Xenobiotic influences on embryonic differentiation, growth and morphology in vitro

Direct in vitro exposure of post-implantation rat embryos to 18 known teratogens induced typical malformations in all cases. Of 21 non-teratogens in vivo, 20 induced, in vitro either no malformations at all, even at high concentrations, or abnormal development could only be observed at concentrations which affected growth and differentiation significantly. Responses of chemically induced exposed embryos in vitro occurred within wide concentration ranges. Actinomycin D, for example, affected embryonic development at a concentration as low as 3 X 10(-4) micrograms/ml, whereas other substances had no effect at concentrations up to 9 X 10(2) micrograms/ml.     

Xenobiotic influences on embryonic differentiation,growth and morphology in vitro

1. Direct in vitro exposure of post-implantation rat embryos to 18 known teratogens induced typical malformations in all cases.

2. Of 21 non-teratogens in vivo, 20 induced, in vitro either no malformations at all, even at high concentrations, or abnormal development could only be observed at concentrations which affected growth and differentiation significantly.

3. Responses of chemically induced exposed embryos in vitro occurred within wide concentration ranges. Actinomycin D, for example, affected embryonic development at a concentration as low as 3 × 10^?4 μg/ml, whereas other substances had no effect at concentrations up to 9 × 10² μg/ml.     

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.     

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.     

三氯化铬对小鼠卵母细胞成熟和体外受精的影响

采用小鼠卵母细胞体外培养 ,体外受精的方法研究了三氯化铬对小鼠卵母细胞成熟和受精能力的影响 .结果表明 ,三氯化铬可以抑制卵母细胞第一极体的释放 ,降低小鼠超排卵数和卵母细胞的存活率和体外受精率 .对小鼠体内生发泡破裂没有影响 ,但可以抑制体外培养卵母细胞的生发泡破裂 ;随着在正常培养液中培养时间的延长 ,卵母细胞的第一极体的释放率和体外受精率 (除了 6.0 mg· kg-1组外 )均有显著提高 ,且与对照组相比已经无显著性差异 .结果提示 ,三氯化铬可以破坏卵母细胞的成熟 ,降低卵母细胞的受精能力 ,具有明显的生殖毒性     

The effect of glucocorticoids on mouse oocyte in vitro maturation and subsequent fertilization and embryo development

Increased glucocorticoid levels, due to medical therapy or stress-related, may affect reproduction via the hypothalamus–pituitary-axis or directly at the oocyte level. We examined the effects of natural (corticosterone) or synthetic (dexamethasone) glucocorticoids on mouse oocyte maturation and underlying changes in extracellular signal-regulated kinase (ERK) phosphorylation patterns. Fertilization and progression up to the blastocyst stage were also evaluated. Oocytes were exposed to corticosterone or dexamethasone (0, 0.25, 2.5, 25 or 250 μM) for 17 h during in vitro maturation. After maturation, ERK-1/2 activation in oocytes was assessed by SDS–PAGE and immunoblotting, and fertilization and developmental capacity were examined in vitro. Corticosterone exposure during oocyte maturation significantly decreased progression to metaphase II, fertilization and embryo development at the highest concentration. Corticosterone caused a concentration-dependent inhibition of ERK-1/2 activation, with the highest concentration resulting in considerable inhibition of oocyte ERK-1/2 phosphorylation and no blastocyst development. In contrast, dexamethasone had no effect on maturation, fertilization and cleavage, and no effect was seen on ERK-1/2 phosphorylation. Based on these in vitro findings, high glucocorticoid levels may have consequences for subsequent development, although a short exposure to physiologic or stress-related glucocorticoid levels may not represent a hazard to meiosis progression of the oocyte.     

