http://informahealthcare.com/dct ISSN: 0148-0545 (print), 1525-6014 (electronic) Drug Chem Toxicol, Early Online: 1–8 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/01480545.2014.1002037
RESEARCH ARTICLE
Apoptotic effects of dillapiole on maturation of mouse oocytes, fertilization and fetal development Yan-Der Hsuuw1 and Wen-Hsiung Chan2,3
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Department of Life Science, National Pingtung University of Science and Technology, Pingtung, Taiwan, 2Department of Bioscience Technology and Center for Nanotechnology, Chung Yuan Christian University, Chung Li, Taiwan, and 3Center for Biomedical Technology, Chung Yuan Christian University, Chung Li, Taiwan Abstract
Keywords
Previously, we reported that dillapiole, a phenylpropanoid with antileishmanial, anti-inflammatory, antifungal and acaricidal activities, is a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, leading to impaired embryonic development and cell viability. In the current study, we investigated the deleterious effects of dillapiole on mouse oocyte maturation, in vitro fertilization (IVF) and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, dillapiole induced significant impairment of mouse oocyte maturation, decrease in the IVF rate and inhibition of subsequent embryonic development in vitro. Pre-incubation of oocytes with dillapiole during in vitro maturation led to an increase in post-implantation embryo resorption and decrease in mouse fetal weight. In an in vivo animal model, 2.5, 5 or 10 mM dillapiole provided in drinking water caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, pre-incubation of oocytes with a caspase-3-specific inhibitor effectively blocked dillapiole-triggered deleterious effects, clearly implying that embryonic injury induced by dillapiole is mediated via a caspasedependent apoptotic mechanism. To the best of our knowledge, this is the first study to establish the impact of dillapiole on maturation of mouse oocytes, fertilization and sequential embryonic development.
Apoptosis, dillapiole, embryonic development, oocyte maturation
Introduction Peperomia pellucida is an annual herb belonging to the Piperaceae family, mainly located in Africa, Central and South America, Australia and Southeast Asia. Traditionally, P. pellucida has been used as a food and medicinal herb (Loc et al., 2010), such as folk medicine for the treatment of gastric ulcer, along with gout arthritis, wounds, high blood cholesterol and skin-related problems (i.e. acne; Abdul Hamid et al., 2007; de Fatima Arrigoni-Blank et al., 2004; Mutee et al., 2010). To date, several plant-derived natural products have been shown to play critical roles for potential application in cancer chemotherapy strategies (Gao et al., 2011). Dillapiole, a phenylpropanoid, is the main component of Piper aduncum (Bernard et al., 1995; de Almeida et al., 2009), with reported antileishmanial (Parise-Filho et al., 2012), anti-inflammatory (Parise-Filho et al., 2011), antifungal (de Almeida et al., 2009) and acaricidal (Araujo et al., 2012) bioactivities. However, the cytotoxic effects of this organic compound are not well documented. A recent study demonstrated that Address for correspondence: Dr Wen-Hsiung Chan, Department of Bioscience Technology and Center for Nanotechnology, Chung Yuan Christian University, Chung Li 32023, Taiwan. Fax: +886-3-2653599. E-mail: [email protected]
History Received 18 September 2014 Revised 19 November 2014 Accepted 20 December 2014 Published online 27 February 2015
dillapiole exerts broad cytotoxic effects, supporting its significant potential for development as an anticancer drug against a variety of tumor cell types (Ferreira et al., 2014). Further investigation disclosed that the compound acts as a pro-oxidant to induce intracellular reactive oxygen species (ROS) in MDA-MB-231 cells (Ferreira et al., 2014). Moreover, dillapiole inhibits cell proliferation through cell cycle arrest at the G0/G1 phase and association with disruption of actin filaments. These findings collectively highlight the potential of dillapiole as a promising anticancer or chemotherapeutic agent. However, its side-effects or cytotoxicity in normal tissue cells are yet to be established. Ongoing experiments by our group have revealed that several natural chemical compounds exert cytotoxic effects via induction of apoptosis during early development of mouse embryos (Chan, 2005, 2006; Chan et al., 2007; Chang et al., 2012). More recently, our coworkers showed that dillapiole triggers apoptotic processes in mouse blastocysts and impairs early post-implantation development in vitro and in vivo (Chan, 2014). However, no studies to date have elucidated the potential cytotoxic effects of dillapiole on oocyte maturation, fertilization, and subsequent embryonic development. The microenvironment affects oocyte growth and maturation, with consequent impacts on oocyte viability.
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For instance, oxygen concentration, temperature and glucose content have been identified as key determinants of oocyte viability (Banwell et al., 2007; de Castro & Hansen, 2007; Sartori et al., 2002). A number of previous studies have documented the influence of natural chemical compounds on oocyte maturation in vivo and in vitro (Chang & Chan, 2010; Chang et al., 2012). During normal embryogenesis and early development, programmed cell death (apoptosis) functions to remove abnormal or redundant cells in pre-implantation embryos (Hardy, 1997; Hardy et al., 2003). However, programmed cell death processes do not occur prior to the blastocyst stage during normal mouse embryonic development (Byrne et al., 1999), and induction of cell death during oocyte maturation and early stages of embryogenesis (e.g. upon teratogen exposure) causes embryonic developmental injury (Banwell et al., 2007; Chan, 2006, 2009; Hsuuw et al., 2005; Shiao & Chan, 2009). In the current study, we systematically investigated whether dillapiole has injurious effects on early stage embryo maturation and development by incubating mouse oocytes with the compound for 24 h and comparing subsequent development in an untreated control group. The main purpose of this study was to determine whether short-term exposure to dillapiole at the oocyte stage has a long-term deleterious impact on subsequent embryonic development. Our results clearly demonstrate that exposure to dillapiole at the oocyte stage not only retards oocyte maturation but also causes injurious effects on IVF and subsequent embryonic development.
Materials and methods Chemicals and reagents Dulbecco’s modified Eagle’s medium (DMEM), dillapiole and pregnant mare serum gonadotropin (PMSG) were obtained from Sigma (St. Louis, MO). Human chorionic gonadotropin (hCG) was purchased from Serono (NV Organon Oss, The Netherlands). TUNEL in situ cell death detection kits were acquired from Roche (Mannheim, Germany). CMRL-1066 medium was purchased from Gibco Life Technologies (Grand Island, NY). Cumulus–oocyte complex collection and in vitro maturation ICR mice were acquired from the National Laboratory Animal Center (Taiwan, ROC) and provided food (Teklad Mouse Breeder Diet; Harlan Laboratories, Indianapolis, IN) and water ad libitum. Mice were housed in standard 28 16 11 cm (height) polypropylene cages with wire-grid tops, and maintained under a 12-h day/night regimen. Cumulus–oocyte complexes (COCs) were obtained according to a previous protocol (Banwell et al., 2007). Briefly, COCs were isolated from female hybrid ICR mice (21 days old), injected with hCG (5 IU) 44 h prior to oocyte collection. COCs were collected in HEPES-buffered a-minimum essential medium (a-MEM) containing 50 mg/ml streptomycin sulfate, 75 mg/ml penicillin G and 5% fetal bovine serum (FBS) by gently puncturing visible antral follicles present on the ovary surface. Germinal vesicle stage oocytes containing an intact vestment of cumulus
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cells were collected and pooled from at least eight animals and randomly selected for experiments. For in vitro maturation (IVM) of oocytes, one drop (100 ml) of medium (a-MEM supplemented with 50 mg/ml streptomycin, 75 mg/ml penicillin G, 5% FBS and 50 mIU/ml recombinant human folliclestimulating hormone) containing 10 COCs for each treatment group was added under oil in 35 mm culture dishes. COC maturation was analyzed following incubation with or without different concentrations of dillapiole (2.5, 5 or 10 mM) for 24 h under a 5% O2 and 6% CO2 atmosphere (balance, N2) at 37 C. This research was approved by the Animal Research Ethics Board of Chung Yuan Christian University (Taiwan, ROC). All animals received humane care following Principles of Laboratory Animal Care (National Institutes of Health publication 85-23, revised 1996; Institute of Laboratory Animal Resources, 1996). Maturation status assessment After IVM, COCs of each group were treated with 50 U/ml ovine hyaluronidase and gently pipetted to remove all cumulus cells. Denuded oocytes were collected and washed first with fresh medium and then with phosphate-buffered saline (PBS). Oocytes were fixed in ethanol:glacial acetic acid (1:3) for 48 h and stained with a 1% aceto-orcein solution. Nuclear structures were visualized using phasecontrast microscopy (Olympus BX51, Tokyo, Japan). In vivo maturation In vivo-matured oocytes were obtained by injecting 21-day-old mice with 5 IU equine chorionic gonadotrophin (eCG) and 5 IU hCG, 61 and 13 h prior to fertilization, respectively. Mature ova were collected from the oviduct into HEPES-buffered a-MEM medium. Effects of dillapiole intake on oocyte maturation in an animal model The effects of dillapiole on oocytes were analyzed in 21-dayold ICR virgin albino mice. Female mice were randomly divided into four groups of 20 animals each and administered a standard diet with 0, 2.5, 5 or 10 mM dillapiole in drinking water for 4 days. COCs were collected by pre-treatment with 5 IU hCG for 44 h prior to oocyte collection, and analyzed for oocyte maturation, IVF and embryonic development. In vitro fertilization For IVF, ova were washed twice in bicarbonate-buffered a-MEM medium containing 50 mg/ml streptomycin, 75 mg/ ml penicillin G and 3 mg/ml fatty acid free bovine serum albumin (BSA), and fertilized in the same medium containing fresh sperm (obtained from a CBAB6F1 male donor). After incubation with sperm for 4.5 h, eggs were washed three times in potassium simplex optimized medium (KSOM) without amino acids in the presence of L-alanyl-L-glutamine (1.0 mM). Next, eggs were placed in 20-ml drops of KSOM under oil, and cultured overnight. During cleavage to the 2-cell stage, embryos were transferred to a fresh drop of KSOM under oil, and cultured for an additional 72 h.
