Prevention of methylmercury-triggered ROS-mediated impairment of embryo development by co-culture with adult adipose-derived mesenchymal stem cells.

Methylmercury (MeHg) is a potent toxin that exerts deleterious effects on human health via environmental contamination. Significant effects of MeHg on neuronal development in embryogenesis have been reported. Recently, our group demonstrated that MeHg exerts toxic effects on pre- and post-implantation embryonic development processes from zygote to blastocyst stage. Our results showed that MeHg impairs embryo development by induction of apoptosis through reactive oxygen species (ROS) generation that triggers caspase-3 cleavage and activation, which, in turn, stimulates p21-activated kinase 2 (PAK2) activity. Importantly, ROS were identified as a key upstream regulator of apoptotic events in MeHg-treated blastocysts. Data from the current study further confirmed that MeHg exerts hazardous effects on cell proliferation, apoptosis, implantation, and pre- and post-implantation embryo development. Notably, MeHg-induced injury was markedly prevented by co-culture with adipose-derived mesenchymal stem cells (ADMSCs) in vitro. Furthermore, ADMSC injection significantly reduced MeHg-mediated deleterious effects on embryo, placenta, and fetal development in vivo. Further investigation of the regulatory mechanisms by which co-cultured ADMSCs could prevent MeHg-induced impairment of embryo development revealed that ADMSCs effectively reduced ROS generation and its subsequent downstream apoptotic events, including loss of mitochondrial membrane potential and activation of caspase-3 and PAK2. The collective findings indicate that co-culture with mesenchymal stem cells (MSCs) or utilization of MSC-derived cell-conditioned medium offers an effective potential therapeutic strategy to prevent impairment of embryo development by MeHg.

Role of activated p21-activated kinase 2 in methylmercury-induced embryotoxic effects on mouse blastocysts.

Methylmercury (MeHg), a biotransformation product derived from mercury or inorganic mercury compounds in waterways, is a potent toxin that exerts hazardous effects on human health via environmental contamination. Previous studies have reported MeHg-induced impairment of nerve development in embryogenesis and placental development. However, the potential deleterious effects and regulatory mechanisms of action of MeHg on pre- and post-implantation embryo development are yet to be established. Experiments from the current study clearly demonstrate that MeHg exerts toxic effects on early embryonic development processes, including the zygote to blastocyst stage. Induction of apoptosis and decrease in embryo cell number were clearly detected in MeHg-treated blastocysts. Additionally, intracellular reactive oxygen species (ROS) generation and activation of caspase-3 and p21-activated protein kinase 2 (PAK2) were observed in MeHg-treated blastocysts. Importantly, prevention of ROS generation by pre-treatment with Trolox, a potent antioxidant, significantly attenuated MeHg-triggered caspase-3 and PAK2 activation as well as apoptosis. Notably, the downregulation of PAK2 via transfection of specifically targeted siRNA (siPAK2) led to marked attenuation of PAK2 activity and apoptosis and the deleterious effects of MeHg on embryonic development in blastocysts. Our findings strongly suggest that ROS serve as an important upstream regulator to trigger the activation of caspase-3, which further cleaves and activates PAK2 in MeHg-treated blastocysts. Activated PAK2 promotes apoptotic processes that, in turn, cause sequent impairment of embryonic and fetal development.

Role of caspase-3-cleaved/activated PAK2 in brusatol-triggered apoptosis of human lung cancer A549 cells.

Brusatol, a major quassinoid extract of Bruceae fructus, is an important bioactive component with antineoplastic capacity. Several beneficial pharmacological and biological properties of brusatol have been uncovered to date, including anti-inflammatory, anticolitis, antimalarial, and anticancer activities. To confer anticancer benefits, brusatol is reported to effectively inhibit the Nrf2-mediated antioxidant response and trigger apoptotic signaling. In this study, we investigated the regulatory mechanisms underlying apoptotic processes in brusatol-treated A549 cells in detail. Our experiments showed that brusatol induces cell death through intracellular ROS-triggered mitochondria-dependent apoptotic events and does not involve necrosis. Mechanistically, p21-activated protein kinase 2 (PAK2) was cleaved by caspase-3 to generate an activated p34 fragment involved in brusatol-induced apoptosis of A549 cells. Notably, PAK2 knockdown led to downregulation of caspase-3-mediated PAK2 activity, in turn, effectively attenuating brusatol-induced apoptosis, highlighting a crucial role of caspase-3-activated PAK2 in this process. Moreover, knockdown of PAK2 resulted in significant inhibition of c-Jun N-terminal kinase (JNK) activity in brusatol-treated A549 cells, clearly suggesting that JNK serves as a downstream substrate of caspase-3-cleaved/activated PAK2 in the apoptotic cascade. SP600125, a specific JNK inhibitor, significantly suppressed brusatol-induced JNK activity but only partially prevented apoptosis, implying that JNK serves as only one of a number of substrates for PAK2 in the brusatol-triggered apoptotic cascade. Based on the collective results, we propose a signaling cascade model for brusatol-induced apoptosis in human A549 cells involving ROS, caspases, PAK2, and JNK.

