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.     

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.     

Biomolecules of human female fertility--potential therapeutic targets for pharmaceutical design

Scientists are able to advance the frontiers of human reproduction by employing a variety of molecular biological techniques to understand the biological processes intricately linked to oocyte and ovarian follicle development. Despite an abundance of knowledge concerning essential pathways which may have critical roles in oogenesis and folliculogenesis, the repertoire of medications to treat female fertility problems remains limited to a few classes of drugs involved in the induction or suppression of folliculogenesis and ovulation; many of which are not specific in their drug actions and can give rise to complications during clinical application. This paper aims to review biomolecules and pathways (e.g. PI3K, WNT, MAPK) pertinent to ovarian follicular development and active in human oocytes, including those involved in communication between somatic cells within the ovary (cumulus cells, granulosa cells and thecal cells) and the oocyte itself. These biomolecules which are involved in the nuclear and cytoplasmic maturation of oocytes and the control of ovarian folliculogenesis have potential as targets for improved ovarian stimulation, optimisation of oocyte maturation, and as biomarkers of oocyte viability assessment.     

Pre- and postnatal toxicity of the commercial glyphosate formulation in Wistar rats

Glyphosate is the active ingredient and polyoxyethyleneamine is the surfactant present in the herbicide Roundup formulation commercialized in Brazil. The aim of this study was to assess the reproductive effects of glyphosate-Roundup on male and female offspring of Wistar rats exposed during pregnancy and lactation. Dams were treated orally with water or 50, 150 or 450 mg/kg glyphosate during pregnancy (21-23 days) and lactation (21 days). These doses do not correspond to human exposure levels. The results showed that glyphosate-Roundup did not induce maternal toxicity but induced adverse reproductive effects on male offspring rats: a decrease in sperm number per epididymis tail and in daily sperm production during adulthood, an increase in the percentage of abnormal sperms and a dose-related decrease in the serum testosterone level at puberty, and signs of individual spermatid degeneration during both periods. There was only a vaginal canal-opening delay in the exposed female offspring. These findings suggest that in utero and lactational exposure to glyphosate-Roundup may induce significant adverse effects on the reproductive system of male Wistar rats at puberty and during adulthood.     

Effects of a low oral dose of diethylstilbestrol (DES) on reproductive tract development in F1 female CD-1 mice

The synthetic estrogen diethylstilbestrol (DES) is a model to study the effects on female reproductive tract of endocrine disrupting chemicals interacting with estrogen receptors. Pregnant CD-1 mice were given daily by gavage 10microg/kg bw of DES (the lower range of therapeutic exposure) during gestational days 9-16, critical period for reproductive tract development. Parameters of sexual development were recorded after weaning and at sexual maturation. No signs of general toxicity were observed in dams. In DES-treated group, reduced litter weight during lactation and earlier vaginal patency was observed. Uterus weight was increased in F1 treated females at weaning. Histological analysis showed reduced endometrium thickness and increased polyovular follicles, irregular and oocytes with condensed chromatin in the ovary at sexual maturity. Prenatal DES oral administration induces subtle but significant effects on puberty onset, uterine and ovary morphology.     

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.     

Natural uranium disturbs mouse folliculogenesis in vivo and oocyte meiosis in vitro

We investigated whether uranium intoxication affects female fertility by assessing its effects on ovarian function and on the oocyte. We treated two groups of female mice for 15 weeks with 5, 50 or 400 mg/L of uranyl nitrate in drinking water. In the first group, mice were euthanized immediately after intoxication. Mice of the second group were paired after intoxication with untreated males. Dams and their female pups were euthanized 3 months after the end of intoxication. We assayed the kidneys, femurs and one ovary per female for U content and collected the other ovary for histology. The number and size of all the ovarian follicles were analyzed. Mice from the first group and female pups had significantly fewer large antral follicles (? > 200 microm) than the untreated mice. By contrast, dams in the second group had more secondary and early preantral follicles (? 70-110 microm) than untreated mice. However, U had no effect on follicle atresia. We then analyzed the in vitro effects of U on oocyte maturation and fragmentation. GV-oocytes were cultured in the presence of 1mM uranyl acetate and observed for 72 h. Oocyte maturation was slowed down by U during resumption of meiosis and at metaphase II. However, the rhythm and rate of oocyte fragmentation were similar to those of control mice. Our findings demonstrate that U induces changes in folliculogenesis and oocyte maturation in mice and could consequently represent a risk for women who are chronically exposed.     

