Maternal benzo[a]pyrene exposure is correlated with the meiotic arrest and quality deterioration of offspring oocytes in mice

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon (PAH) in particulate matter that has a diameter of ≤2.5 μm (PM2.5). Studies have demonstrated that BaP exposure causes oocyte meiotic arrest in mice. However, whether BaP exposure also affects oocyte maturation in offspring remains unclear. To test this, female mice were administered BaP before pregnancy to generate BaP-exposed offspring. Our findings showed that BaP exposure reduced the in vitro maturation and increased the abnormalities of meiotic apparatus in offspring oocytes. In addition, BaP exposure reduced the mitochondrial content and intracellular ATP generation, induced early apoptosis, increased reactive oxidative species accumulation and the genomic DNA 5-methylcytosine (5mc) level in offspring oocytes. Along with the abovementioned defective parameters, maternal BaP exposure further compromised the embryo developmental competence of offspring oocytes. In summary, our study demonstrated that maternal BaP exposure compromised offspring oocyte maturation and quality.     

Zearalenone exposure affects mouse oocyte meiotic maturation and granulosa cell proliferation

Zearalenone (ZEN) is a metabolite of Fusarium and is a common contaminant of grains and foodstuffs. ZEN acts as a xenoestrogen and is considered to be cytotoxic, tissue toxic, and genotoxic, which causes abortions and stillbirths in humans and animals. Since estrogens affect oocyte maturation during meiosis, in this study we investigated the effects of ZEN on mouse oocyte meiotic maturation and granulosa cell proliferation. Our results showed that ZEN‐treated oocyte maturation rates were decreased, which might be due to the disrupted cytoskeletons: (1) ZEN treatment resulted in significantly more oocytes with abnormal spindle morphologies; (2) actin filament expression and distribution were also disrupted after ZEN treatment, which was confirmed by the aberrant distribution of actin regulatory proteins. In addition, cortical granule‐free domains (CGFDs) were disrupted after ZEN treatment, which indicated that ZEN may affect mouse oocyte fertilization capability. ZEN reduced mouse granulosa cell proliferation in a dose‐dependent manner as determined by MTT assay and TUNEL apoptosis analysis, which may be another cause for the decreased oocyte maturation. Thus, our results demonstrated that exposure to zearalenone affected oocyte meiotic maturation and granulosa cell proliferation in mouse. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1226–1233, 2015.     

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.     

MDR-1 function protects oocyte mitochondria against the transgenerational effects of nitrogen mustard exposure

Oocytes are vulnerable to alkylating agents like nitrogen mustard (NM), which can cause mitochondrial dysfunction associated with increased oxidative stress. Because mitochondria are maternally inherited, NM exposure affects oocyte mitochondrial physiology and compromises future progeny. Multidrug resistance transporters (MDRs) are transmembrane proteins that efflux such cytotoxic substances; MDR-1 is expressed in oocyte plasma and mitochondrial membranes and protects against oxidative stress. Our objective was to investigate how loss of MDR-1 can modulate oocyte response to NM transgenerationally. Wild Type (WT) and Mdr1a mutant female mice were injected intraperitoneally with sterile saline (control) or 0.1 mg/kg NM. 48 h post-injection, females were either sacrificed for F0 studies or mated with control males to yield F1 pups. After weaning, F1 females were sacrificed or mated to yield F2 pups. Germinal vesicle oocytes were assessed for mitochondrial membrane potential and reactive oxygen species (ROS) levels. NM exposed oocytes of both genotypes exhibited significantly higher ROS than controls in F0 and F1. NM F2 oocytes of neither genotype exhibited significantly higher ROS, though variation in Mdr1a mutants led to an upward trend. NM oocytes of both genotypes exhibited significantly disrupted mitochondrial membrane potential in F0. WT regained normalcy by F1 whereas Mdr1a mutants were unable to by F2. Our data suggest that Mdr1a mutants exhibit transgenerational mitochondrial dysfunction following toxic challenge that persists, implying that MDR-1 protects against toxicant-induced mitochondrial stress. Women without functional MDR-1 exposed to environmental toxicants could therefore be at risk for passing on compromised mitochondria to future offspring.     