Low-dose silver nanoparticles plus methyl mercury exert embryotoxic effects on mouse blastocysts via endoplasmic reticulum stress and mitochondrial apoptosis.

The health and environmental impacts of the increasing commercial use of silver nanoparticles (AgNPs) are a growing concern. Methyl mercury (MeHg) is a potent toxin that biotransforms from mercury or inorganic mercury compounds in waterways and causes dangerous environmental contamination. However, the potential interactions and combined effects of AgNPs and MeHg are yet to be established. In the current study, we showed that low/non-embryotoxic doses of AgNPs and MeHg interact synergistically to induce embryotoxicity and further explored the underlying mechanisms affecting mouse embryo development. Notably, co-treatment with noncytotoxic concentrations of AgNPs (10 µM) and MeHg (0.1 µM) triggered apoptotic processes and embryotoxicity in mouse blastocysts and evoked intracellular reactive oxygen species (ROS) generation, which was effectively blocked by preincubation with 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), a classic antioxidant. Further experiments demonstrated that ROS serve as a key upstream inducer of endoplasmic reticulum (ER) stress and mitochondria-dependent apoptotic processes in AgNP/MeHg-induced injury of mouse embryo implantation and pre- and postimplantation development. Our results collectively indicate that AgNP and MeHg at non-embryotoxic concentrations can synergistically evoke ROS, ultimately causing embryotoxicity through promotion of ER stress and mitochondria-dependent apoptotic signaling cascades.

High levels estradiol affect blastocyst implantation and post-implantation development directly in mice.

BACKGROUND: Previous studies have demonstrated that high levels of estradiol (E2) impair blastocyst implantation through effects on the endometrium; however, whether high E2 directly affects blastocysts is not well established. The present study sought to clarify the direct impacts of high E2 levels on blastocysts in vitro. METHODS: ICR virgin albino mice were used. Using an in-vitro 8-day blastocyst culture model, immunofluorescence staining for the estrogen receptor (ER), blastocyst outgrowth assays, differential staining and TUNEL assays of blastocysts, and embryo transfer, we investigated the main outcomes of exposure to different E2 concentrations (10-7 to 10-4 M) in vitro and in vivo. RESULTS: ERα and ERß expression were detected in pre-implantation stage embryos. In vitro exposure of blastocysts to 10-4 M E2 for 24 h followed by 7 days culture in the absence of E2 caused severe inhibition of implantation and post-implantation development. The late adverse effects of E2 on post-implantation development still occurred at concentrations of 10-7 to 10-5 M. In addition, blastocyst proliferation was reduced and apoptotic cells were increased following exposure to 10-4 M E2. Using an in vivo embryo-transfer model, we also showed that treatment with high E2 resulted in fewer implantation sites (38% vs. 72% in control) and greater resorption of implanted blastocysts (81% vs. 38% in control). CONCLUSION: Exposure to high E2 concentrations in vitro is deleterious to blastocyst implantation and early post-implantation development, mainly owing to direct impacts of E2 on implanting blastocysts. In clinical assisted reproductive technique (ART), high serum E2 concentrations not only affects the endometrium, but also affects blastocysts directly at the period of implantation.

Enniatin B induces dosage-related apoptosis or necrosis in mouse blastocysts leading to deleterious effects on embryo development.