Oxidative stress as a damage mechanism in porcine cumulus‐oocyte complexes exposed to malathion during in vitro maturation

Malathion is one of the most commonly used insecticides. Recent findings have demonstrated that it induces oxidative stress in somatic cells, but there are not enough studies that have demonstrated this effect in germ cells. Malathion impairs porcine oocyte viability and maturation, but studies have not shown how oxidative stress damages maturation and which biochemical mechanisms are affected in this process in cumulus‐oocyte complexes (COCs). The aims of the present study were to determine the amount of oxidative stress produced by malathion in porcine COCs matured in vitro, to define how biochemical mechanisms affect this process, and determine whether trolox can attenuate oxidative damage. Sublethal concentrations 0, 750, and 1000 µM were used to evaluate antioxidant enzyme expressions, reactive oxygen species (ROS production), protein oxidation, and lipid peroxidation, among other oxidation products. COCs viability and oocyte maturation decreased in a concentration‐dependent manner. Malathion increased Cu, Zn superoxide dismutase (SOD1), glutathione‐S‐transferase (GST), and glucose 6 phosphate dehydrogenase (G6PD) protein level and decreased glutathione peroxidase (GSH‐Px) and catalase (CAT) protein level. Species reactives of oxygen (ROS), protein oxidation and Thiobarbituric acid reactive substances (TBARS) levels increased in COCs exposed to the insecticide, but when COCs were pre‐treated with the trolox (50 µM) 30 min before and during malathion exposure, these parameters decreased down to control levels. This study showed that malathion has a detrimental effect on COCs during in vitro maturation, inducing oxidative stress, while trolox attenuated malathion toxicity by decreasing oxidative damage.     

Germ cell studies in mice after prolonged exposure to nitrous oxide

Male and female Swiss Webster (SW) mice, age 13 to 14 weeks, were exposed by inhalation for 4 hr per day, 5 days per week, for 14 weeks, to either room air, 0.5% nitrous oxide, 5.0% nitrous oxide, or 50% nitrous oxide. Murine germ cells were examined for evidence of injury after this exposure. A group of male mice were treated with methyl methanesulfonate (MMS) as a positive control for sperm abnormalities while a group of female mice were treated with 3-methylcholanthrene (3-MC) as a positive control for oocyte destruction. There were no significant differences among the four inhalation exposure groups in testes weight, percentage of abnormally shaped sperm, sperm count, or histologic appearance of the testes; the mean percentage (+/- SE) of abnormal sperm ranged from 8.9 +/- 2.4 (5.0% nitrous oxide) to 13.5 +/- 0.5 (50% nitrous oxide) with a concurrent control value of 10.4 +/- 2.3%. In the positive control experiment, 25.2 +/- 4.1% of sperm from mice treated with MMS were abnormal compared with 2.5 +/- 0.3% of sperm from mice treated with saline (p less than 0.001), indicating that sperm of SW mice are sensitive to chemical damage. There was no significant difference between the mean number of oocytes in mice treated with 50% nitrous oxide (33.3 +/- 14.4) and in control mice (29.8 +/- 8.0). In the positive control experiment, mice treated with 3-MC had significantly fewer (p less than 0.001) primordial oocytes, 67.2 +/- 19.5 compared with control mice, 222.4 +/- 21.9, indicating that this strain is sensitive to chemical damage of the ovary. Thus, murine germ cells showed no evidence of toxic effects due to prolonged exposure to nitrous oxide.     

Exposure to Aroclor‐1254 impairs spindle assembly during mouse oocyte maturation

Polychlorinated biphenyls (PCBs), as typical environmental estrogen disruptors, are a structurally‐related group of halogenated aromatic hydrocarbons that are composed of 209 isomers and present as a mixture in the environment. PCBs congener with different numbers and positions of chlorine atoms substituted on the biphenyl moiety. Aroclor‐1254 is a mixture of more than 60 PCB congeners. Previous studies have provided the evidence that PCBs have severe negative effects on reproductive functions, but the effects of PCBs on spindle assembly during mouse oocyte maturation in vitro have not been reported. In the present study, female ICR mouse immature oocytes were cultured in M2 medium with 1 and 10 μg mL^?1 Aroclor‐1254 separately in vitro. The percentage of germinal vesicle breakdown (GVBD) and the first polar body extrusion were recorded. The results showed no significant difference in the percentage of GVBD or the first polar body extrusion between control oocytes and Aroclor‐1254‐treated oocytes. Further studies showed that the normal localization of γ‐tubulin and Aurora‐A kinase was interfered and α‐tubulin assembling into spindle was affected when mouse oocytes were exposed to Aroclor‐1254. The length of spindle from 10 μg mL^?1 Aroclor‐1254‐treated oocytes was longer than that from control oocytes, and the spindle area in the Aroclor‐1254‐treated groups were decreased. Furthermore, the percentage of DNA damage in cumulus cells revealed an increase after exposed to Aroclor‐1254. These results will provide the important reference for the prevention of reproductive disorders caused by PCBs. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1652–1662, 2016.     