Chronic cadmium exposure induced hepatic cellular stress and inflammation in aged female mice

Previous studies have revealed that acute cadmium (Cd) exposure led to inflammation in different organs through an oxidative stress mechanism. However, whether chronic Cd exposure induces inflammation in liver and the mechanistic link between inflammation and cell stress remains unclear. In the present study, we investigated the effects of chronic Cd exposure on hepatic cellular stress and inflammatory responses. Female CD1 mice were administrated with CdCl₂ (10 and 100 mg/L) in drinking water for 57 weeks. Our results showed that the mRNA levels of Inos and the protein content of HO‐1, markers of oxidative stress, were markedly increased in Cd‐treated mice. In addition, the protein level of GRP78, the chaperone of endoplasmic reticulum (ER) stress, was significantly increased in Cd‐treated mice. The expression of the proteins CHOP and peIF2α, two proteins downstream of ER stress, was also upregulated in the Cd‐100 mg/L and Cd‐10 mg/L group, respectively. Moreover, there were increased inflammatory cells existing in liver after Cd administration. Besides, there was a significant elevation in the mRNA level of Mip‐2, Il‐10 and Il‐12 in the Cd‐100 mg/L group. The mRNA level of Tgf‐β was also upregulated in Cd‐treated mice. Moreover, we also found that the number of Ki67‐positive hepatic cells was increased in the Cd‐10 mg/L group. Hence, our results indicated that chronic Cd exposure induced oxidative stress, ER stress, inflammatory responses and proliferation in the liver of aged female mice.     

Mechanisms and risk of chemically induced aneuploidy in mammalian germ cells

Aneuploidy is a pathological condition that affects 35% of human spontaneous abortions and 0.3% of livebirths. In spite of the increasing knowledge about molecular mechanisms of meiosis and chromosome segregation, maternal age remains the only ascertained aetiological factor. Genetically modified mouse models have been produced that show increased incidence of aneuploid gametes or abnormalities in meiotic recombination and synapsis. They suggest that genetic polymorphisms might also be involved in the aetiology of human germ cell aneuploidy. Experimental studies in the mouse have identified chemicals that can induce aneuploidy in male and female germ cells. Compounds affecting spindle assembly/dynamics are potent aneugens for oocytes and less so also for spermatocytes. They are active at acute doses during a short time interval preceding the metaphase-to-anaphase transition. Topoisomerase inhibitors are also meiotic aneugens which act on the recombination process; for the first time, the production of viable aneuploid mouse progeny was shown after paternal treatment with etoposide. A comparison between in vitro and in vivo effects of suspect aneugens demonstrates that there are biological mechanisms protecting mammalian oocytes from acute exposures to exogenous chemicals. Endocrine disruptors are a novel group of compounds that might affect chromosome segregation at meiosis. Data on bisphenol-A suggest that such chemicals could be active at low chronic exposure levels, but this hypothesis needs to be confirmed by further experiments. Experiments on cultured mouse oocytes treated with inhibitors of biochemical reactions involved in the regulation of chromosome segregation point to possible new mechanisms of action of environmental aneugens.     

Grape Seed Proanthocyanidin Ameliorates FB1-Induced Meiotic Defects in Porcine Oocytes

Fumonisin B₁ (FB₁), as the most prevalent and toxic fumonisin, poses a health threat to humans and animals. The cytotoxicity of FB₁ is closely related to oxidative stress and apoptosis. The purpose of this study is to explore whether Grape seed proanthocyanidin (GSP), a natural antioxidant, could alleviate the meiotic maturation defects of oocytes caused by FB₁ exposure. Porcine cumulus oocyte complexes (COCs) were treated with 30 μM FB₁ alone or cotreated with 100, 200 and 300 μM GSP during in vitro maturation for 44 h. The results show that 200 μM GSP cotreatment observably ameliorated the toxic effects of FB₁ exposure, showing to be promoting first polar body extrusion and improving the subsequent cleavage rate and blastocyst development rate. Moreover, 200 μM GSP cotreatment restored cell cycle progression, reduced the proportion of aberrant spindles, improved actin distribution and protected mitochondrial function in FB₁-exposed oocytes. Furthermore, reactive oxygen species (ROS) generation was significantly decreased and the mRNA levels of CAT, SOD2 and GSH-PX were obviously increased in the 200 μM GSP cotreatment group. Notably, the incidence of early apoptosis and autophagy level were also significantly decreased after GSP cotreatment and the mRNA expression levels of BAX, CASPASE3, LC3 and ATG5 were markedly decreased, whereas BCL2 and mTOR were observably increased in the oocytes after GSP cotreatment. Together, these results indicate that GSP could exert significant preventive effects on FB₁-induced oocyte defects by ameliorating oxidative stress through repairing mitochondrial dysfunction.     