Effects of dillapiole on oocyte maturation
DOI: 10.3109/01480545.2014.1002037
All fertilization embryo culture steps were carried out in a 5% O2/6% CO2 atmosphere (balance, N2) at 37 C. Fertilization assessment Fertilization was assessed by incubating ova with sperm for 4.5 h, followed by 3 h of culture in fresh medium. Zygotes were assessed for the presence of the male pronucleus by orcein staining, as described previously (Banwell et al., 2007).
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Cell proliferation Cell proliferation was analyzed by dual differential staining, which facilitates the counting of cell numbers in the inner cell mass (ICM) and trophectoderm (TE; Chan, 2007; Huang et al., 2007; Pampfer et al., 1990). The zona pellucida was removed by incubating blastocysts with 0.4% pronase in M2BSA medium (M2 medium containing 0.1% BSA). Denuded blastocysts were exposed to 1 mM trinitrobenzenesulfonic acid (TNBS) in BSA-free M2 medium containing 0.1% polyvinylpyrrolidone (PVP) at 4 C for 30 min, and washed with M2 (Hardy et al., 1989). Blastocysts were then incubated with an anti-dinitrophenol–BSA complex antibody in M2BSA (30 mg/ml) at 37 C for 30 min, followed by incubation with M2 supplemented with 10% whole guinea pig serum (source of complement), together with 20 mg/ml bisbenzimide and 10 mg/ml propidium iodide (PI) at 37 C for 30 minutes. Immunolyzed blastocysts were gently transferred to slides and protected from light before observation. Under UV light, ICM cells (which take up bisbenzimidine but exclude PI) appeared blue, whereas TE cells (which take up both fluorochromes) appeared orange-red. Since multinucleated cells are not common in pre-implantation embryos (Gardner & Davies, 1993), the number of nuclei represent an accurate measurement of cell number. TUNEL assay of blastocysts For terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, embryos were washed in dillapiole-free medium, fixed, permeabilized and labeled using an in situ cell death detection kit (Roche Molecular Biochemicals, Indianapolis, IN), according to the manufacturer’s protocol. Images were obtained with a fluorescence microscope under bright-field illumination (Olympus BX70, Tokyo, Japan).
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embryos and eight dillapiole-treated embryos were transferred to the left and right uterine horns, respectively, of day 4 pseudopregnant mice. Forty surrogate mice per group were analyzed and killed on day 13 post-transfer (18-day fetuses); the frequency of implantation was calculated as the number of implantation sites per number of embryos transferred. The incidence rates of surviving fetuses (number of surviving fetus/number of implantations) and resorbed fetuses ([number of implantations – number of surviving fetuses]/number of implantations) were calculated. The weights of surviving fetuses and placenta were measured immediately after dissection. Statistical analysis Data were analyzed using one-way analysis of variance (ANOVA) and t-tests, and presented as means ± SD. Data were considered statistically significant at p50.05.
Results Impact of dillapiole on oocyte maturation status, fertilization rate and in vitro embryo development Although recent evidence has shown that dillapiole induces apoptosis and developmental injury in mouse blastocysts (Chan, 2014), its effects on oocyte maturation and subsequent development are currently unclear. To evaluate the effects of dillapiole on oocyte nuclear maturation status, oocytes were incubated with the compound, as described in the ‘‘Materials and methods’’ section, in experiments examining 10 independent experimental replicates with 200–250 oocytes per group. Overall, 93.7% of oocytes reached the metaphase II (MII) stage of maturation after IVM in the untreated control group. Notably, pre-incubation with dillapiole prior to IVM induced a dose-dependent decrease in the maturation rate and significant decrease in the ability of oocytes to be fertilized by fresh sperm, measured as male pronucleus formation in oocytes (Figure 1). We further investigated in vitro embryo development from the zygote to the 2-cell and blastocyst stages. Dillapiole pretreatment significantly decreased the cleavage rate of oocytes
Blastocyst development following embryo transfer The ability of expanded blastocysts to implant and develop in vivo was assessed by transferring generated embryos to recipient mice. Albino ICR female mice (6–8 weeks old) were mated with vasectomized male C57BL/6 J mice National Laboratory Animal Center, Taiwan, ROC) to produce pseudopregnant dams as recipients for embryo transfer. To ensure that all fetuses in pseudopregnant mice were derived from embryo transfer (white color) and not fertilization by C57BL/6 J (black color), we examined skin color at day 18 post-coitus. The impact of dillapiole on post-implantation growth in vivo was assessed by exposing COCs to 0–10 mM dillapiole for 24 h, followed by fertilization and IVM to the blastocyst stage. Subsequently, eight untreated control
Figure 1. Effects of dillapiole on mouse oocyte maturation and embryo development in vitro. Oocytes were collected from 21-day-old mice, cultured for 24 h in IVM medium containing dillapiole (2.5, 5 or 10 mM), fertilized in vitro and transferred to in vitro culture (IVC) medium. Oocyte maturation, IVF, cleavage and blastocyst development were analyzed. Values are presented as means ± SD of eight determinations. Data are based on 200–250 samples per group. ***p50.001 versus untreated control group.
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to the 2-cell stage, indicative of a deleterious effect (Figure 1). In addition, the ratio of embryos that cleaved and developed to form blastocysts in the dillapiole-treated group was markedly lower than in the untreated control group (Figure 1).
