in parallel into pairs of the uterine horns of day 4 pseudo-pregnant mice. The control blastocysts (ginkgolide B-free group) were transferred to the right uterine horn and ginkgolide B-treated blastocysts were transferred to the left uterine horn in each pseudo-pregnant mouse. The surrogate mice were killed on day 18 of pregnancy, and the frequency of implantation was calculated as the number of implantation sites per number of embryos transferred. The frequency of resorption or surviving fetuses was calculated as the number of resorptions or surviving fetuses per number of implantations. The weights of the surviving fetuses and placentas were measured immediately after dissection.
Effect of ginkgolide B on embryos in an animal model
Female mice were fed for the duration of the experiment (total 5 days) with either a standard diet or one supplemented with ginkgolide B at 25 g/kg of feed, representing a dose of ~7.6 mmoles/kg body weight/day. Twenty-four hours later, these mice were mated overnight with a single fertile male of the same strain. Blastocysts were obtained by flushing the uterine horn on day 4 after mating, and cell apoptosis and proliferation were analysed.
Statistics
The data were analysed using one-way analysis of variance (ANOVA) and t-tests and are presented as the mean ± SD. P < 0.05 was considered significant.
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Results |
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The effects of ginkgolide B on ESC-B5 cells
Although we previously showed that ginkgolides induce apoptosis and decrease cell numbers in mouse blastocysts (Chan, 2005
), the precise regulatory mechanisms governing this effect remain unclear. To begin identifying the apoptotic signalling pathway(s) involved in ginkgolide B-induced cell death, we first studied the effect of ginkgolide B treatment on ESCs (ESC-B5) in vitro. We found that ginkgolide B reduced ESC-B5 cell viability in a dose-dependent manner (Figure 1A). We then investigated whether this ginkgolide B-induced cell death was due to apoptosis. An ELISA method that quantitatively determines histone-associated oligonucleosomal DNA fragments revealed that 5 and 10 µM ginkgolide B induced 2.1- to 2.5-fold increases in DNA fragmentation in these cells (Figure 1B). We further found no differences or changes in the morphology of ESC-B5 cells between ginkgolide B-treated and untreated control group (Figure 1C). Using DCF-DA as the detection reagent, we tested whether ROS formation occurred in ginkgolide B-treated ESC-B5 cells. As shown in Figure 2A, ginkgolide B increased the intracellular ROS content in ESC-B5 cells by ~10-fold when compared with the levels found in untreated control cells. Because JNK activation and MMP changes are directly associated with apoptosis, we studied JNK activity during ginkgolide B-induced apoptosis in ESC-B5 cells. Our results revealed that treatment of ESC-B5 cells with 5–10 µM ginkgolide B significantly activated JNK (Figure 2B), indicating that JNK may be involved in ginkgolide B-induced apoptosis. We then investigated the effect of ginkgolide B on MMP in ESC-B5 cells. We found that ginkgolide B treatment dose dependently decreased the uptake of DiOC6(3) and TMRE into the mitochondria of ESC-B5 cells, indicating a significant and dose-dependent loss of MMP (Figure 2C). Moreover, using an in vitro ELISA assay, we assessed the effect of ginkgolide B on caspase-3 activation. Our results revealed that treatment of ESC-B5 cells with 5 or 10 µM ginkgolide B significantly activated capase-3 (Figure 2D). To determine whether G. biloba extract also could cause cell injury, we incubated ESC-B5 cells and blastocysts in a medium containing 5 or 10% natural extract of G. biloba leaves at 37°C for 24 h and measured the apoptosis. We found that G. biloba extract induced ESC-B5 cell and blastocyst apoptosis in a dose-dependent manner (Figure 2E and F).