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.     

The toxic effects of Fluorene‐9‐bisphenol on porcine oocyte in vitro maturation

Fluorene‐9‐bisphenol (9,9‐bis(4‐hydroxyphenyl)‐fluorene [BHPF]) is a bisphenol A (BPA) substitute used in the production of “BPA‐free” plastics, now has been identified is harmful to living organisms. Our previous study showed that BHPF impaired mouse denuded oocyte in vitro maturation. However, there is a question that whether BHPF is still able to affect oocyte maturation in the presence of dense cumulus cells. In the present study, we checked the toxic effects of BHPF on porcine oocyte maturation which is derived from COCs in vitro culture. Our results showed that BHPF (50 μM) inhibited the expansion of cumulus cells, led to a significant decrease in polar body extrusion (PBE). Importantly, BHPF resulted in abnormal spindle assembly, ATP level decrease, reactive oxygen species (ROS) accumulation and early apoptosis in porcine oocytes, which are all negative to oocyte maturation. Furthermore, BHPF also declined porcine oocyte quality by disturbing the cortical granules (CGs) distribution. In conclusion, our study showed that BHPF still inhibited oocyte maturation even in the presence of cumulus cells leading to abnormal spindle assembly, ATP decrease, increased ROS level, early apoptosis, and disturbed CGs distribution in porcine oocytes, and also indicates that BHPF has a wide range toxic effects on oocyte in different species.     

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.     

Exposure to first line anti-tuberculosis drugs in prepubertal age reduces the quality and functional competence of spermatozoa and oocytes in Swiss albino mice

Prepubertal Swiss albino mice of both sex were administered with first-line anti-tuberculosis drugs (ATDs) viz; rifampicin, isoniazid, pyrazinamide, streptomycin and ethambutol intraperitoneally, for 4 weeks. Two weeks after the completion of treatment, male mice were sacrificed to collect caudal spermatozoa and female mice were superovulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) to collect metaphase II (MII) oocytes from oviduct. Administration of ATDs not only decreased the count, motility and, nuclear maturity and also, increased the head abnormalities, mitochondrial damage and DNA damage in epididymal spermatozoa. Reduction in number of ovulated oocytes, increased degeneration rate and altered distribution pattern of cytoplasmic organelles was observed in oocytes of female mice. Presence of ATDs in in vitro maturation (IVM) medium increased abnormalities in meiotic resulted in abnormal spindle organization (except ethambutol) without affecting nuclear maturation. In conclusion, the result of this study indicates that ATDs have considerable adverse effects on the functional competence of male and female gametes, however, with varied degree of toxicity.     

Effects of mono(2-ethylhexyl)phthalate on cytoplasmic maturation of oocytes – The bovine model

Phthalates are known reproductive toxicants, but their intracellular disruptive effects on oocyte maturation competence are less known. We studied the potential risk associated with acute exposure of oocytes to mono(2-ethylhexyl)phthalate (MEHP). First, bovine oocytes were matured in vitro with or without 50 μM MEHP and examined for mitochondrial features associated with DNA fragmentation. MEHP increased reactive oxygen species levels and reduced the proportion of highly polarized mitochondria along with alterations in genes associated with mitochondrial oxidative phosphorylation (CYC1, MT-CO1 and ATP5B). In a second set of experiments, we associated the effects of MEHP on meiotic progression with those on cytoplasmic maturation. MEHP impaired reorganization of cytoplasmic organelles in matured oocytes reflected by reductions in category I mitochondria, type III cortical granules and class I endoplasmic reticulum. These alterations are associated with the previously reported reduced developmental competence of MEHP-treated bovine oocytes, and reveal the risk associated with acute exposure.     