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Effects of dillapiole on cell proliferation and apoptosis of embryos during maturation of oocytes in vitro For further evaluation of whether the hazardous effects of dillapiole on oocyte maturation and sequent development are exerted through inhibition of cell proliferation or induction of apoptosis, proliferation was determined by counting total blastocyst cell numbers during in vitro maturation of oocytes in dillapiole-treated and untreated groups. Differential staining for ICM and TE cells was used to assist in the assessment of cell proliferation. Pre-incubation of oocytes with dillapiole led to significantly decreased blastocyst cell numbers, compared with control oocytes (Figure 2A). In addition, the number of ICM cells in blastocysts was decreased during IVM in the dillapiole-pretreated group (Figure 2A). However, dillapiole had no significant effect on the number of TE cells in blastocysts (Figure 2A). TUNEL staining data showed a concentration-dependent increase in apoptosis in blastocysts derived from the dillapiolepre-incubated oocyte group (Figure 2B). Quantitative analyses further revealed a 7.8- to 13.6-fold increase in apoptotic cells in blastocysts derived from dillapiole-pretreated oocytes, compared with the untreated control group (Figure 2C). Developmental potential of blastocysts from oocytes and effects of dillapiole intake on oocyte development For further evaluation of the developmental potential of blastocysts derived from dillapiole-treated oocytes, embryonic development was measured using an embryo transfer assay. Embryos were transferred to 50 recipients per group (8 per horn). In embryo transfer assays, a total of 40 recipients per group were found to be pregnant in at least one horn on day 18. The implantation rate of blastocysts derived from the dillapiole (10 mM)-treated oocyte group (28%) was significantly lower than that observed for control blastocysts (81%) during IVM (Figure 3A). Implanted embryos that failed to develop were subsequently resorbed in the uterus. About 73% of implanted embryos failed to develop normally in the group treated with 10 mM dillapiole, a rate significantly higher than that for the untreated control group (32%). Consistently, the resorption rate for dillapiole-pre-treated group was higher than that for the untreated control group (Figure 3A). In addition, the embryo survival rate, calculated based on survival to day 13 post-transfer (18-day fetuses), was markedly higher for the control group (68.2%) than the dillapiole (10 mM)-treated group (27.4%; Figure 3A). Interestingly, the placental weights of blastocysts derived from dillapiole-treated oocytes matured in vitro were not significantly different from those of the control group (Figure 3B), while fetal weights were lower in dillapiole (5 or 10 mM)-treated groups, compared with the untreated control group. Specifically, only 8.9% of fetuses in the dillapiole (10 mM)-pre-treated group weighed more than 600 mg, whereas 48% of control fetuses exceeded this
Figure 2. Effects of dillapiole on cell number and apoptosis in embryos during in vitro maturation of oocytes. Oocytes were cultured for 24 h in IVM medium containing dillapiole (2.5, 5 or 10 mM), fertilized in vitro and transferred to in vitro culture (IVC) medium for in vitro development. (A) Total cell number and numbers of TE lineage and ICM cells were counted in blastocysts. (B) Apoptotic cells were examined at the blastocyst stage using TUNEL staining followed by light microscopy. Black cells correspond to TUNEL-positive cells. (C) The mean number of apoptotic (TUNEL-positive) cells per blastocyst was calculated. Values are presented as means ± SD of eight determinations. Data are based on at least 200 samples in each group. ***p50.001 versus the untreated control group.
threshold, an important indicator of successful embryonic and fetal development (Figure 3C). Our results collectively indicate that exposure of oocytes to dillapiole during IVM causes a decrease in post-implantation development potential. To establish whether dillapiole exerts deleterious effects on oocyte maturation and embryo development in vivo, we
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DOI: 10.3109/01480545.2014.1002037
Effects of dillapiole on oocyte maturation
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Figure 3. Effects of dillapiole treatment or dietary dillapiole on embryo development during oocyte IVM. Oocytes were cultured for 24 h in IVM medium containing dillapiole (2.5, 5 or 10 mM), fertilized in vitro and transferred to in vitro culture (IVC) medium for development. (A) Fetus implantation, resorption and survival were analyzed as described in the ‘‘Materials and methods’’ section. The implantation percentage represents the number of implantations per number of transferred embryos 100. The percentage of resorption or surviving fetuses represents the number of resorptions or surviving fetuses per number of implantations 100. (B) Placental weights of 40 recipient mice were measured. (C) Weight distribution of surviving fetuses at day 13 post-transfer (18-day fetuses). Surviving fetuses were obtained via embryo transfer of control and dillapiole-pretreated groups, as described in the ‘‘Materials and methods’’ section (320 total blastocysts from 40 recipients). (D) Random female mice (21 days old) were fed a standard diet and drinking water with or without dillapiole (2.5, 5 or 10 mM) for 4 days. Oocytes were collected for IVM, IVF, cleavage and blastocyst development analyses. Data are based on at least 320 samples in each group. ***p50.001 versus the dillapiole-free group.
analyzed the impact of dillapiole intake in an animal model. Female mice were supplied drinking water containing dillapiole (5 or 10 mM) for 4 days or untreated drinking water, prior to COC collection, followed by evaluation of oocyte maturation status, fertilization rate and in vitro embryo development. Compared with the control group, dillapiole intake significantly decreased oocyte maturation and fertilization and inhibited subsequent embryonic development from the zygote to blastocyst stage (Figure 3D). Impact of dillapiole intake on the developmental potential of blastocyst-stage embryos The deleterious effects of dillapiole intake on embryo implantation and subsequent post-implantation development in vivo were measured using an embryo transfer assay. In this animal model, female mice were fed a standard diet and supplied drinking water with or without dillapiole (2.5, 5 or 10 mM) for 4 days before transfer of blastocyst-stage embryos to the uterine horn. Forty recipients were pregnant at day 18 (day 14 post-embryo transfer). Notably, the implantation rate of blastocysts was significantly lower in the dillapiole-intake group than the untreated control group (Figure 4A). Moreover, the resorption rate was higher and embryo survival rate lower, compared to the dillapiole-free control group (Figure 4A). Interestingly, placental weights of blastocysts derived from the dillapiole-intake group were not
significantly different from those of the control group (data not shown). However, fetal weights were lower in the dillapiole-intake (10 mM) than the untreated control group (Figure 4B). Our results clearly demonstrate that pretreatment or exposure of embryos to dillapiole reduces implantation potential and subsequent post-implantation development. Effects of dillapiole-induced apoptosis on oocyte maturation status, fertilization rate and embryo development during IVM To identify the regulatory mechanisms of dillapiole activity, we pre-incubated oocytes with 100 mM Ac-DEVD-cho, a caspase-3-specific inhibitor, with the aim of preventing dillapiole-induced embryo cell apoptosis during IVM. AcDEVD-cho effectively prevented apoptosis of blastocysts derived from oocytes pre-treated with 10 mM dillapiole (Figure 5A). Notably, inhibition of dillapiole-induced apoptosis further blocked the deleterious effects on oocyte maturation, fertilization rate and subsequent embryo development during IVM (Figure 5B). In addition, pre-incubation with Ac-DEVD-cho prevented dillapiole-induced reduction in post-implantation development potential following embryo transfer, as reflected by improvements in the embryo implantation and fetal survival rates and fetal development status (i.e. fetal weight) (Figure 5C). These findings indicate
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Figure 4. Effects of dietary dillapiole on embryo development in mouse blastocysts. Random female mice (21 days old) were fed a standard diet and drinking water with or without dillapiole (2.5, 5 or 10 mM) for 4 days before embryo transfer to the uterus. (A) Fetus implantation, resorption and survival were analyzed as described in the ‘‘Materials and methods’’ section. The implantation percentage represents the number of implantations per number of transferred embryos 100. The percentage of resorption or surviving fetuses represents the number of resorptions or surviving fetuses per number of implantations 100. (B) Weight distribution of surviving fetuses at day 14 post-transfer (18-day fetuses). Surviving fetuses were obtained via embryo transfer in the control and dillapiole-intake groups (320 total blastocysts from 40 recipients). ***p50.001 versus the dillapiole-free group.
that dillapiole-induced disruption of oocyte development during IVM involves an apoptotic mechanism.