The current study has focused on the effects of enniatin B (ENN B, a major mycotoxin produced by Fusarium fungi) on early embryonic development. In in vitro analysis, mouse blastocysts were incubated in medium with ENN B (0-40 µM) or 0.5% DMSO (control group) for 24 hours. In an animal study, blastocysts were collected from mice which were intravenously injected with ENN B (1, 3, 5, and 7mg/kg body weight/day) for 4 days in order to analyze apoptosis and necrosis via Annexin V/PI staining assay; and proliferation using dual differential staining. Exposure to low ENN B concentration (10 µM in vitro and 3 mg/kg/day in vivo) promoted Reactive Oxygen Species (ROS) generation and apoptosis in the Inner Cell Mass (ICM), the mass of cells inside the blastocyst, impairing post-implantation development alone. On the other hand, exposure to a higher ENN B concentration (40 µM in vitro and 7 mg/kg/day in vivo) induced ROS generation and decreased in intracellular ATP which encouraged necrotic processes in both trophectoderm (TE) and ICM of blastocysts leading to impaired implantation and post-implantation development. Moreover, 5 and 7 mg/kg/day ENN B intraperitoneal injection to female mice for 4 days has caused downregulation of CXCL1, IL-1ß and IL-8 expressions and increased ROS generation in the liver of newborn mice. Over all, ENN B can induce apoptosis and/or necrosis depending on the treatment dosage in mouse blastocysts. ENN B-induced necrosis in blastocysts may exert long-term harmful effects on next-generation newborns.

Non-embryotoxic dosage of alternariol aggravates ochratoxin A-triggered deleterious effects on embryonic development through ROS-dependent apoptotic processes.

Alternariol (AOH) and ochratoxin A (OTA), two mycotoxins found in many foods worldwide, exhibit cytotoxicity and embryotoxicity, triggering apoptosis and cell cycle arrest in several mammalian cells and mouse embryos. The absorption rate of AOH from dietary foodstuff is low, meaning that the amount of AOH obtained from the diet rarely approaches the cytotoxic threshold. Thus, the potential harm of dietary consumption of AOH is generally neglected. However, previous findings from our group and others led us to question whether a low dosage of AOH could aggravate the cytotoxicity of other mycotoxins. In the present study, we examined how low dosages of AOH affected OTA-triggered apoptosis and embryotoxicity and investigated the underlying regulatory mechanism in mouse blastocysts. Our results revealed that non-cytotoxic concentrations of AOH (1 and 2 µM) could enhance OTA (8 µM)-triggered apoptotic processes and embryotoxicity in mouse blastocysts. We also found that AOH can enhance OTA-evoked intracellular reactive oxygen species (ROS) generation and that this could be prevented by pretreatment with the potent ROS scavenger, N-acetylcysteine. Finally, we observed that this ROS generation acts as a key inducer of caspase-dependent apoptotic processes and subsequent impairments of embryo implantation and pre- and post-implantation embryonic development. In sum, our results show that non-cytotoxic dosages of AOH can aggravate OTA-triggered apoptosis and embryotoxicity through ROS- and caspase-dependent signaling pathways.

Alternariol exerts embryotoxic and immunotoxic effects on mouse blastocysts through ROS-mediated apoptotic processes.

Alternariol (AOH), a mycotoxin belonging to the genus Alternaria, has been shown to induce cytotoxicity, including apoptosis and cell cycle arrest, in several mammalian cell types. However, its effects on early-stage embryonic development require further investigation. Here, we have shown that AOH exerts embryotoxic effects on mouse blastocyst-stage embryos and long-term adverse effects on immunity in one-day-old newborn mice of the next generation. Significant apoptosis and decrease in total cell number, predominantly through loss of inner cell mass (ICM), and to a minor extent, trophectoderm (TE) cells, were observed in AOH-treated blastocysts. Moreover, AOH exerted detrimental effects on pre- and post-implantation embryo development potential and induced a decrease in fetal weight in in vitro development and embryo transfer assays. Injection of pregnant mice with AOH (1, 3 and 5 mg/kg body weight/day) for 4 days resulted in apoptosis of blastocyst-stage embryos and injurious effects on embryonic development from the zygote to blastocyst stage or embryo degradation and a further decrease in fetal weight. Furthermore, AOH exerted a long-term impact on the next generation, triggering a significant increase in total oxidative stress content and expression of genes encoding antioxidant proteins. Lower expression of CXCL1, IL-1ß and IL-8 related to innate immunity was detected in liver tissue extracts obtained from one-day-old newborns of AOH-injected pregnant mice (5 mg/kg body weight/day) relative to their non-treated counterparts. In addition, ROS served as an upstream regulator of AOH-triggered apoptotic processes and impairment of embryonic development. Our collective results highlight the potential of AOH as an embryotoxic and immunotoxic risk factor during embryo and infant development stages in mice.