Mancozeb adversely affects meiotic spindle organization and fertilization in mouse oocytes

In this study the effects of mancozeb, a widely used ethylenebisdithiocarbamate fungicide, on mouse oocyte meiotic maturation and fertilization were analyzed. Oocyte cumulus cell-complexes were matured in vitro with or without increasing concentrations of the fungicide (from 0.001 to 1 microg/ml) that, due to its different stability in organic solvents and in water, was resuspended either in dimethyl sulfoxide or in culture medium. Although, about 95% of oocytes reached the metaphase II stage; mancozeb-exposed oocytes showed a dose-dependent increase of alterations in spindle morphology, and this negative effect was more evident when the fungicide was resuspended in culture medium. Under the latter culture condition, oocytes matured in the presence of 0.1 and 1 microg/ml mancozeb showed a significant reduction also in the formation of male and female pronuclei. These results indicate that mancozeb can adversely affect mammalian reproductive performance, likely by perturbing microtubular organization during meiotic maturation.     

Perfluorooctanoic acid disrupts gap junction intercellular communication and induces reactive oxygen species formation and apoptosis in mouse ovaries

Perfluorooctanoic acid (PFOA) is a member of the perfluoroalkyl acid family of compounds. Due to the presence of strong carbon–fluorine bonds, it is practically nonbiodegradable and highly persistent in the environment. PFOA has been detected in the follicular fluid of women, and positively associated with reduced fecundability and infertility. However, there are no reports concerning the experimental evaluation of PFOA on oocyte toxicity in mammals. The aim of the present study was to determine if PFOA is able to induce oxidative stress in fetal ovaries and cause apoptosis in oocytes in vitro. In addition, since inhibition of the gap junction intercellular communication (GJIC) by PFOA has been demonstrated in liver cells in vivo and in vitro, the effect of PFOA on the GJIC between the oocyte and its supportive cumulus cells was studied. Results show that PFOA induced oocyte apoptosis and necrosis in vitro (medium lethal concentration, LC₅₀ = 112.8 μM), as evaluated with Annexin‐V‐Alexa 508 in combination with BOBO‐1 staining. Reactive oxygen species (ROS) levels, as assessed by DCFH‐DA, increased significantly in fetal ovaries exposed to ¼ LC₅₀ (28.2 μM, a noncytotoxic and relevant occupational exposure concentration) and LC₅₀ PFOA ex vivo. This perfluorinated compound also caused the blockage of GJIC in cumulus cells‐oocyte complexes (COCs) obtained from female mice exposed in vivo, as evaluated by calcein transfer from cumulus cells to the oocyte. The ability of PFOA of disrupting the GJIC in COCs, generating ROS in the fetal ovary and causing apoptosis and necrosis in mammal's oocytes, might account for the reported association between increasing maternal plasma concentrations of PFOA with reduced fertility in women.     

Reversal of cadmium-induced vascular dysfunction and oxidative stress by meso-2,3-dimercaptosuccinic acid in mice

Cadmium (Cd) is a heavy metal which causes concern as an environmental toxicant. Therapy with chelating agents is considered to be the rational treatment against metal poisoning. This study was designed to evaluate whether meso-2,3-dimercaptosuccinic acid (DMSA) could alleviate oxidative stress and vascular dysfunction in mice with subchronic exposure to Cd. Male ICR mice received CdCl₂ (100 mg/L) via drinking water for 8 weeks. After Cd exposure, DMSA at a dose of 25 mg/kg or 50 mg/kg was intragastrically administered once daily for 5 consecutive days at the end of Cd treatment. It was found that Cd-induced hypertension and markedly blunted vascular responses to vasoactive agents, including acetylcholine, phenylephrine and sodium nitroprusside. Treatment with DMSA significantly restored blood pressure and improved vascular responsiveness when compared with Cd-treated controls. Moreover, DMSA protected against Cd-induced severe oxidative stress by normalization of the redox ratios of glutathione to glutathione disulfide and suppression of plasma malondialdehyde, plasma protein carbonyl, urinary nitrate/nitrite, and superoxide production from thoracic aorta. DMSA partially reduced Cd contents in the blood, heart, liver and kidneys. In conclusion, our present study provides the first evidence of the therapeutic efficacy of DMSA against oxidative stress and vascular dysfunction in Cd-intoxicated mice.     