Discussion Deleterious effects of chemical or physical injury may affect normal progression of the complex and precisely orchestrated process of oocyte maturation and early stage embryonic development, leading to malformation or miscarriage of the embryo. Therefore, it is important to establish the possible effects of various chemical agents or physical factors on these processes. One such agent is the phenylpropanoid dillapiole, the main component of Piper aduncum that is also present in other Piper species. A recent study demonstrated for the first time that dillapiole induces cell death through the mitochondria-dependent apoptotic pathway by triggering oxidative stress and exerts anti-proliferative effects on MDA-MB-231 cells (Ferreira et al., 2014). The broad cytotoxic effects of dillapiole against a variety of tumor cells have been highlighted (Ferreira et al., 2014). Safrole, a phenylpropanoid structure related to dillapiole also identified in Piper (genus; Andrade et al., 2008), is reported to trigger apoptosis in human oral cancer cells (Yu et al., 2011), as well as cell cycle arrest and apoptosis in human leukemia cells via a ROSmediated endoplasmic reticulum (ER) stress-involved cell regulatory signaling cascade (Yu et al., 2012). These findings support the potential utility of dillapiole for development as a chemopreventive and/or chemotherapeutic agent in human
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cancers. Importantly, a recent study by our coworkers disclosed that 5 and 10 mM dillapiole has hazardous effects on pre- and post-implantation embryonic development, leading to induction of apoptosis, retardation of early stage embryonic development and negative effects on postimplantation progress. Based on these results, we conclude that dillapiole at concentrations of 5 and 10 mM induces apoptosis and negatively affects mouse embryonic development, both in vitro and in vivo (Chan, 2014). Here, we further examined the possible cytotoxic effects of dillapiole on oocyte maturation, fertilization and early and post-implantation embryonic development. Our results consistently suggest that apoptotic effects play a regulatory role in dillapioleinduced oocyte developmental injury. Chemical compounds or physical factors have significant potential to cause deleterious effects on the complex processes of oocyte maturation, fertilization and early stage embryonic development, leading to developmental problems or embryonic malformation. Experiments from the current study demonstrated for the first time that dillapiole impairs mouse oocyte maturation, fertilization and subsequent embryonic development (Figure 1). Pre-incubation of oocytes with dillapiole led to decreased cell numbers and increased apoptosis (Figure 2A and B). The trophoblasts of blastocysts develop into TE. These cells contribute to the placenta and are required for mammalian conceptus development in post-implantation stage embryos (Cross et al., 1994). Previous studies have demonstrated that a decrease in TE and/or ICM lineage cell numbers inhibits embryo implantation and reduces viability (Kelly et al., 1992; Pampfer et al., 1990). TE cells as well as ICM and total blastocyst cell numbers are positively correlated with embryo implantation and subsequent development (Lane & Gardner, 1997). In our experiments, dillapiole treatment during oocyte maturation had no impact on the number of blastocyst TE cells, but decreased cell numbers in the ICM (Figure 2). These results indicate that pre-incubation with dillapiole during IVM triggers mortality and/or deleterious effects on development in post-implantation mouse embryos. Moreover, the mechanisms underlying these actions involve ICM cell death and/or inhibition of proliferation (Figures 2 and 3). Blastocysts derived from dillapiole-treated oocytes had lower implantation rates, and exhibited higher embryo resorption and reduced fetal survival (Figure 3A). However, the placental weights of dillapiole-treated and control animals were not significantly different. TE cells in embryos are major contributors to implantation and subsequent placental development. Interestingly, dillapiole had no deleterious effects on blastocyst TE cells or the related placental development process. Based on these findings, we propose that reduction in the ICM cell number induced by dillapiole during oocyte maturation is the major factor responsible for inhibition of embryonic development. Further investigation of the regulatory mechanisms underlying the deleterious effects of dillapiole on oocyte maturation and subsequent embryonic development revealed a decrease in cell number, increased apoptosis and delayed postimplantation development of blastocysts in dillapiole-treated oocytes, compared with untreated controls. These negative effects were attenuated upon suppression of blastocyst
Effects of dillapiole on oocyte maturation
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DOI: 10.3109/01480545.2014.1002037
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Figure 5. Effects of inhibition of dillapiole-induced cell apoptosis on in vitro maturation of oocytes. Oocytes were collected from 21-day-old mice and cultured for 24 h in IVM medium alone or IVM containing 100 mM Ac-DEVD-cho and 10 mM dillapiole, fertilized in vitro and transferred to in vitro culture (IVC) medium for in vitro development. (A) Apoptotic cells were examined at the blastocyst stage via TUNEL staining. (B) Oocyte maturation, IVF, cleavage, and blastocyst development were analyzed. (C) Fetus implantation, resorption and survival were analyzed using the embryo transfer assay, as described for Figure 3. ***p50.001 versus the dillapiole-free group. #p50.001 versus the dillapiole-treated group.
apoptosis via pre-treatment of oocytes with a caspase-3 inhibitor (Figure 5). Pre-treatment with the caspase-3 inhibitor additionally prevented dillapiole-induced reduction of post-implantation development potential following embryo transfer, and led to improvements in the embryo implantation rate, fetal survival rate and fetal development status. These results collectively suggest that dillapiole induces improper apoptotic processes in early stage embryos, leading to loss of embryonic cells and prevents post-implantation development, thereby acting as a teratogen through induction of programmed cell death in early stage cells. Evidence from the current study that dillapiole exerts injurious effects on oocyte maturation, fertilization and subsequent embryonic development supports the theory that dillapiole is a risk factor for normal embryonic development that may lead to reduced oocyte maturation in infertile couples. Our results provide an important framework for further determining the possible teratogenic effects of dillapiole on human oocytes and embryo development, with a view to assessing the safety of its application as an anti-inflammatory or anti-cancer drug in the clinic. Therefore, further studies to determine the possible teratogenic effects of dillapiole on human oocyte and embryo development are essential.
Declaration of interest This work was supported by grants (NSC101-2311-B-033001-MY3, NSC101-2632-M-033-001-MY2, NSC102-2627M-033-001 and NSC102-2632-M-033-001-MY3) from the National Science Council of Taiwan, ROC.
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Chang MH, Chang SC, Chan WH. (2012). Injurious effects of emodin on maturation of mouse oocytes, fertilization and fetal development via apoptosis. Int J Mol Sci 13:13911–13925. Chang YJ, Chan WH. (2010). Methylglyoxal has injurious effects on maturation of mouse oocytes, fertilization, and fetal development, via apoptosis. Toxicol Lett 193:217–223. Cross JC, Werb Z, Fisher SJ. (1994). Implantation and the placenta: key pieces of the development puzzle. Science 266:1508–1518. de Almeida RR, Souto RN, Bastos CN, et al. (2009). Chemical variation in Piper aduncum and biological properties of its dillapiole-rich essential oil. Chem Biodivers 6:1427–1434. de Castro EPLA, Hansen PJ. (2007). Interactions between oxygen tension and glucose concentration that modulate actions of heat shock on bovine oocytes during in vitro maturation. Theriogenology 68: 763–770. de Fatima Arrigoni-Blank M, Dmitrieva EG, Franzotti EM, et al. (2004). Anti-inflammatory and analgesic activity of Peperomia pellucida (L.) HBK (Piperaceae). J Ethnopharmacol 91:215–218. Ferreira AK, de-Sa-Junior PL, Pasqualoto KF, et al. (2014). Cytotoxic effects of dillapiole on MDA-MB-231 cells involve the induction of apoptosis through the mitochondrial pathway by inducing an oxidative stress while altering the cytoskeleton network. Biochimie 99:195–207. Gao J, Morgan WA, Sanchez-Medina A, Corcoran O. (2011). The ethanol extract of Scutellaria baicalensis and the active compounds induce cell cycle arrest and apoptosis including upregulation of p53 and Bax in human lung cancer cells. Toxicol Appl Pharmacol 254: 221–228. Gardner RL, Davies TJ. (1993). Lack of coupling between onset of giant transformation and genome endoreduplication in the mural trophectoderm of the mouse blastocyst. J Exp Zool 265:54–60. Hardy K. (1997). Cell death in the mammalian blastocyst. Mol Hum Reprod 3:919–925. Hardy K, Handyside AH, Winston RM. (1989). The human blastocyst: cell number, death and allocation during late preimplantation development in vitro. Development 107:597–604. Hardy K, Stark J, Winston RM. (2003). Maintenance of the inner cell mass in human blastocysts from fragmented embryos. Biol Reprod 68: 1165–1169. Hsuuw YD, Chang CK, Chan WH, Yu JS. (2005). Curcumin prevents methylglyoxal-induced oxidative stress and apoptosis in mouse embryonic stem cells and blastocysts. J Cell Physiol 205:379–386.