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To further study the impact of ginkgolide B on early embryo development in a stem cell assay model, we incubated cells with or without ginkgolide B and tested them for their abilities to form EBs in vitro. Our results revealed that EB formation was significantly decreased in cells that had been pretreated with ginkgolide B (Figure 3A). To determine whether the expression levels of OCT 4 and phosphorylated STAT3, two well-known pluripotent markers, are affected by ginkgolide B, we treated stem cells with the compound for 24 h or left them untreated. Incubation of mouse ESC-B5 stem cells with 5 or 10 µM ginkgolide B for 24 h had no significant effects on the expression of OCT 4 and phosphorylated STAT3, compared with that in the untreated control group, as revealed by immunoblotting (Figure 3B). Moreover, immunoblotting assays reveal that pretreatment with 5 or 10 µM ginkgolide B effectively inhibited NGF-induced expression of MAP-2 (Figure 3C). Taken together, these results indicate that ginkgolide B inhibits early embryo development in mouse ESCs and additionally appears to induce apoptosis in these cells through mechanisms involving ROS generation, JNK activation, decreased MMP and the activation of caspase-3.
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Effect of ginkgolide B on mouse blastocyst development in vitro
To determine whether ginkgolide B treatment not only induces ESC injury but also impairs subsequent growth of these cells, we incubated blastocysts with 0, 5 or 10 µM ginkgolide B and then transferred the cells to ginkgolide B-free medium in fibronectin-coated dishes for 72 h. In control dishes (no ginkgolide B pretreatment), most of the blastocysts (177/185; 95.7%) had attached and outgrown, whereas fewer of the 10 µM ginkgolide B-treated blastocysts (175/190; 92.1%) had attached and outgrown. As outgrowth continues in vitro, the attached blastocysts develop a compact and structured ICM at the centre of the TE layer; this TE eventually expands to surround a relatively small cluster of ICM cells. We observed that significantly fewer outgrowths derived from ginkgolide B-pretreated blastocysts reached this stage versus control blastocysts (Figure 4). In addition, the ginkgolide B-pretreated embryo outgrowths showed significantly fewer nuclei per outgrowth versus control outgrowth embryos (164 ± 13.4 versus 119 ± 14.7) (Figure 5A). TUNEL analysis and bisbenzimide revealed significantly more apoptotic cells in the ICM and TE of ginkgolide B-pretreated blastocysts (Figure 5B and C), indicating that the decrease in cell number was at least partially due to increased apoptosis.
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Effect of ginkgolide B on mouse blastocyst development in vivo
To examine the effect of ginkgolide B on blastocyst development in vivo, we performed multiple embryo transfers and examined uterine contents 13 days after transfer (day 18 of pregnancy). The implantation ratio of control blastocysts was
72.5% (116 of 160 embryos in 20 recipients), whereas that in the ginkgolide B-pretreated group was only 57.5% (92 of 160 embryos in 20 recipients) (Figure 6A). The proportion of implanted embryos that failed to develop normally (i.e. were resorbed) was significantly higher in the ginkgolide B-pretreated group (65 of 92 implanted embryos; 70.65%) versus the control group (50 of 116 implanted embryos; 43.1%). The stage of embryonic resorption may be identified as early (a small, dark mole without distinct structure) or late (placenta without fetal structure). We found that the ginkgolide B-pretreated group had a higher late resorption rate versus the untreated control group. Interestingly, the surviving fetuses had no gross morphological alterations in either group (data not shown), and there were no differences in placental weights between the two groups (Figure 6B). However, the fetal weight was lower in the ginkgolide B-pretreated group versus the controls (548 ± 78 versus 508 ± 74); the ginkgolide B-pretreated group had 23% fewer fetuses weighing over 600 mg, which is an important indicator for embryo and fetus development (Figure 6C).
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The ability of ginkgolide B to trigger apoptosis and developmental injury in vitro led us to suspect that it might have the same activity in vivo. The results of the animal model study revealed that dietary ginkgolide B significantly induced apoptosis (Figure 7A) and decreased cell proliferation (Figure 7B) in mouse blastocysts.
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Collectively, these studies suggest that ginkgolide B can induce apoptosis and inhibit proliferation of embryonic cells and appears to cause embryonic developmental injury in vitro and in vivo.
