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.     

Apoptotic effects of dillapiole on maturation of mouse oocytes,fertilization and fetal development

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?μM 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 caspase-dependent 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.     

Injury effects of ginkgolide B on maturation of mouse oocytes,fertilization, and fetal development in vitro and in vivo

Ginkgolide B (GKB), the major active component of Ginkgo biloba extracts, exerts both stimulatory and inhibitory effects on apoptotic signaling. Previous studies by our group demonstrated that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell number, hinders early postimplantation blastocyst development, and increases early-stage blastocyst death. Here, we further investigate the effects of GKB on oocyte maturation, and subsequent pre- and postimplantation development in vitro and in vivo. In our experiments, GKB induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with 1–6 μM GKB during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased placental and fetal weights. Data obtained using an in vivo mouse model further disclosed that consumption of drinking water containing 3–6 μM GKB led to decreased oocyte maturation and in vitro fertilization, as well as early embryo developmental injury, specifically, inhibition of development to the blastocyst stage in vivo. To our knowledge, this is the first study to investigate the impact of GKB on maturation of mouse oocytes, fertilization, and sequential embryonic development.     

Methylglyoxal has injurious effects on maturation of mouse oocytes,fertilization, and fetal development,via apoptosis

Methylglyoxal (MG) is a metabolite of glucose. The serum MG level is increased in diabetic patients, and MG is implicated in diabetic complications related to embryonic development injury. We previously reported cytotoxic effects of MG on mouse embryonic stem cells and blastocysts, and a further association with defects in subsequent development. Here, we further investigate the effects of MG on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, MG induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with MG during in vitro maturation (IVM) led to increased resorption of post-implantation embryos and decreased fetal weight. Experiments with an in vivo mouse model disclosed that consumption of drinking water containing 10–20 μM MG led to decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. Finally, pretreatment with a caspase-3-specific inhibitor effectively prevented MG-triggered injury effects, suggesting that embryo impairment by MG occurs via a caspase-dependent apoptotic process.     

Hazardous effects of sanguinarine on maturation of mouse oocytes,fertilization, and fetal development through apoptotic processes

Previously, we reported that sanguinarine, a phytoalexin with antimicrobial, anti‐oxidant, anti‐inflammatory and pro‐apoptotic effects, is a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, causing decreased embryonic development and cell viability. In the current study, we investigated the deleterious effects of sanguinarine on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent pre‐ and postimplantation development both in vitro and in vivo. Notably, sanguinarine significantly impaired mouse oocyte maturation, decreased IVF rates, and inhibited subsequent embryonic development in vitro. Preincubation of oocytes with sanguinarine during in vitro maturation induced an increase in postimplantation embryo resorption and a decrease in mouse fetal weight. In an in vivo animal model, 1 to 5 μM sanguinarine, provided in drinking water, caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, preincubation of oocytes with a caspase‐3‐specific inhibitor effectively blocked sanguinarine‐triggered deleterious effects, clearly implying that embryonic injury induced by sanguinarine is mediated by a caspase‐dependent apoptotic mechanism. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 946–955, 2015.     

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.     

Effects of citrinin on maturation of mouse oocytes, fertilization, and fetal development in vitro and in vivo

The mycotoxin citrinin (CTN), a natural contaminant in foodstuffs and animal feeds, exerts cytotoxic and genotoxic effects on various mammalian cells. An earlier study by our group shows that CTN has cytotoxic effects on mouse embryonic stem cells and blastocysts, and is associated with defects in their subsequent development, both in vitro and in vivo. Here, we further investigate the effects of CTN on oocyte maturation, and subsequent pre- and postimplantation development in vitro and in vivo. CTN induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryo development. Treatment of oocytes with 5 microM CTN during in vitro maturation (IVM) led to increased resorption of postimplantation embryos, and decreased placental and fetal weight. Using an in vivo mouse model, we show that consumption of drinking water containing 5 microM CTN results in decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. To our knowledge, this is the first study investigating the impact of CTN on maturation of mouse oocytes, fertilization, and sequential embryonic development.     