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Huang LH, Shiao NH, Hsuuw YD, Chan WH. (2007). Protective effects of resveratrol on ethanol-induced apoptosis in embryonic stem cells and disruption of embryonic development in mouse blastocysts. Toxicology 242:109–122. Institute of Laboratory Animal Resources. (1996). Commission on Life Sciences NRC. Washington, DC: National Academy Press. Kelly SM, Robaire B, Hales BF. (1992). Paternal cyclophosphamide treatment causes postimplantation loss via inner cell mass-specific cell death. Teratology 45:313–318. Lane M, Gardner DK. (1997). Differential regulation of mouse embryo development and viability by amino acids. J Reprod Fertil 109: 153–164. Loc NH, Bach NH, Kim TG, Yang MS. (2010). Tissue culture and expression of Escherichia coli heat-labile enterotoxin B subunit in transgenic Peperomia pellucida. Protein Expr Purif 72:82–86. Mutee AF, Salhimi SM, Yam MF, et al. (2010). In vivo antiinflammatory and in vitro antioxidant activities of Peperomia pellucida. Int J Pharmacol 6:686–690. Pampfer S, de Hertogh R, Vanderheyden I, et al. (1990). Decreased inner cell mass proportion in blastocysts from diabetic rats. Diabetes 39: 471–476. Parise-Filho R, Pasqualoto KF, Magri FM, et al. (2012). Dillapiole as antileishmanial agent: discovery, cytotoxic activity and preliminary SAR studies of dillapiole analogues. Arch Pharm 345: 934–944. Parise-Filho R, Pastrello M, Pereira Camerlingo CE, et al. (2011). The anti-inflammatory activity of dillapiole and some semisynthetic analogues. Pharm Biol 49:1173–1179. Sartori R, Sartor-Bergfelt R, Mertens SA, et al. (2002). Fertilization and early embryonic development in heifers and lactating cows in summer and lactating and dry cows in winter. J Dairy Sci 85:2803–2812. Shiao NH, Chan WH. (2009). Injury effects of ginkgolide B on maturation of mouse oocytes, fertilization, and fetal development in vitro and in vivo. Toxicol Lett 188:63–69. Yu CS, Huang AC, Yang JS, et al. (2012). Safrole induces G0/G1 phase arrest via inhibition of cyclin E and provokes apoptosis through endoplasmic reticulum stress and mitochondrion-dependent pathways in human leukemia HL-60 cells. Anticancer Res 32: 1671–1679. Yu FS, Yang JS, Yu CS, et al. (2011). Safrole induces apoptosis in human oral cancer HSC-3 cells. J Dent Res 90:168–174.
RESEARCH ARTICLE
Apoptotic effects of dillapiole on maturation of mouse oocytes, fertilization and fetal development Yan-Der Hsuuw1 and Wen-Hsiung Chan2,3
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Department of Life Science, National Pingtung University of Science and Technology, Pingtung, Taiwan, 2Department of Bioscience Technology and Center for Nanotechnology, Chung Yuan Christian University, Chung Li, Taiwan, and 3Center for Biomedical Technology, Chung Yuan Christian University, Chung Li, Taiwan Abstract
Keywords
Previously, we reported that dillapiole, a phenylpropanoid with antileishmanial, anti-inflammatory, antifungal and acaricidal activities, is a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, leading to impaired embryonic development and cell viability. In the current study, we investigated the deleterious effects of dillapiole on mouse oocyte maturation, in vitro fertilization (IVF) and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, dillapiole induced significant impairment of mouse oocyte maturation, decrease in the IVF rate and inhibition of subsequent embryonic development in vitro. Pre-incubation of oocytes with dillapiole during in vitro maturation led to an increase in post-implantation embryo resorption and decrease in mouse fetal weight. In an in vivo animal model, 2.5, 5 or 10 mM dillapiole provided in drinking water caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, pre-incubation of oocytes with a caspase-3-specific inhibitor effectively blocked dillapiole-triggered deleterious effects, clearly implying that embryonic injury induced by dillapiole is mediated via a caspasedependent apoptotic mechanism. To the best of our knowledge, this is the first study to establish the impact of dillapiole on maturation of mouse oocytes, fertilization and sequential embryonic development.
Apoptosis, dillapiole, embryonic development, oocyte maturation
Introduction Peperomia pellucida is an annual herb belonging to the Piperaceae family, mainly located in Africa, Central and South America, Australia and Southeast Asia. Traditionally, P. pellucida has been used as a food and medicinal herb (Loc et al., 2010), such as folk medicine for the treatment of gastric ulcer, along with gout arthritis, wounds, high blood cholesterol and skin-related problems (i.e. acne; Abdul Hamid et al., 2007; de Fatima Arrigoni-Blank et al., 2004; Mutee et al., 2010). To date, several plant-derived natural products have been shown to play critical roles for potential application in cancer chemotherapy strategies (Gao et al., 2011). Dillapiole, a phenylpropanoid, is the main component of Piper aduncum (Bernard et al., 1995; de Almeida et al., 2009), with reported antileishmanial (Parise-Filho et al., 2012), anti-inflammatory (Parise-Filho et al., 2011), antifungal (de Almeida et al., 2009) and acaricidal (Araujo et al., 2012) bioactivities. However, the cytotoxic effects of this organic compound are not well documented. A recent study demonstrated that Address for correspondence: Dr Wen-Hsiung Chan, Department of Bioscience Technology and Center for Nanotechnology, Chung Yuan Christian University, Chung Li 32023, Taiwan. Fax: +886-3-2653599. E-mail: [email protected]
History Received 18 September 2014 Revised 19 November 2014 Accepted 20 December 2014 Published online 27 February 2015
dillapiole exerts broad cytotoxic effects, supporting its significant potential for development as an anticancer drug against a variety of tumor cell types (Ferreira et al., 2014). Further investigation disclosed that the compound acts as a pro-oxidant to induce intracellular reactive oxygen species (ROS) in MDA-MB-231 cells (Ferreira et al., 2014). Moreover, dillapiole inhibits cell proliferation through cell cycle arrest at the G0/G1 phase and association with disruption of actin filaments. These findings collectively highlight the potential of dillapiole as a promising anticancer or chemotherapeutic agent. However, its side-effects or cytotoxicity in normal tissue cells are yet to be established. Ongoing experiments by our group have revealed that several natural chemical compounds exert cytotoxic effects via induction of apoptosis during early development of mouse embryos (Chan, 2005, 2006; Chan et al., 2007; Chang et al., 2012). More recently, our coworkers showed that dillapiole triggers apoptotic processes in mouse blastocysts and impairs early post-implantation development in vitro and in vivo (Chan, 2014). However, no studies to date have elucidated the potential cytotoxic effects of dillapiole on oocyte maturation, fertilization, and subsequent embryonic development. The microenvironment affects oocyte growth and maturation, with consequent impacts on oocyte viability.
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For instance, oxygen concentration, temperature and glucose content have been identified as key determinants of oocyte viability (Banwell et al., 2007; de Castro & Hansen, 2007; Sartori et al., 2002). A number of previous studies have documented the influence of natural chemical compounds on oocyte maturation in vivo and in vitro (Chang & Chan, 2010; Chang et al., 2012). During normal embryogenesis and early development, programmed cell death (apoptosis) functions to remove abnormal or redundant cells in pre-implantation embryos (Hardy, 1997; Hardy et al., 2003). However, programmed cell death processes do not occur prior to the blastocyst stage during normal mouse embryonic development (Byrne et al., 1999), and induction of cell death during oocyte maturation and early stages of embryogenesis (e.g. upon teratogen exposure) causes embryonic developmental injury (Banwell et al., 2007; Chan, 2006, 2009; Hsuuw et al., 2005; Shiao & Chan, 2009). In the current study, we systematically investigated whether dillapiole has injurious effects on early stage embryo maturation and development by incubating mouse oocytes with the compound for 24 h and comparing subsequent development in an untreated control group. The main purpose of this study was to determine whether short-term exposure to dillapiole at the oocyte stage has a long-term deleterious impact on subsequent embryonic development. Our results clearly demonstrate that exposure to dillapiole at the oocyte stage not only retards oocyte maturation but also causes injurious effects on IVF and subsequent embryonic development.