双酚A对大鼠卵泡体外生长和卵母细胞成熟的影响

目的观察双酚A(BPA)对大鼠卵泡体外生长发育及卵母细胞成熟的影响。方法采用大鼠卵泡体外长期培养方法,从12～14d龄的雌性大鼠卵巢中机械性分离腔前卵泡(140～170μm),隔天分别换一半含BPA 0,50,100和150μmol·L-1的培养液,连续培养10d。倒置相差显微镜下观察卵泡发育的形态,计算卵泡存活率、有腔卵泡形成率和卵丘-卵母细胞复合体(COC)排出率,测定卵泡直径,显微镜下观察卵泡的排卵情况以及卵母细胞成熟情况,计算生发泡(GV)、生发泡破裂(GVBD)和第一极体(PB)的形成率;分别于培养2,6和10d时采用磁性酶联免疫法测定培养基中雌二醇和孕酮的分泌量。结果正常对照组卵泡在10d培养过程中,大多数正常对照组卵泡都经历了腔前卵泡、有腔卵泡以及成熟卵泡阶段。与正常对照组相比,BPA 100和150μmol·L-1组的卵泡存活率、有腔卵泡形成率、COC排出率、GVBD率以及PB率均明显降低(P<0.05)。BPA 50μmol·L-1组培养10d时的卵泡直径以及培养6和10d时BPA100和150μmol·L-1组卵泡和卵母细胞的直径均明显降低(P<0.05);与正常对照组相比,BPA 100和150μmol·L-1组卵泡膜细胞和颗粒细胞的增殖受到明显抑制(P<0.05)。与正常对照组相比,BPA 100和150μmol·L-1组在培养6和10d时雌二醇和孕酮含量均显著减少(P<0.01)。结论 BPA 100和150μmol·L-1可抑制大鼠卵泡的生长和卵母细胞的成熟。     

Tretinoin-loaded lipid-core nanocapsules decrease reactive oxygen species levels and improve bovine embryonic development during in vitro oocyte maturation

In vitro oocyte maturation (IVM) protocols can be improved by adding chemical supplements to the culture media. Tretinoin is considered an important retinoid in embryonic development and its association with lipid-core nanocapsules (TTN-LNC) represents an innovative way of improving its solubility, and chemical stability, and reducing its toxicity. The effects of supplementing IVM medium with TTN-LNC was evaluated by analyzing production of reactive oxygen species (ROS), S36-phosphorilated-p66Shc levels and caspase activity in early embryonic development, and expression of apoptosis and pluripotency genes in blastocysts. The lowest concentration tested (0.25 μM) of TTN-LNC generated higher blastocyst rate, lower ROS production and S36-p66Shc amount. Additionally, expression of BAX and SHC1 were lower in both non-encapsulated tretinoin (TTN) and TTN-LNC-treated groups. Nanoencapsulation allowed the use of smaller concentrations of tretinoin to supplement IVM medium thus reducing toxic effects related with its use, decreasing ROS levels and apoptose frequency, and improving the blastocyst rates.     

Fluoride impairs oocyte maturation and subsequent embryonic development in mice

The damage caused by fluorosis is permanent, and has been recognized as a public health problem in a number of regions of the world. Although multiple studies provided evidence that sodium fluoride (NaF) elicits adverse effects on reproductive function, the effect of fluoride on female germ cell development is not well understood. Therefore, the present study aimed at evaluating the effect of fluoride treatments on in vivo maturation and developmental potential of mouse oocytes, in which female ICR mice were treated with a range of doses (0, 30, 60, and 150 mg/L) of NaF. After treatment, mice were superovulated to collect ovulated oocytes. The effects of NaF on oocyte quality, fertilization potential and early embryonic development were evaluated, as well as the underlying mechanisms were primarily investigated. The findings of this study showed that NaF treatment resulted in abnormal spindle configuration, actin cap formation, and cortical granule‐free domain formation. Additionally, overexposure of mice to NaF notably reduced ATP production and mitochondrial membrane potential, further influencing in vitro fertilization and subsequent embryonic development. These results indicated that NaF treatment impairs the subsequent embryonic developmental potential of the oocytes. In conclusion, overexposure to fluoride in vivo was associated with a significant disruption of cytoskeletal dynamics and decreased oocyte quality, affecting the oocyte's subsequent fertilization and embryonic development. Results of this study provide a rationale for treating reproductive diseases such as infertility or miscarriage caused by environmental contaminants. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1486–1495, 2016.     

未刺激的未成熟卵体外培养在多囊卵巢综合征患者中的应用

目的探讨未刺激的未成熟卵体外培养成熟（IVM）治疗难治性多囊卵巢综合征（PCOS）患者的疗效。方法2005年10月至2006年10月本中心对50例难治性PCOS患者65个周期的IVM治疗。结果9个周期取消,取消率为13．85％;65个周期共获卵1338枚,平均每周期20．6枚,培养48h后总成熟率为68．5％。正常受精率为70．2％,卵裂率为91．1％,每穿刺周期和移植周期的临床妊娠率分别为37．5％和40．6％,28例临床妊娠中,6例多胎,多胎妊娠率为21．4％。结论未经刺激的IVM技术用于难治性PCOS是一种有效的方法,可获得与常规体外受精-胚胎移植（IVF-ET）相似的临床妊娠率。     

Preconceptional paternal glycidamide exposure affects embryonic gene expression: single embryo gene expression study following in vitro fertilization