Dose-dependent beneficial and harmful effects of berberine on mouse oocyte maturation and fertilization and fetal development.

Previous studies have shown that berberine, an isoquinoline alkaloid isolated from several traditional Chinese herbal medicines, suppresses growth and induces apoptosis in some tumor cell lines. It has also been shown that berberine possesses anti-atherosclerosis and antioxidant activities in hyperlipidemic model rats. Our previous study in mice found that berberine causes harmful effects on preimplantation and postimplantation embryonic development, both in vitro and in vivo, by triggering reactive oxygen species (ROS)-mediated apoptotic cascades in mouse blastocysts. In the current investigation, we further showed that berberine treatment has distinct dose-dependent effects on oocyte maturation and subsequent development. Preincubation of oocytes with 2.5 µM berberine significantly enhanced maturation and in vitro fertilization (IVF) rates, with subsequent beneficial effects on embryonic development. In contrast, preincubation with 10 µM berberine negatively impacted mouse oocyte maturation, decreased IVF rates and impaired subsequent embryonic development. Similar dose-dependent effects were also demonstrated in vivo. Specifically, intravenous injection of berberine significantly enhanced mouse oocyte maturation, IVF rate and early-stage embryo development after fertilization at a dose of 1 mg/kg body weight but significantly impaired oocyte maturation and IVF rates and caused harmful effects on early embryonic development at a dose of 5 mg/kg. Mechanistically, we found that berberine enhanced intracellular ROS production and apoptosis of oocytes at a concentration of 10 µM but actually significantly decreased total intracellular ROS content and had no apoptotic effect at a concentration of 2.5 µM. Moreover, pretreatment of oocytes with Ac-DEVD-cho, a caspase-3-specific inhibitor, effectively blocked berberine-induced negative impacts on oocyte maturation, fertilization and subsequent development. Collectively, these findings establish the dose-dependent beneficial versus deleterious effects of berberine and suggest that the mechanism underlying the deleterious effects of berberine involves a caspase-3-dependent apoptotic process acting downstream of an increase in intracellular ROS levels.

Dosage-related beneficial and deleterious effects of ginsenoside Rb1 on mouse oocyte maturation and fertilization and fetal development.

Ginsenoside Rb1 (GRb1), the major saponin component of ginseng root, has a wide range of therapeutic applications for various diseases. Previously, our group showed that GRb1 triggers ROS-mediated apoptotic cascades in mouse blastocysts, leading to decreased cell viability and impairment of pre- and postimplantation embryonic development, both in vitro and in vivo. In this study, we further found that GRb1 exerted dose-dependent effects on oocyte maturation and sequent development in vitro. Oocytes preincubated with 25 µg/mL GRB1 displayed significantly enhanced maturation and in vitro fertilization (IVF) rates, along with progression of subsequent embryonic development. In contrast, treatment with 50 and 100 µg/mL GRB1 led to impairment of mouse oocyte maturation, decreased IVF rates, and injurious effects on subsequent embryonic development. In vivo, intravenous injection of 1 mg/kg body weight GRb1 significantly promoted mouse oocyte maturation, IVF, and early-stage embryo development after fertilization while administration of 5 mg/kg body weight GRb1 led to a marked decrease in oocyte maturation and IVF rates concomitant with impairment of early embryonic development in our animal model. In terms of the mechanisms underlying the regulatory effects of GRb1 demonstrated increased intracellular reactive oxygen species (ROS) production and apoptosis in the 100 µg/mL GRb1 treatment group. However, we observed a significant decrease in total intracellular ROS content and inhibition of apoptosis events in the 25 µg/mL GRb1 treatment group, signifying that the intracellular ROS content serves as a key upstream regulator of GRb1 that influences its dose-dependent beneficial or deleterious effects on oocyte maturation and sequent embryonic development. For further clarification of the mechanisms underlying GRb1-triggered injurious effects, oocytes were pretreated with Ac-DEVD-CHO, a caspase-3-specific inhibitor, which effectively blocked injury to oocyte maturation, fertilization, and sequent development. In sum, study findings highlight the potential involvement of p53-, p21-, and caspase-3-dependent regulatory signaling cascades in GRb1-mediated apoptotic processes.