Materials and methods Chemicals and reagents Dulbecco’s modified Eagle’s medium (DMEM), dillapiole and pregnant mare serum gonadotropin (PMSG) were obtained from Sigma (St. Louis, MO). Human chorionic gonadotropin (hCG) was purchased from Serono (NV Organon Oss, The Netherlands). TUNEL in situ cell death detection kits were acquired from Roche (Mannheim, Germany). CMRL-1066 medium was purchased from Gibco Life Technologies (Grand Island, NY). Cumulus–oocyte complex collection and in vitro maturation ICR mice were acquired from the National Laboratory Animal Center (Taiwan, ROC) and provided food (Teklad Mouse Breeder Diet; Harlan Laboratories, Indianapolis, IN) and water ad libitum. Mice were housed in standard 28 16 11 cm (height) polypropylene cages with wire-grid tops, and maintained under a 12-h day/night regimen. Cumulus–oocyte complexes (COCs) were obtained according to a previous protocol (Banwell et al., 2007). Briefly, COCs were isolated from female hybrid ICR mice (21 days old), injected with hCG (5 IU) 44 h prior to oocyte collection. COCs were collected in HEPES-buffered a-minimum essential medium (a-MEM) containing 50 mg/ml streptomycin sulfate, 75 mg/ml penicillin G and 5% fetal bovine serum (FBS) by gently puncturing visible antral follicles present on the ovary surface. Germinal vesicle stage oocytes containing an intact vestment of cumulus
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cells were collected and pooled from at least eight animals and randomly selected for experiments. For in vitro maturation (IVM) of oocytes, one drop (100 ml) of medium (a-MEM supplemented with 50 mg/ml streptomycin, 75 mg/ml penicillin G, 5% FBS and 50 mIU/ml recombinant human folliclestimulating hormone) containing 10 COCs for each treatment group was added under oil in 35 mm culture dishes. COC maturation was analyzed following incubation with or without different concentrations of dillapiole (2.5, 5 or 10 mM) for 24 h under a 5% O2 and 6% CO2 atmosphere (balance, N2) at 37 C. This research was approved by the Animal Research Ethics Board of Chung Yuan Christian University (Taiwan, ROC). All animals received humane care following Principles of Laboratory Animal Care (National Institutes of Health publication 85-23, revised 1996; Institute of Laboratory Animal Resources, 1996). Maturation status assessment After IVM, COCs of each group were treated with 50 U/ml ovine hyaluronidase and gently pipetted to remove all cumulus cells. Denuded oocytes were collected and washed first with fresh medium and then with phosphate-buffered saline (PBS). Oocytes were fixed in ethanol:glacial acetic acid (1:3) for 48 h and stained with a 1% aceto-orcein solution. Nuclear structures were visualized using phasecontrast microscopy (Olympus BX51, Tokyo, Japan). In vivo maturation In vivo-matured oocytes were obtained by injecting 21-day-old mice with 5 IU equine chorionic gonadotrophin (eCG) and 5 IU hCG, 61 and 13 h prior to fertilization, respectively. Mature ova were collected from the oviduct into HEPES-buffered a-MEM medium. Effects of dillapiole intake on oocyte maturation in an animal model The effects of dillapiole on oocytes were analyzed in 21-dayold ICR virgin albino mice. Female mice were randomly divided into four groups of 20 animals each and administered a standard diet with 0, 2.5, 5 or 10 mM dillapiole in drinking water for 4 days. COCs were collected by pre-treatment with 5 IU hCG for 44 h prior to oocyte collection, and analyzed for oocyte maturation, IVF and embryonic development. In vitro fertilization For IVF, ova were washed twice in bicarbonate-buffered a-MEM medium containing 50 mg/ml streptomycin, 75 mg/ ml penicillin G and 3 mg/ml fatty acid free bovine serum albumin (BSA), and fertilized in the same medium containing fresh sperm (obtained from a CBAB6F1 male donor). After incubation with sperm for 4.5 h, eggs were washed three times in potassium simplex optimized medium (KSOM) without amino acids in the presence of L-alanyl-L-glutamine (1.0 mM). Next, eggs were placed in 20-ml drops of KSOM under oil, and cultured overnight. During cleavage to the 2-cell stage, embryos were transferred to a fresh drop of KSOM under oil, and cultured for an additional 72 h.
Effects of dillapiole on oocyte maturation
DOI: 10.3109/01480545.2014.1002037
All fertilization embryo culture steps were carried out in a 5% O2/6% CO2 atmosphere (balance, N2) at 37 C. Fertilization assessment Fertilization was assessed by incubating ova with sperm for 4.5 h, followed by 3 h of culture in fresh medium. Zygotes were assessed for the presence of the male pronucleus by orcein staining, as described previously (Banwell et al., 2007).
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Cell proliferation Cell proliferation was analyzed by dual differential staining, which facilitates the counting of cell numbers in the inner cell mass (ICM) and trophectoderm (TE; Chan, 2007; Huang et al., 2007; Pampfer et al., 1990). The zona pellucida was removed by incubating blastocysts with 0.4% pronase in M2BSA medium (M2 medium containing 0.1% BSA). Denuded blastocysts were exposed to 1 mM trinitrobenzenesulfonic acid (TNBS) in BSA-free M2 medium containing 0.1% polyvinylpyrrolidone (PVP) at 4 C for 30 min, and washed with M2 (Hardy et al., 1989). Blastocysts were then incubated with an anti-dinitrophenol–BSA complex antibody in M2BSA (30 mg/ml) at 37 C for 30 min, followed by incubation with M2 supplemented with 10% whole guinea pig serum (source of complement), together with 20 mg/ml bisbenzimide and 10 mg/ml propidium iodide (PI) at 37 C for 30 minutes. Immunolyzed blastocysts were gently transferred to slides and protected from light before observation. Under UV light, ICM cells (which take up bisbenzimidine but exclude PI) appeared blue, whereas TE cells (which take up both fluorochromes) appeared orange-red. Since multinucleated cells are not common in pre-implantation embryos (Gardner & Davies, 1993), the number of nuclei represent an accurate measurement of cell number. TUNEL assay of blastocysts For terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, embryos were washed in dillapiole-free medium, fixed, permeabilized and labeled using an in situ cell death detection kit (Roche Molecular Biochemicals, Indianapolis, IN), according to the manufacturer’s protocol. Images were obtained with a fluorescence microscope under bright-field illumination (Olympus BX70, Tokyo, Japan).
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embryos and eight dillapiole-treated embryos were transferred to the left and right uterine horns, respectively, of day 4 pseudopregnant mice. Forty surrogate mice per group were analyzed and killed on day 13 post-transfer (18-day fetuses); the frequency of implantation was calculated as the number of implantation sites per number of embryos transferred. The incidence rates of surviving fetuses (number of surviving fetus/number of implantations) and resorbed fetuses ([number of implantations – number of surviving fetuses]/number of implantations) were calculated. The weights of surviving fetuses and placenta were measured immediately after dissection. Statistical analysis Data were analyzed using one-way analysis of variance (ANOVA) and t-tests, and presented as means ± SD. Data were considered statistically significant at p50.05.