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Discussion |
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We previously showed that ginkgolides could trigger cell death and decreased cell proliferation in blastocysts (Chan, 2005
). Here, we show for the first time that ginkgolide B can induce apoptosis in both mouse stem cells and mouse blastocysts. Our finding that ginkgolide B-induced apoptosis of ESCs in a dose-dependent manner (Figure 1) is consistent with previous reports that G. biloba extract and its derivative components can induce apoptosis and inhibit cell proliferation in other cell types, including oral cavity cancer, human breast cancer, colorectal cancer and hepatoma cells (Webster et al., 1996; Mutoh et al., 2000; Papadopoulos et al., 2002; Kim et al., 2005).
We then examined the nature of ginkgolide B-triggered apoptotic signalling in ESC-B5 cells. Oxidative stress has been demonstrated to act as a stimulator of various cell responses, including apoptosis (Buttke and Sandstrom, 1994; Slater et al., 1995). We previously demonstrated that oxidative stress was involved in environmental stress-induced apoptosis (Tang et al., 1998; Chan et al., 1999, 2000), and studies have shown that many natural compounds prevent apoptosis via anti-oxidative properties (Chan et al., 2003; Chan and Wu, 2004). JNK also plays important roles in many cell processes, including the induction of apoptosis in various cell types in response to certain apoptotic triggers (Xia et al., 1995; Verheij et al., 1996; Seimiya et al., 1997). Mitochondria have been shown to act as important signalling conduits during programmed cell death, and loss of MMP is induced by many key regulators of apoptosis (Kroemer et al., 1997; Li et al., 1997; Zou et al., 1997; Green and Reed, 1998; Weber et al., 2003). These signalling components may act in combination to regulate apoptotic signalling in response to divergent cellular stresses; for example, heat shock, DNA damage and oxidative stress all trigger caspase activation through mitochondrial-mediated processes (Liu et al., 1996; Green and Reed, 1998). Similarly, numerous reports have demonstrated that ROS generation is an important upstream controller of JNK activation and subsequent apoptotic biochemical changes, including MMP changes and caspase-3 activation (Chan et al., 2003; Chan and Wu, 2004). In this work, we showed for the first time that ginkgolide B treatment triggers ROS generation, JNK activation, MMP loss and caspase-3 activation in ESC-B5 cells (Figure 2). These results in combination with our previous studies strongly suggest that ginkgolide B induces apoptosis via ROS generation and JNK activation, two important regulators of apoptotic processes.
On the basis of the pro-apoptotic effects of ginkgolide B in mouse ESCs, we next sought to examine the possible effects of ginkgolide B on embryonic development. The solid ICM of the late blastocyst becomes the hollow egg cylinder during the morphogenesis of a post-implantation mouse embryo (Huang and Lin, 2001). Previous studies have shown that when the number of cells in the ICM of a blastocyst is reduced by 30% or more, there is a high risk of fetal loss or developmental injury, even in cases where the implantation rate and TE cell numbers are normal (Tam, 1988). In addition, previous studies have suggested that the ICM cell number is essential for proper implantation, and reduction in the ICM cell lineage may reduce embryonic viability (Pampfer et al., 1990; Kelly et al., 1992). We previously showed that ginkgolides have significant negative effects on the ICM cells of blastocysts (Chan, 2005). Here, we demonstrate that ginkgolide B induces embryo development injury, both in a stem cell model and in blastocyst assay (Figures 3–
6). These findings collectively suggest that ginkgolide B may significantly decrease proper mouse embryonic development, implantation and embryogenesis.