Prevention of ochratoxin A-induced oxidative stress-mediated apoptotic processes and impairment of embryonic development in mouse blastocysts by liquiritigenin.

Ochratoxin A (OTA), a mycotoxin constituent of a range of food commodities, including coffee, wine, beer, grains, and spices, exerts toxicological and pathological effects in vivo, such as nephrotoxicity, hepatotoxicity, and immunotoxicity. In a previous report, we highlighted the potential of OTA to induce apoptosis via reactive oxygen species (ROS) generation in mouse blastocysts that led to impaired preimplantation and postimplantation embryo development in vitro and in vivo. Here, we have shown that liquiritigenin (LQ), a type of flavonoid isolated from Glycyrrhiza radix, effectively protects against OTA-mediated apoptosis and inhibition of cell proliferation in mouse blastocysts. Preincubation of blastocysts with LQ clearly prevented OTA-triggered impairment of preimplantation and postimplantation embryonic development and fetal weight loss, both in vitro and in vivo. Detailed investigation of regulatory mechanisms revealed that OTA mediated apoptosis and embryotoxicity through ROS generation, loss of mitochondrial membrane potential (MMP), and activation of caspase-9 and caspase-3, which were effectively prevented by LQ. The embryotoxic effects of OTA were further validated in an animal model in vivo. Intravenous injection of dams with OTA (3 mg/kg/day) led to apoptosis of blastocysts, impairment of embryonic development from zygote to blastocyst stage and decrease in day 18 fetal weight. Notably, preinjection of dams with LQ (5 mg/kg/day) effectively prevented OTA-induced apoptosis and toxic effects on embryo development. Our collective results clearly demonstrate that OTA exposure via injection has the potential to damage preimplantation and postimplantation embryonic development against which LQ has a protective effect.

Enniatin B1 exerts embryotoxic effects on mouse blastocysts and induces oxidative stress and immunotoxicity during embryo development.

Enniatins are mycotoxins of Fusarium fungi that naturally exist as mixtures of cyclic depsipeptides. Previous reports have documented hazardous effects of enniatins on cells, such as apoptosis. However, their effects on pre- and post-implantation embryonic development require further clarification. Here, we showed for the first time that enniatin B1 (ENN B1) exerts cytotoxic effects on mouse blastocyst-stage embryos and induces intracellular oxidative stress and immunotoxicity in mouse fetuses. Co-incubation of blastocysts with ENN B1 triggered significant apoptosis and led to a decrease in total cell number predominantly through loss of inner cell mass. In addition, ENN B1 appeared to exert hazardous effects on pre and postimplantation embryo development potential in an in vitro development assay. Treatment of blastocysts with 1-10 µM ENN B1 led to increased resorption of post-implantation embryos and decreased fetal weight in the embryo transfer assay in a dose-dependent manner. Importantly, in an in vivo model, intravenous injection with ENN B1 (1, 3, and 5 mg/kg body weight/d) for 4 days resulted in apoptosis of blastocyst-stage embryos and impairment of embryonic development from the zygote to blastocyst stage, subsequent degradation of embryos, and further decrease in fetal weight. Intravenous injection with 5 mg/kg body weight/d ENN B1 additionally induced a significant increase in total reactive oxygen species (ROS) content and transcription levels of genes encoding antioxidant proteins in mouse fetal liver. Moreover, ENN B1 triggered apoptosis through ROS generation and strategies to prevent apoptotic processes effectively rescued ENN B1-mediated hazardous effects on embryonic development. Transcription levels of CXCL1, IL-1ß, and IL-8 related to innate immunity were downregulated after intravenous injection of ENN B1. These results collectively highlight the potential of ENN B1 to exert cytotoxic effects on embryos as well as oxidative stress and immunotoxicity during mouse embryo development.

Hazardous impacts of silver nanoparticles on mouse oocyte maturation and fertilization and fetal development through induction of apoptotic processes.