Effects of estrogenic compounds on neonatal oocyte development

In the mouse, oocytes develop in germline cysts that undergo breakdown resulting in primordial follicles, consisting of a single oocyte surrounded by granulosa cells. During this process, approximately two-thirds of the oocytes die. Exposure of female mice to environmental estrogens can alter oocyte development, limiting the number of primordial follicles that can be used for reproduction. Here we asked whether exposure to synthetic estrogens, diethylstilbestrol, ethinyl estradiol and bisphenol A affected perinatal oocyte development. Neonatal mice were injected with a low or high dose of each compound on postnatal days (PND) 1-4 and ovaries analyzed on PND5. Cyst breakdown, oocyte survival and follicle development were altered. The percentage of single oocyte was reduced from 84% in controls to 50-75%. The oocyte number per section was increased from 8 to 12-16. Follicle activation was reduced with 62% primordial follicles in controls to over 80% in most cases.     

Cadmium: toxic effects on the reproductive system and the embryo

The heavy metal cadmium (Cd) is a pollutant associated with several modern industrial processes. Cd is absorbed in significant quantities from cigarette smoke, and is known to have numerous undesirable effects on health in both experimental animals and humans, targeting the kidneys, liver and vascular systems in particular. However, a wide spectrum of deleterious effects on the reproductive tissues and the developing embryo has also been described. In the testis, changes due to disruption of the blood-testis barrier and oxidative stress have been noted, with onset of widespread necrosis at higher dosage exposures. Incorporation of Cd into the chromatin of the developing gamete has also been demonstrated. Ovarian Cd concentration increases with age, and has been associated with failure of progression of oocyte development from primary to secondary stage, and failure to ovulate. A further mechanism by which ovulation could be rendered ineffective is by failure of pick-up of the oocyte by the tubal cilia due to suboptimal expansion of the oocyte-cumulus complex and mis-expression of cell adhesion molecules. Retardation of trophoblastic outgrowth and development, placental necrosis and suppression of steroid biosynthesis, and altered handling of nutrient metals by the placenta all contribute to implantation delay and possible early pregnancy loss. Cd has been shown to accumulate in embryos from the four-cell stage onwards, and higher dosage exposure inhibits progression to the blastocyst stage, and can cause degeneration and decompaction in blastocysts following formation, with apoptosis and breakdown in cell adhesion. Following implantation, exposure of experimental animals to oral or parenteral Cd causes a wide range of abnormalities in the embryo, depending on the stage of exposure and dose given. Craniofacial, neurological, cardiovascular, gastrointestinal, genitourinary, and limb anomalies have all been described in placentates, with axial abnormalities and defects in somite structure noted in fish and ventral body wall defect and vertebral malformation occurring in the chick. In this paper, we examine the mechanisms by which Cd can affect reproductive health, and consider the use of micronutrients in prevention of these problems.     

The effects of the endocrine disruptors dithiocarbamates on the mammalian ovary with particular regard to mancozeb

Many human-made chemicals are called endocrine disruptors (EDs) because they have the potential to disrupt endocrine functions in exposed organisms. Many EDs can disrupt hormonal homeostasis by interfering with hormone receptor recognition, binding and activation, while others act by still unknown mechanisms. Among the EDs specifically affecting the female reproductive system, those with steroidogenic/antisteroidogenic effects have been extensively studied and the mechanisms of toxicity clarified also at molecular level. For many others, information is restricted to few epidemiological data and in vivo/in vitro experiments with animal models. This is the case of the dithiocarbamates, and in particular of the fungicide mancozeb, an ethylenedithiocarbamate widely used to protect fruit and vegetables, ginseng included, because of its low acute toxicity in humans. Although the mechanism(s) by which mancozeb may specifically act on female reproductive organs are largely unknown, data on experimental animals in vivo have demonstrated that the fungicide can induce several disturbances on estrus cycle. When used in vitro at concentrations considered too low to cause human health injuries, the fungicide impairs mouse embryo development and meiotic spindle assembly. The possibility that the female germ cell (the oocyte) could be a specific target of mancozeb suggests a role for this fungicide as probable inductor of infertility also in exposed human populations.