Recognition of early determinants of disease onset has sparked an interest in paternally transmitted factors and their impact on the developing embryo. Acrylamide (AA), a widely distributed xenobiotic compound, is converted to its active metabolite glycidamide (GA) by the CYP2E1 enzyme. Based on its capacity to induce dominant lethal mutations, we hypothesized that paternal GA exposure would have a negative impact on embryonic genome activation, via GA-DNA and protamine adducts persisting in the fertilizing sperm. Using a combination of in vitro fertilization (IVF) techniques and RT-qPCR single embryo gene expression (SEGE), we studied the expression of key DNA repair genes and genes important for embryo development, at the 1-, 2-, 4- and 8-cell stage of the developing mouse embryo. Compared to controls paternal GA-exposure gave rise to an altered pattern of embryonic gene expression, with an initial reduced expression at early stages followed by increased expression at the 8-cell stage.     

银杏内酯B促进体外培养胚胎大鼠脊髓神经细胞生长发育的研究

目的探讨GB对体外培养的胚鼠脊髓神经元存活和生长发育的作用。方法胚胎大鼠脊髓神经细胞原代培养,相差显微镜下进行细胞计数和显微测量,观察GB对神经元存活和生长发育的作用。对培养7d细胞行NSE免疫组化染色,光镜下观察GB对NSE染色阳性神经元生长发育的影响。结果GB及NGF能够促进体外培养胚胎大鼠脊髓神经细胞及NSE染色阳性神经元的存活,促进其细胞及突起的生长。其促进脊髓神经细胞的存活、胞体及突起发育的作用与NGF一致,而其促进神经突起分化与生长的作用强于NGF。结论NGF和GB能促进培养大鼠发育期脊髓神经元存活、分化和生长,并且表现出各自作用的特异性。     

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.     

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.     

In vitro fertilization and childhood retinoblastoma

AIMS: To estimate the frequency of childhood retinoblastoma among children conceived by in vitro fertilization (IVF). METHODS: Using the United Kingdom-based General Practice Research Database (GPRD), we identified all live births, cases of retinoblastoma and IVF procedures occurring between January 1989 and December 2001. RESULTS: We identified 0 cases of retinoblastoma among 176 children conceived by IVF (Risk = 0/176, one-sided 97.5% CI 0, 0.02) and 24 cases of retinoblastoma diagnosed before age 5 years among 358,094 children not conceived by IVF (6.7 cases per 100,000 births [95% CI 4.5, 10]). CONCLUSIONS: These data provide some reassurance that children born as a result of IVF are not at markedly increased risk of retinoblastoma.     

Nitric oxide in oocyte maturation,ovulation, fertilization,cleavage and implantation: a little dab'll do ya

Nitric oxide synthases, the enzymes that generate NO gas, may be involved in reproduction and development of multicellular organisms at many levels and thus provide important targets for design of drugs to intervene in reproductive processes. This review focuses on the role of nitric oxide in key events of reproduction including gamete activation, fertilization, early cell divisions and implantation. A general trend highlighted by the studies reviewed is that NO plays a biphasic role in reproduction. That is, a narrow range of NO concentrations, usually low, will stimulate or enhance these early events in reproduction, but either a lack of NO or too much NO has negative consequences. One of the shortcomings of the field currently is the lack of molecular detail concerning the mechanism of NO action. This has been due in part to lack of technology for effective detection of NO and its molecular targets. A few targets of NO have been indirectly implicated and advances in this area of research will provide substrates for development of drugs to control reproductive function. Work from both invertebrate and vertebrate model systems is presented and implications for control of reproductive physiology discussed. Ubiquity of NO signaling in animals may mean that effective control of reproduction must target mediators of NO action and not NOS enzymes themselves.     

Impact of bisphenol-A on early embryonic development and reproductive maturation

Bisphenol-A (BPA) is a synthetic estrogen and monomer component of polycarbonate plastics and epoxy resins that are widely used in the production of food and beverage containers. It leaches into our food and drink at concentrations shown to have biological consequences. Here we show that exposure to low levels of BPA accelerated early embryonic development within 24 h of exposure, attenuated body growth, and advanced the times of hatching and reproductive maturation in medaka fish (Oryzias latipes). The acceleration in embryonic development and time to hatch were blocked by the thyroid-hormone receptor (TH-R) antagonist amiodarone, suggesting that BPA alters global developmental timing through a thyroid-hormone pathway. Our results are likely to have broad implications regarding the effects of plastic-derived contaminants on embryonic and reproductive 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.