Silver nanoparticles (AgNPs) are antibacterial materials widely used in numerous products and medical supplies. Previously, we showed that AgNPs trigger apoptotic processes in mouse blastocysts, leading to a decrease in cell viability and impairment of preimplantation and postimplantation embryonic development in vitro and in vivo. In the present study, we further investigated the hazardous effects of AgNPs on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent preimplantation and postimplantation development in vitro and in vivo. Data from in vitro experiments revealed that AgNPs impair mouse oocyte maturation, decrease IVF rates, and induce injury effects on subsequent embryonic development to a significant extent. In an animal model, intravenous injection of AgNPs (5 mg/kg body weight) led to a significant decrease in mouse oocyte maturation and IVF concomitant with impairment of early embryonic development in vivo. Importantly, pretreatment with N-acetylcysteine effectively prevented AgNP-triggered reactive oxygen species (ROS) production and apoptosis, clearly suggesting a critical role of ROS as an upstream initiator or key regulator of AgNP-induced hazardous effects on oocyte maturation and sequent embryonic development. Furthermore, preincubation of oocytes with Ac-DEVD-cho, a caspase-3-specific inhibitor, effectively prevented hazardous effects, highlighting the potential involvement of caspase-dependent apoptotic signaling cascades in AgNP-mediated events. Expression levels of p53 and p21 of blastocysts were upregulated upon preincubation of mouse oocytes with AgNPs. Our collective results imply that cell apoptosis in mouse blastocysts derived from the AgNP-pretreated oocytes via intracellular ROS generation, which is further mediated through p53-, p21-, and caspase-3-dependent regulatory mechanisms.

Berberine impairs embryonic development in vitro and in vivo through oxidative stress-mediated apoptotic processes.

Berberine, an isoquinoline alkaloid isolated from several traditional Chinese herbal medicines, has been shown to suppress growth and induce apoptosis in some tumor cell lines. However, berberine has also been reported to attenuate H2 O2 -induced oxidative injury and apoptosis. The basis for these ambiguous effects of berberine-triggering or preventing apoptosis-has not been well characterized to date. In the current investigation, we examined whether berberine exerts cytotoxic effects on mouse embryos at the blastocyst stage and affects subsequent embryonic development in vitro and in vivo. Treatment of blastocysts with berberine (2.5-10 µM) induced a significant increase in apoptosis and a corresponding decrease in trophectoderm cell number. Moreover, the implantation success rate of blastocysts pretreated with berberine was lower than that of their control counterparts. Pretreatment with berberine was also associated with increased resorption of postimplantation embryos and decreased fetal weight. In an animal model, intravenous injection of berberine (2, 4, or 6 mg/kg body weight/d) for 4 days resulted in apoptosis of blastocyst cells and early embryonic developmental injury. Berberine-induced injury of mouse blastocysts appeared to be attributable to oxidative stress-triggered intrinsic apoptotic signaling processes that impaired preimplantation and postimplantation embryonic development. Taken together, our results clearly demonstrate that berberine induces apoptosis and retards early preimplantation and postimplantation development of mouse embryos, both in vitro and in vivo.

Protective Effects of Liquiritigenin against Citrinin-Triggered, Oxidative-Stress-Mediated Apoptosis and Disruption of Embryonic Development in Mouse Blastocysts.

The mycotoxin citrinin (CTN), a natural contaminant in foodstuffs and animal feeds, exerts cytotoxic and genotoxic effects on various mammalian cells and embryos. A previous investigation by our group revealed potentially hazardous effects of CTN on mouse oocyte maturation and pre- and post-implantation embryo development via the induction of apoptosis. The present study showed that CTN induces apoptosis and inhibits cell proliferation in the inner cell mass of mouse blastocysts. Notably, we observed for the first time that both these effects are suppressed by liquiritigenin (LQ). LQ is a type of flavonoid isolated from Glycyrrhiza radix with several biochemical and pharmacological activities, including antioxidant and anti-inflammatory properties. The preincubation of blastocysts with LQ clearly prevented CTN-induced disruption of pre- and post-implantation embryonic development and fetal weight loss, both in vitro and in vivo. CTN-induced damage processes directly promoted reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential (MMP) and activation of caspase-9 and caspase-3, which were effectively blocked by LQ. Moreover, in an animal model, intravenous injection of dams with CTN (3 mg/kg/day) triggered apoptosis of blastocysts, disruption of embryonic development from the zygote to the blastocyst stage and a decrease in fetal weight. Pre-injection with LQ (5 mg/kg/day) effectively reduced apoptosis and impaired the cytotoxic effects of CTN on development. Our in vivo findings further confirm that CTN exposure via injection has the potential to impair pre- and post-implantation development, leading to apoptosis and the suppression of sequent embryonic development, which can be effectively prevented by LQ.