N-acetyl-l-cysteine protects porcine oocytes undergoing meiotic resumption from heat stress

Heat stress (HS) is a notable risk factor for female reproductive performance. In particular, impaired oocyte maturation was thought to contribute largely to the HS-induced reproductive dysfunctions. In this study, we confirmed that oocytes undergoing GVBD were much susceptible to HS, and thus compromising subsequent embryonic development. Using N-acetyl-l-cysteine (NAC), we found supplementation of a relatively high dose NAC during in vitro maturation, can protect oocytes from HS-induced complications, and thus rescuing impaired embryonic development. Further analysis indicated that mechanisms responsible for protecting GVBD oocytes from HS by NAC may include: (1) reversing disorganized spindle assembly and inhibited extracellular signal–regulated kinase (ERK) signaling; (2) correcting erroneous H3K27me3 modification and dysregulated expression of imprinted genes; (3) alleviating increased intraoocyte reactive oxygen species accumulation and apoptosis initiation. Our study, focusing on the oocyte meiotic maturation, may provide a safe and promising strategy for protecting reproductive sows under environmental hyperthermal conditions.     

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.     

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.     

Reproductive system impairment of mice fed diets containing beluga whale blubber from the St Lawrence estuary and arctic populations

The toxic potential of naturally relevant mixtures of PCBs and other organohalogens on the reproductive system of C57Bl/6 female mice was assessed. Mice were fed diets in which lipids were replaced by blubber of beluga whales from a highly contaminated population of the Saint Lawrence River, and a less contaminated population from the Arctic Ocean. Ratios of blubber from both sources were mixed in order to perform a dose-response study. Control mice were fed diets for 90 d in which fat was replaced by corn oil or beef tallow. There were no significant effects of diets on body, liver, spleen or thymus weights. Similarly ovulation occurred in all control and experimental groups. However, Graafian follicles from ovaries of mice fed contaminated diets showed abnormal development of oocytes. Cumulus granulosa cells bind normally to the oocyte prior to ovulation and are essential for sperm penetration and fertilization. These cells were absent in both Graafian follicles and ovulated oocytes in the oviduct of all groups fed contaminated diets. Oviducts of these mice revealed evidence of epithelial degeneration. These results suggest the female mouse reproductive system is sensitive to organohalogens and illustrate the toxic potential of contaminant mixtures as found in the less contaminated Arctic population.     

Effects of trifluralin on the mouse ovary

Trifluralin, a herbicide used to protect many arable and horticultural crops, was evaluated for its potential toxicity on the mammalian ovary. To this end, adult female mice were fed or not (control) with a trifluralin‐enriched diet (150 mg/kg body weight/day) during gestation and lactation. After weaning, 3‐week‐old female mice from either trifluralin‐treated or control groups were used to evaluate whether the exposure to this herbicide in utero and during lactation could induce stress responses in the ovary. It was found that trifluralin exposure caused a significantly higher level of p53, but not of pRb, in the whole ovary, and in particular in granulosa cells. TUNEL staining showed that herbicide treatment did not increase the apoptotic index of the somatic compartment. Also oocyte fertilizability was unaffected, as metaphase II oocytes retrieved from treated mice were capable of forming male and female pronuclei after in vitro fertilization as control mice. However, trifluralin determined a slightly higher number of oocytes with cytoplasmic degeneration compared with control animals. In conclusion, our results suggest that exposure to a low trifluralin dose during pregnancy and lactation does not impair oocyte quality, but can induce a stress response in ovarian somatic cells. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2013.     

Chronic cadmium exposure causes oocyte meiotic arrest by disrupting spindle assembly checkpoint and maturation promoting factor

Cadmium (Cd) is a bioaccumulative heavy metal element with potential toxicity on the female reproductive system, but the exact molecular mechanisms have not yet been clearly defined. In this study, female mice were exposed to 0.5 mg/kg/day of CdCl₂ for 60 consecutive days. We found that chronic Cd exposure significantly decreased the fecundity of female mice by affecting oocyte meiotic progression as indicated by disrupted spindle assembly, chromosome alignment and kinetochore-microtubule attachments, consequently resulting in aneuploid oocytes. Further studies showed that the periodic fluctuations of MPF activity and cyclin B1 expression were disturbed in Cd-exposed oocytes probably by affecting the spindle assembly checkpoint protein Bub3. In addition, Cd exposure induced oxidative stress as indicated by an increased level of reactive oxygen species and apoptosis in oocytes, leading to oocyte quality deterioration. Taken together, these data suggest that Cd exposure causes disrupted molecular events of meiotic progression and deterioration of oocyte quality via oxidative stress, leading to decrease of female fertility.     