Results Impact of dillapiole on oocyte maturation status, fertilization rate and in vitro embryo development Although recent evidence has shown that dillapiole induces apoptosis and developmental injury in mouse blastocysts (Chan, 2014), its effects on oocyte maturation and subsequent development are currently unclear. To evaluate the effects of dillapiole on oocyte nuclear maturation status, oocytes were incubated with the compound, as described in the ‘‘Materials and methods’’ section, in experiments examining 10 independent experimental replicates with 200–250 oocytes per group. Overall, 93.7% of oocytes reached the metaphase II (MII) stage of maturation after IVM in the untreated control group. Notably, pre-incubation with dillapiole prior to IVM induced a dose-dependent decrease in the maturation rate and significant decrease in the ability of oocytes to be fertilized by fresh sperm, measured as male pronucleus formation in oocytes (Figure 1). We further investigated in vitro embryo development from the zygote to the 2-cell and blastocyst stages. Dillapiole pretreatment significantly decreased the cleavage rate of oocytes
Blastocyst development following embryo transfer The ability of expanded blastocysts to implant and develop in vivo was assessed by transferring generated embryos to recipient mice. Albino ICR female mice (6–8 weeks old) were mated with vasectomized male C57BL/6 J mice National Laboratory Animal Center, Taiwan, ROC) to produce pseudopregnant dams as recipients for embryo transfer. To ensure that all fetuses in pseudopregnant mice were derived from embryo transfer (white color) and not fertilization by C57BL/6 J (black color), we examined skin color at day 18 post-coitus. The impact of dillapiole on post-implantation growth in vivo was assessed by exposing COCs to 0–10 mM dillapiole for 24 h, followed by fertilization and IVM to the blastocyst stage. Subsequently, eight untreated control
Figure 1. Effects of dillapiole on mouse oocyte maturation and embryo development in vitro. Oocytes were collected from 21-day-old mice, cultured for 24 h in IVM medium containing dillapiole (2.5, 5 or 10 mM), fertilized in vitro and transferred to in vitro culture (IVC) medium. Oocyte maturation, IVF, cleavage and blastocyst development were analyzed. Values are presented as means ± SD of eight determinations. Data are based on 200–250 samples per group. ***p50.001 versus untreated control group.
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to the 2-cell stage, indicative of a deleterious effect (Figure 1). In addition, the ratio of embryos that cleaved and developed to form blastocysts in the dillapiole-treated group was markedly lower than in the untreated control group (Figure 1).
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Effects of dillapiole on cell proliferation and apoptosis of embryos during maturation of oocytes in vitro For further evaluation of whether the hazardous effects of dillapiole on oocyte maturation and sequent development are exerted through inhibition of cell proliferation or induction of apoptosis, proliferation was determined by counting total blastocyst cell numbers during in vitro maturation of oocytes in dillapiole-treated and untreated groups. Differential staining for ICM and TE cells was used to assist in the assessment of cell proliferation. Pre-incubation of oocytes with dillapiole led to significantly decreased blastocyst cell numbers, compared with control oocytes (Figure 2A). In addition, the number of ICM cells in blastocysts was decreased during IVM in the dillapiole-pretreated group (Figure 2A). However, dillapiole had no significant effect on the number of TE cells in blastocysts (Figure 2A). TUNEL staining data showed a concentration-dependent increase in apoptosis in blastocysts derived from the dillapiolepre-incubated oocyte group (Figure 2B). Quantitative analyses further revealed a 7.8- to 13.6-fold increase in apoptotic cells in blastocysts derived from dillapiole-pretreated oocytes, compared with the untreated control group (Figure 2C). Developmental potential of blastocysts from oocytes and effects of dillapiole intake on oocyte development For further evaluation of the developmental potential of blastocysts derived from dillapiole-treated oocytes, embryonic development was measured using an embryo transfer assay. Embryos were transferred to 50 recipients per group (8 per horn). In embryo transfer assays, a total of 40 recipients per group were found to be pregnant in at least one horn on day 18. The implantation rate of blastocysts derived from the dillapiole (10 mM)-treated oocyte group (28%) was significantly lower than that observed for control blastocysts (81%) during IVM (Figure 3A). Implanted embryos that failed to develop were subsequently resorbed in the uterus. About 73% of implanted embryos failed to develop normally in the group treated with 10 mM dillapiole, a rate significantly higher than that for the untreated control group (32%). Consistently, the resorption rate for dillapiole-pre-treated group was higher than that for the untreated control group (Figure 3A). In addition, the embryo survival rate, calculated based on survival to day 13 post-transfer (18-day fetuses), was markedly higher for the control group (68.2%) than the dillapiole (10 mM)-treated group (27.4%; Figure 3A). Interestingly, the placental weights of blastocysts derived from dillapiole-treated oocytes matured in vitro were not significantly different from those of the control group (Figure 3B), while fetal weights were lower in dillapiole (5 or 10 mM)-treated groups, compared with the untreated control group. Specifically, only 8.9% of fetuses in the dillapiole (10 mM)-pre-treated group weighed more than 600 mg, whereas 48% of control fetuses exceeded this
Figure 2. Effects of dillapiole on cell number and apoptosis in embryos during in vitro maturation of oocytes. Oocytes were cultured for 24 h in IVM medium containing dillapiole (2.5, 5 or 10 mM), fertilized in vitro and transferred to in vitro culture (IVC) medium for in vitro development. (A) Total cell number and numbers of TE lineage and ICM cells were counted in blastocysts. (B) Apoptotic cells were examined at the blastocyst stage using TUNEL staining followed by light microscopy. Black cells correspond to TUNEL-positive cells. (C) The mean number of apoptotic (TUNEL-positive) cells per blastocyst was calculated. Values are presented as means ± SD of eight determinations. Data are based on at least 200 samples in each group. ***p50.001 versus the untreated control group.
threshold, an important indicator of successful embryonic and fetal development (Figure 3C). Our results collectively indicate that exposure of oocytes to dillapiole during IVM causes a decrease in post-implantation development potential. To establish whether dillapiole exerts deleterious effects on oocyte maturation and embryo development in vivo, we
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DOI: 10.3109/01480545.2014.1002037
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Figure 3. Effects of dillapiole treatment or dietary dillapiole on embryo development during oocyte IVM. Oocytes were cultured for 24 h in IVM medium containing dillapiole (2.5, 5 or 10 mM), fertilized in vitro and transferred to in vitro culture (IVC) medium for development. (A) Fetus implantation, resorption and survival were analyzed as described in the ‘‘Materials and methods’’ section. The implantation percentage represents the number of implantations per number of transferred embryos 100. The percentage of resorption or surviving fetuses represents the number of resorptions or surviving fetuses per number of implantations 100. (B) Placental weights of 40 recipient mice were measured. (C) Weight distribution of surviving fetuses at day 13 post-transfer (18-day fetuses). Surviving fetuses were obtained via embryo transfer of control and dillapiole-pretreated groups, as described in the ‘‘Materials and methods’’ section (320 total blastocysts from 40 recipients). (D) Random female mice (21 days old) were fed a standard diet and drinking water with or without dillapiole (2.5, 5 or 10 mM) for 4 days. Oocytes were collected for IVM, IVF, cleavage and blastocyst development analyses. Data are based on at least 320 samples in each group. ***p50.001 versus the dillapiole-free group.