Rhein Induces Oxidative Stress and Apoptosis in Mouse Blastocysts and Has Immunotoxic Effects during Embryonic Development.

Rhein, a glucoside chemical compound found in a traditional Chinese medicine derived from the roots of rhubarb, induces cell apoptosis and is considered to have high potential as an antitumor drug. Several previous studies showed that rhein can inhibit cell proliferation and trigger mitochondria-related or endoplasmic reticulum (ER) stress-dependent apoptotic processes. However, the side effects of rhein on pre- and post-implantation embryonic development remain unclear. Here, we show that rhein has cytotoxic effects on blastocyst-stage mouse embryos and induces oxidative stress and immunotoxicity in mouse fetuses. Blastocysts incubated with 5-20 µM rhein showed significant cell apoptosis, as well as decreases in their inner cell mass cell numbers and total cell numbers. An in vitro development assay showed that rhein affected the developmental potentials of both pre- and post-implantation embryos. Incubation of blastocysts with 5-20 µM rhein was associated with increased resorption of post-implantation embryos and decreased fetal weight in an embryo transfer assay. Importantly, in an in vivo model, intravenous injection of dams with rhein (1, 3, and 5 mg/kg body weight/day) for four days resulted in apoptosis of blastocyst-stage embryos, early embryonic developmental injury, and decreased fetal weight. Intravenous injection of dams with 5 mg/kg body weight/day rhein significantly increased the total reactive oxygen species (ROS) content of fetuses and the transcription levels of antioxidant proteins in fetal livers. Additional work showed that rhein induced apoptosis through ROS generation, and that prevention of apoptotic processes effectively rescued the rhein-induced injury effects on embryonic development. Finally, the transcription levels of the innate-immunity related genes, CXCL1, IL-1ß and IL-8, were down-regulated in the fetuses of dams that received intravenous injections of rhein. These results collectively show that rhein has the potential to induce embryonic cytotoxicity and induce oxidative stress and immunotoxicity during the development of mouse embryos.

Impairment of preimplantation and postimplantation embryonic development through intrinsic apoptotic processes by ginsenoside Rg1 in vitro and in vivo.

Ginsenoside Rg1, which is the most abundant compound found in Asian ginseng (Panax ginseng), has demonstrated various pharmacological actions, including neuroprotective, immune-stimulatory, and antidiabetic effects. Pregnant women, especially in the Asian community, consume ginseng as a nutritive supplement. Thus, the effects of ginsenoside-Rg1 on embryonic development need to be investigated, such as in a mouse model. As previous investigations have found that ginsenoside Rg1 appears to either trigger or prevent apoptosis in different cell lines, the effects of this agent on apoptosis remain to be clarified. In this study, we investigated whether ginsenoside Rg1 exerts a hazardous effect on mouse blastocysts and/or affects subsequent embryonic development in vitro and in vivo. Blastocysts treated with 25-100 µM ginsenoside Rg1 exhibited significant induction of apoptosis and a corresponding decrease in the inner cell mass (ICM) cell number. Importantly, the implantation rate was lower among ginsenoside Rg1-treated blastocysts compared to untreated controls. Moreover, embryo transfer assays revealed that blastocysts treated with 100 µM ginsenoside Rg1 exhibited increased resorption of postimplantation embryos and decreased weight among surviving fetuses. In vivo, intravenous injection of mice with ginsenoside Rg1 (2, 4, or 6 mg/kg body weight/day) for 4 days was associated with increased apoptosis of blastocyst-stage embryos and negatively impacted early embryonic development. Further experiments revealed that these effects may reflect the ability of ginsenoside Rg1 to trigger oxidative stress-mediated intrinsic apoptotic signaling. Our in vitro results indicate that ginsenoside　Rg1 treatment increases intracellular oxidative stress, decreases mitochondrial membrane potential, increases the Bax/Bcl-2 ratio, and activates caspase-9 and caspase-3, but not caspase-8. Taken together, our study results strongly suggest that ginsenoside Rg1 induces apoptosis and impairs the early preimplantation and postimplantation development of mouse embryos, both in vitro and in vivo.