The effect of glucocorticoids on ERK-1/2 phosphorylation during maturation of lamb oocytes and their subsequent fertilization and cleavage ability in vitro

High levels of glucocorticoids may alter reproduction, but little is known about their direct actions on oocyte maturation, fertilization and subsequent development. Earlier work suggested negative effects of cortisol or dexamethasone on oocyte maturation but differences were noted between animal models. Both glucocorticoids reduce the p34^cdc2-cyclin B1 complex but it is unknown if other signaling pathways important for meiosis progression are affected. In this study, using sheep oocytes as a model system, we assessed in vitro the effects of increasing concentration of glucocorticoids (0–250 μM) on oocyte maturation and underlying changes in the MAP kinase pathway, and the ability of oocytes to undergo fertilization and embryo development. Cortisol decreased oocyte maturation but only at the highest concentration, whereas dexamethasone had no effect. Fertilization and cleavage were not affected. On the other hand, both cortisol and dexamethasone inhibited ERK-1/2 activation in a concentration-dependent manner. It thus seems that oocytes can overcome deleterious effects of glucocorticoids during maturation despite the decrease in ERK-1/2 activity, but repercussions in vivo should be further explored.     

In vitro fertilization after in vivo treatment of rats with three reproductive toxicants

One objective of these experiments was to establish a sensitive assay to evaluate fertilizing potential of rat gametes in vitro. A second objective was to evaluate this in vitro fertilization (IVF) assay as a method to detect in vivo effects of reproductive toxicants on male and female gametes using three known reproductive toxicants as model systems. The IVF assay with zona-free oocytes was more precise than the assay with cumulus-intact oocytes in these studies (coefficients of variation of 8.7 and 14.4%, respectively). Sperm fertilizing potential for zona-free oocytes was reduced by treatment of rats with m-dinitrobenzene (10-10 000 microg/kg) and ethylene glycol monomethyl ether (50-100 mg/kg) that had no effect on sperm motility. Molinate (60 mg/kg for 5 days) reduced sperm fertilizing potential concurrently with reductions in sperm motility. Neither molinate (60 mg/kg for 5 days) nor dinitrobenzene (0.002% in the drinking water for 14 days) administered to females seemed to affect the fertilizability of their oocytes. Ethylene glycol monomethyl ether treatment (0.15-0.25% in the drinking water for 14 days) reduced the number of ovulated oocytes. IVF is a means to evaluate toxicant effects on female gametes and demonstrates sperm's ability to interact with the oocyte plasma membrane.     

Chlorpyrifos and endosulfan affect buffalo oocyte maturation,fertilization, and embryo development in vitro directly and through cumulus cells

This study was undertaken to examine the effect of 10 different levels (0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.5, 1.0, 2.0, and 4.0 μg/mL) of two pesticides (chlorpyrifos and endosulfan) on buffalo oocyte viability, maturation, fertilization, and developmental competences in vitro. Studies were conducted to test the development of oocytes cultured with pesticides during maturation, fertilization, and during different embryo development stages. We also conducted experiments to test the hypotheses that the effects of these pesticides are hormones and somatic cells mediated. We observed a dose dependant decline in viability and developmental competence rates of oocytes. Chlorpyrifos and endosulfan had a negative impact on oocytes at 0.02 and 0.1 μg/mL levels, respectively. These pesticides reduced the oocyte nuclear maturation by a direct effect on oocytes, cumulus cell‐mediated action, and by blocking the action of hormones. Chlorpyrifos was found to be more ovotoxic and embryotoxic than endosulfan. This study will provide information on dose‐response relationship and risk assessment in domestic buffaloes. © 2009 Wiley Periodicals, Inc. Environ Toxicol 26: 57–67, 2011.     

Oocyte maturation, fertilization and embryonic growth in vitro

As the oocyte develops into an embryo, cytological and metabolic events follow one another in an accurate and successive sequence. Meiosis resumes in the ovarian follicle, parallel to cytoplasmic and membrane maturation, from the onset of the ovulatory LH discharge. Only a fully mature oocyte will be recognized and penetrated by a fertilizing sperm, to ensure rapid and synchronous male and female pronuclear growth and early embryonic development. In vitro, the resumption meiosis is easily obtained once the oocyte is withdrawn from the inhibitory influence of the follicle. Cytoplasmic and membrane maturation may however be impaired, leading to fertilization failures or anomalies such as triploidy and even impaired embryo viability. Human in vitro fertilization is nowadays routinely carried out with a high success rate, but in vitro embryonic growth to the blastocyst is still unsatisfactory even with oocytes matured in the ovary, and major improvements are needed to reach optimal viability. Many studies have now been published on human oocyte maturation, fertilization and the growth of embryos in vitro. We give only a brief account of them, due to limited space, and have therefore included topics of most relevance to assisted conception as opposed to those more involved with academic research.