analyzed the impact of dillapiole intake in an animal model. Female mice were supplied drinking water containing dillapiole (5 or 10 mM) for 4 days or untreated drinking water, prior to COC collection, followed by evaluation of oocyte maturation status, fertilization rate and in vitro embryo development. Compared with the control group, dillapiole intake significantly decreased oocyte maturation and fertilization and inhibited subsequent embryonic development from the zygote to blastocyst stage (Figure 3D). Impact of dillapiole intake on the developmental potential of blastocyst-stage embryos The deleterious effects of dillapiole intake on embryo implantation and subsequent post-implantation development in vivo were measured using an embryo transfer assay. In this animal model, female mice were fed a standard diet and supplied drinking water with or without dillapiole (2.5, 5 or 10 mM) for 4 days before transfer of blastocyst-stage embryos to the uterine horn. Forty recipients were pregnant at day 18 (day 14 post-embryo transfer). Notably, the implantation rate of blastocysts was significantly lower in the dillapiole-intake group than the untreated control group (Figure 4A). Moreover, the resorption rate was higher and embryo survival rate lower, compared to the dillapiole-free control group (Figure 4A). Interestingly, placental weights of blastocysts derived from the dillapiole-intake group were not
significantly different from those of the control group (data not shown). However, fetal weights were lower in the dillapiole-intake (10 mM) than the untreated control group (Figure 4B). Our results clearly demonstrate that pretreatment or exposure of embryos to dillapiole reduces implantation potential and subsequent post-implantation development. Effects of dillapiole-induced apoptosis on oocyte maturation status, fertilization rate and embryo development during IVM To identify the regulatory mechanisms of dillapiole activity, we pre-incubated oocytes with 100 mM Ac-DEVD-cho, a caspase-3-specific inhibitor, with the aim of preventing dillapiole-induced embryo cell apoptosis during IVM. AcDEVD-cho effectively prevented apoptosis of blastocysts derived from oocytes pre-treated with 10 mM dillapiole (Figure 5A). Notably, inhibition of dillapiole-induced apoptosis further blocked the deleterious effects on oocyte maturation, fertilization rate and subsequent embryo development during IVM (Figure 5B). In addition, pre-incubation with Ac-DEVD-cho prevented dillapiole-induced reduction in post-implantation development potential following embryo transfer, as reflected by improvements in the embryo implantation and fetal survival rates and fetal development status (i.e. fetal weight) (Figure 5C). These findings indicate
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Figure 4. Effects of dietary dillapiole on embryo development in mouse blastocysts. Random female mice (21 days old) were fed a standard diet and drinking water with or without dillapiole (2.5, 5 or 10 mM) for 4 days before embryo transfer to the uterus. (A) Fetus implantation, resorption and survival were analyzed as described in the ‘‘Materials and methods’’ section. The implantation percentage represents the number of implantations per number of transferred embryos 100. The percentage of resorption or surviving fetuses represents the number of resorptions or surviving fetuses per number of implantations 100. (B) Weight distribution of surviving fetuses at day 14 post-transfer (18-day fetuses). Surviving fetuses were obtained via embryo transfer in the control and dillapiole-intake groups (320 total blastocysts from 40 recipients). ***p50.001 versus the dillapiole-free group.
that dillapiole-induced disruption of oocyte development during IVM involves an apoptotic mechanism.
Discussion Deleterious effects of chemical or physical injury may affect normal progression of the complex and precisely orchestrated process of oocyte maturation and early stage embryonic development, leading to malformation or miscarriage of the embryo. Therefore, it is important to establish the possible effects of various chemical agents or physical factors on these processes. One such agent is the phenylpropanoid dillapiole, the main component of Piper aduncum that is also present in other Piper species. A recent study demonstrated for the first time that dillapiole induces cell death through the mitochondria-dependent apoptotic pathway by triggering oxidative stress and exerts anti-proliferative effects on MDA-MB-231 cells (Ferreira et al., 2014). The broad cytotoxic effects of dillapiole against a variety of tumor cells have been highlighted (Ferreira et al., 2014). Safrole, a phenylpropanoid structure related to dillapiole also identified in Piper (genus; Andrade et al., 2008), is reported to trigger apoptosis in human oral cancer cells (Yu et al., 2011), as well as cell cycle arrest and apoptosis in human leukemia cells via a ROSmediated endoplasmic reticulum (ER) stress-involved cell regulatory signaling cascade (Yu et al., 2012). These findings support the potential utility of dillapiole for development as a chemopreventive and/or chemotherapeutic agent in human
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cancers. Importantly, a recent study by our coworkers disclosed that 5 and 10 mM dillapiole has hazardous effects on pre- and post-implantation embryonic development, leading to induction of apoptosis, retardation of early stage embryonic development and negative effects on postimplantation progress. Based on these results, we conclude that dillapiole at concentrations of 5 and 10 mM induces apoptosis and negatively affects mouse embryonic development, both in vitro and in vivo (Chan, 2014). Here, we further examined the possible cytotoxic effects of dillapiole on oocyte maturation, fertilization and early and post-implantation embryonic development. Our results consistently suggest that apoptotic effects play a regulatory role in dillapioleinduced oocyte developmental injury. Chemical compounds or physical factors have significant potential to cause deleterious effects on the complex processes of oocyte maturation, fertilization and early stage embryonic development, leading to developmental problems or embryonic malformation. Experiments from the current study demonstrated for the first time that dillapiole impairs mouse oocyte maturation, fertilization and subsequent embryonic development (Figure 1). Pre-incubation of oocytes with dillapiole led to decreased cell numbers and increased apoptosis (Figure 2A and B). The trophoblasts of blastocysts develop into TE. These cells contribute to the placenta and are required for mammalian conceptus development in post-implantation stage embryos (Cross et al., 1994). Previous studies have demonstrated that a decrease in TE and/or ICM lineage cell numbers inhibits embryo implantation and reduces viability (Kelly et al., 1992; Pampfer et al., 1990). TE cells as well as ICM and total blastocyst cell numbers are positively correlated with embryo implantation and subsequent development (Lane & Gardner, 1997). In our experiments, dillapiole treatment during oocyte maturation had no impact on the number of blastocyst TE cells, but decreased cell numbers in the ICM (Figure 2). These results indicate that pre-incubation with dillapiole during IVM triggers mortality and/or deleterious effects on development in post-implantation mouse embryos. Moreover, the mechanisms underlying these actions involve ICM cell death and/or inhibition of proliferation (Figures 2 and 3). Blastocysts derived from dillapiole-treated oocytes had lower implantation rates, and exhibited higher embryo resorption and reduced fetal survival (Figure 3A). However, the placental weights of dillapiole-treated and control animals were not significantly different. TE cells in embryos are major contributors to implantation and subsequent placental development. Interestingly, dillapiole had no deleterious effects on blastocyst TE cells or the related placental development process. Based on these findings, we propose that reduction in the ICM cell number induced by dillapiole during oocyte maturation is the major factor responsible for inhibition of embryonic development. Further investigation of the regulatory mechanisms underlying the deleterious effects of dillapiole on oocyte maturation and subsequent embryonic development revealed a decrease in cell number, increased apoptosis and delayed postimplantation development of blastocysts in dillapiole-treated oocytes, compared with untreated controls. These negative effects were attenuated upon suppression of blastocyst
Effects of dillapiole on oocyte maturation
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DOI: 10.3109/01480545.2014.1002037
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Figure 5. Effects of inhibition of dillapiole-induced cell apoptosis on in vitro maturation of oocytes. Oocytes were collected from 21-day-old mice and cultured for 24 h in IVM medium alone or IVM containing 100 mM Ac-DEVD-cho and 10 mM dillapiole, fertilized in vitro and transferred to in vitro culture (IVC) medium for in vitro development. (A) Apoptotic cells were examined at the blastocyst stage via TUNEL staining. (B) Oocyte maturation, IVF, cleavage, and blastocyst development were analyzed. (C) Fetus implantation, resorption and survival were analyzed using the embryo transfer assay, as described for Figure 3. ***p50.001 versus the dillapiole-free group. #p50.001 versus the dillapiole-treated group.
apoptosis via pre-treatment of oocytes with a caspase-3 inhibitor (Figure 5). Pre-treatment with the caspase-3 inhibitor additionally prevented dillapiole-induced reduction of post-implantation development potential following embryo transfer, and led to improvements in the embryo implantation rate, fetal survival rate and fetal development status. These results collectively suggest that dillapiole induces improper apoptotic processes in early stage embryos, leading to loss of embryonic cells and prevents post-implantation development, thereby acting as a teratogen through induction of programmed cell death in early stage cells. Evidence from the current study that dillapiole exerts injurious effects on oocyte maturation, fertilization and subsequent embryonic development supports the theory that dillapiole is a risk factor for normal embryonic development that may lead to reduced oocyte maturation in infertile couples. Our results provide an important framework for further determining the possible teratogenic effects of dillapiole on human oocytes and embryo development, with a view to assessing the safety of its application as an anti-inflammatory or anti-cancer drug in the clinic. Therefore, further studies to determine the possible teratogenic effects of dillapiole on human oocyte and embryo development are essential.
Declaration of interest This work was supported by grants (NSC101-2311-B-033001-MY3, NSC101-2632-M-033-001-MY2, NSC102-2627M-033-001 and NSC102-2632-M-033-001-MY3) from the National Science Council of Taiwan, ROC.
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