Oxidative stresses-mediated apoptotic effects of ginsenoside Rb1 on pre- and post-implantation mouse embryos in vitro and in vivo.

Ginsenoside Rb1, the major saponin component of ginseng root, has a wide range of therapeutic application. Previous studies have established that ginsenoside Rb1 inhibits the cell cycle and induces apoptosis. However, its side-effects, particularly those on embryonic development, have not been well characterized to date. In the current study, we examined whether ginsenoside Rb1 exerts a cytotoxic effect on mouse embryos at the blastocyst stage, and affects subsequent embryonic development in vitro and in vivo. Blastocysts treated with 25-100 µg mL-1 ginsenoside Rb1 exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rate of blastocysts pretreated with ginsenoside Rb1 was lower than that of their control counterparts. Moreover, in vitro treatment with 25-100 µg mL-1 ginsenoside Rb1 was associated with increased resorption of post-implantation embryos and decreased fetal weight. In an in vivo model, intravenous injection with ginsenoside Rb1 (1, 3, 5 mg kg-1 body weight/day) for 4 days resulted in apoptosis of blastocyst stage embryos and early embryonic developmental injury. In addition, ginsenoside Rb1 appeared to induce injury in mouse blastocysts through oxidative stresses-triggered intrinsic apoptotic signaling processes to impair sequent embryonic development. The collective results strongly indicate that ginsenoside Rb1 induces apoptosis and retards early pre- and post-implantation development of mouse embryos, both in vitro and in vivo. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1990-2003, 2017.

Apoptotic effects on maturation of mouse oocytes, fertilization and fetal development by puerarin.

Previously we identified puerarin, an isoflavone compound, as a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, leading to retardation of embryonic development and cell viability. In the current study, we investigated whether puerarin exerts deleterious effects on mouse oocyte maturation, in vitro fertilization (IVF) and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, puerarin caused significant impairment of these processes in vitro. Pre-incubation of oocytes with puerarin during in vitro maturation led to increased post-implantation embryo resorption and decreased mouse fetal weight. In an in vivo animal model, intravenous injection with or without puerarin (1, 3 and 5 mg/kg body weight/day) for 4 days 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 puerarin-triggered deleterious effects, clearly implying that embryonic injury induced by puerarin is mediated by a caspase-dependent apoptotic mechanism. These results clearly demonstrate that puerarin has deleterious effects on mouse oocyte maturation, fertilization and subsequent embryonic development in vitro and in vivo.

Effects of ochratoxin a on mouse oocyte maturation and fertilization, and apoptosis during fetal development.

We previously reported that ochratoxin A (OTA), a mycotoxin found in many foods worldwide, causes nephrotoxicity, hepatotoxicity, and immunotoxicity, and is a risk factor for abnormal embryonic development. More specifically, OTA triggers apoptotic processes in the inner cell mass of mouse blastocysts, decreasing cell viability and embryonic development. In the current study, we investigated the deleterious effects of OTA on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent pre- and postimplantation development both in vitro and in vivo. Notably, OTA significantly impaired mouse oocyte maturation, decreased IVF rates, and inhibited subsequent embryonic development in vitro. Preincubation of oocytes with OTA during in vitro maturation increased postimplantation embryonic resorption and decreased mouse fetal weight. In an in vivo animal model, provision of 1-10 µM OTA in the drinking water or intravenous injection of 1 or 2 mg/kg body weight of OTA decreased oocyte maturation and IVF, and had deleterious effects on early embryonic development. Importantly, preincubation of oocytes with a caspase-3-specific inhibitor effectively blocked these OTA-triggered deleterious effects, suggesting that the embryonic injury induced by OTA is mediated via a caspase-dependent apoptotic mechanism. Furthermore, OTA upregulated the levels of p53 and p21 in blastocyst cells derived from OTA-pretreated oocytes, indicating that such cells undergo apoptosis via p53-, p21-, and caspase-3-dependent regulatory mechanisms. This could have deleterious effects on embryonic implantation and fetal survival rates, as seen in our animal models. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 724-735, 2016.