Oocyte number in newborn mice after prenatal octylphenol exposure

The aim of the present study was to investigate whether prenatal exposure to the suspected estrogenic compound 4-tert-octylphenol (OP), influences oocyte number in newborn female mice. In addition, effects on the percentage distribution of prefollicular, follicular, and atretic oocytes were investigated. Pregnant mice were subcutaneously injected with OP (1 or 250 mg/kg) or vehicle alone on embryonic day 11.5-16.5 (plug = embryonic day 0.5). As a positive control for estrogenic effects, a group of animals was injected with the synthetic estrogen diethylstilbestrol (DES, 100 microg/kg). Ovaries from the offspring were collected on the day of birth and a stereologic method, the optical fractionator, was used to estimate the number of prefollicular, follicular, and atretic oocytes in the ovaries. The total number of oocytes was calculated as the sum of the three subpopulation estimates. Neither OP nor DES exposure could be observed to affect the total number of oocytes or the percentage distribution of atretic, prefollicular, and follicular oocytes. Thus, prenatal OP exposure does not appear to cause a serious threat to fetal female germ cell proliferation and survival, or early follicle formation.     

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.     

Lack of effects of 2,4-dichlorophenoxyacetic acid administration on markers of oxidative stress during early pregnancy in mice

Induction of oxidative stress by 2,4-dichlorophenoxyacetic acid (2,4-D) both as a pure compound and in commercial formulation was investigated during early pregnancy in mice. Pregnant animals were exposed to increasing doses of the herbicide (0.01, 0.1 and 100mg/kg/d) during gestation days 0-9, after which animals were euthanized and their blood analyzed for catalase activity, thiobarbituric acid reactive substances (TBARs) and total antioxidant capacity (TAC). Number of corpora lutea and uterine implantations and resorptions were also determined. Herbicide exposure did not cause any overt signs of maternal toxicity at any of the doses administered; neither did it cause an effect on developmental parameters. Catalase activity and TBARs were not modified by herbicide exposure although TAC was significantly decreased at 100mg/kg/d of both pure and formulated compound. Thus, 2,4-D does not seem to induce oxidative stress during early pregnancy in mice at the doses administered, indicating that this mechanism is probably not involved in mediating herbicide toxicity at these dose levels. Furthermore, since no manifestations of developmental toxicity were observed after administration of the herbicide, it is also possible that 2,4-D may not produce any early developmental toxicity at the low environmentally relevant doses tested in this animal model.     

Bone lead mobilization in lactating mice and lead transfer to suckling offspring

Drinking water containing 200 μg/ml lead (labeled with ²¹⁰Pb) was provided to adult female mice for 105 days prior to the date of mating or for 105 days prior to mating and during the periods of gestation and lactation (160 days total exposure). During lactation there was an increased rate of lead elimination from adult mice exposed prior to mating compared to nonlactating female mice. The magnitude of the additional lead elimination during lactation was similar to the magnitude of the decrease in femur ash weights during lactation. These data support the hypothesis that lead mobilization during lactation is the result of bone mineral mobilization. Approximately 3% of the lead body burden of mothers exposed to lead prior to mating was retained in litters of suckling pups as a result of lead transferred in milk. The magnitude of lead mobilized from maternal bone during lactation was not increased by a diet which was moderately calcium deficient. Continuous ingestion of lead by mothers during lactation significantly increased the net transfer of lead to suckled pups. Lead transferred from mother to offspring during lactation greatly exceeded transfer during gestation for all treatment groups. These data indicate that both prior and current maternal lead exposure should be considered in assessing potential lead exposure of suckling infants.     

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.     

Effects of inorganic mercury on reproductive performance of mice.

Effects of mercuric chloride (MC) on the reproductive performance of mice were evaluated. Both male and female mice were divided into four groups that were subsequently exposed to 0.00, 0.25, 0.50, and 1.00 mg/kg/day of MC, respectively. At the end of pre-mating dosing, males were paired with females receiving the same dose. Dosing continued for males throughout mating, while dosing in females continued throughout mating, gestation, and lactation. The males were necropsied at the conclusion of mating and the females were necropsied at the conclusion of lactation. Fertility indices, parturition, gestation, live birth litter size, survival indices, and implantation efficiency were recorded. Subsequently, these data were statistically analyzed. Fertility and survival indices were significantly reduced in the treated groups. Exposure of mice to MC did not affect their litter size. No evidence of mercury induced target organ toxicity was seen in either the clinical pathology parameters or histomorphologic evaluations. However, in MC treated females, ovary weights were significantly different from the control. There were no histomorphologic or clinical pathology effects induced by MC. These results suggested that oral exposure to 0.25-1.00 mg/kg/day of MC produced adverse effects on the reproductive performance of mice in the absence of overt mercury toxicity.