Incubation of mouse ESC-B5 stem cells with 5 or 10 µM ginkgolide B for 24 h had no significant effects on the expression of OCT 4 and phosphorylated STAT3, compared with that in the untreated control group (Figure 3B). The results imply that cell apoptosis (Figures 1 and 2) and early embryo development injury (Figure 3A), but not pluripotent properties, are affected by ginkgolide B administered over a short-term period (24 h). Earlier studies demonstrate that ginkgolide B effectively inhibits -amyloid- or prion-induced neurotoxicity at concentrations of 0.1–10 µM (Bate et al., 2004). In our experiments, treatment with 5–10 µM ginkgolide B led to cell apoptosis and embryonic development injury in mouse ESCs and blastocysts. These results imply that treatment conditions, including cell lines in analysis models, determine the precise effects of ginkgolide B. The concentrations of ginkgolides in natural extracts of G. biloba leaves and their biological effects on embryogenesis upon direct ingestion in both animal models and humans require further research.
Although we previously showed that ginkgolide B negatively affects blastocyst development in vitro (Chan, 2005), we had not previously examined the possibility of in utero maternal compensation for these effects. In this study, we find that pretreatment with ginkgolide B inhibited cell proliferation in cultured blastocysts, reduced the ability of blastocysts to develop into outgrowths with structured ICM clusters, reduced the number of nuclei in outgrowths and, finally, could not be rescued by embryo transfer to the maternal environment (Figures 4–6). These results are the first to elucidate the injury effects of ginkgolide B on mouse blastocysts. Specifically, our embryo transfer analysis revealed that pretreatment with ginkgolide B led to a significantly higher incidence of embryonic resorption (Figure 6). These findings collectively suggest that ginkgolide B had a risk effect on embryonic health.
Interestingly, the rate of implantation was not affected by ginkgolide B pretreatment (Figures 4 and 6). Although the major effects of ginkgolide B were detected in the ICM in the present work, we previously demonstrated that the TE and trophoblast cells are sensitive to ginkgolide B-induced apoptosis (Chan, 2005). This could explain the relatively large effect of ginkgolide B on TE outgrowth observed in the present work. During development, the TE arises from the trophoblast at the blastocyst stage and then develops into a sphere of epithelial cells surrounding the ICM and the blastocoele; these cells contribute to the placenta and are required for the development of the mammalian conceptus (Cross et al., 1994). Because the present work and our previous findings indicate that ginkgolide B decreases the number of ICM cells to a larger degree than that of trophoblast cells (Chan, 2005), we hypothesize that the high rate of resorption in ginkgolide B-treated embryos is due to a deficiency of ICM cells, which increases the in vitro risk of fetal death and embryogenic arrest despite the presence of normal TE invasion.
Given the ability of ginkgolide B to induce apoptosis or injury in mouse ESCs, blastocysts and outgrowths in vitro, we examined the in vivo effects of ginkgolide B in an animal pregnancy model. Our results revealed that dietary ginkgolide B significantly increased apoptosis and decreased cell proliferation in blastocysts from pregnant female mice (Figure 7). This suggests that even short-term exposure of pregnant females to ginkgolide B can negatively affect embryonic development. Future work will be required to compare the absorption rate of ginkgolide B in mice and humans and to examine the effects of long-term ginkgolide B treatment in the animal model, to determine whether these findings are relevant to pregnant human females. However, the present results suggest that such work is warranted in an effort to determine the safety of dietary levels of ginkgolide B or G. biloba extracts in pregnant women.
In conclusion, this report shows that ginkgolide B treatment resulted in injury to mouse blastocysts in vitro, especially in the ICM, leading to resorption of post-implantation blastocysts or the retardation of surviving fetuses in vivo. These results support the notion that the ICM, which is the major target of the effects of ginkgolide B, plays a very important role in post-implantation development. The present results and those of our previous studies combine to suggest that dietary use of G. biloba extracts may possibly have an adverse effect on embryogenesis. Future work will be required to further elucidate the possible teratogenic action of ginkgolide B on human embryogenesis.
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This work was supported by grants (NSC94-2320-B-033-003 and NSC94-2745-M-033-002-URD) from the National Science Council of Taiwan, ROC.
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Submitted on February 16, 2006; resubmitted on April 29, 2006; resubmitted on May 22, 2006; accepted on May 27, 2006.
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