Effects of perinatal exposure to bisphenol A on induction of prostate cancer in Sprague Dawley rats by MNU and testosterone.

Perinatal and neonatal exposure to bisphenol A (BPA) has been linked to enhancement of prostate carcinogenesis in rats induced by combined treatment with estradiol and testosterone, but human data are lacking. This study aimed to determine the effects of perinatal BPA exposure on induction of prostate cancer in rats by sequential treatment with N-methyl-N-nitrosamine (MNU) and continuous low dose administration of testosterone. Pregnant Sprague Dawley rats were exposed to BPA administered by subcutaneous Alzet minipumps at doses of 2.5 or 25 µg/kg body weight/day from gestational day 9 until postnatal day 28 when pups were weaned providing exposure of offspring in utero and via the mother's milk. At 10-12 weeks of age, one male offspring per litter was treated with an intraperitoneal injection of MNU after hormonal stimulation of prostatic cell proliferation followed two weeks later by subcutaneous insertion of Silastic implants containing testosterone until the termination of the study 57-58 weeks after MNU injection. The perinatal BPA exposure did not significantly affect the incidence of prostate carcinomas which was slightly lower in exposed rats (33-23 %) than in control animals (40 %). Carcinomas in all accessory sex glands combined were also insignificantly less frequent in exposed (46-48 %) than in control rats (60 %). The incidence of malignant tumors at any site in the body was significantly lower in exposed rats (81-65 %) than in controls (93 %). In conclusion, perinatal BPA exposure did not significantly modify prostate cancer induction by MNU plus testosterone in rats, unlike the enhancement of prostate carcinogenesis induced by treatments involving estradiol administration. Which of the two models of prostate carcinogenesis is more relevant for the human situation is unclear at present.

Chilean dentists' knowledge of hearing loss generated by occupational noise exposure / Conocimiento de odontólogos chilenos sobre la pérdida auditiva generada por exposición ocupacional al ruido

Abstract Introduction: Dentists are a population at high risk of hearing loss due to their constant exposure to instruments that can generate noise of up to 100 dB during their practice. Objective: To determine the level of knowledge of dentists working in Chile regarding hearing loss caused by exposure to noise generated by dental instruments. Materials and methods: A cross-sectional study was conducted in 114 dentists, who completed a virtual survey of 22 questions regarding the perception and level of knowledge about hearing loss due to exposure to loud noises and about national regulations on occupational noise exposure. Differences between perception and knowledge levels were evaluated taking into account the years of professional practice and the average weekly workload in dental treatment rooms. Descriptive and inferential statistics (Chi-squared test) were used for data analysis. Results: Most participants were Chilean (99.1%); 58.8% were women, and 72.8% had less than 10 years of professional experience. In addition, 97.4% were unaware of national regulations on occupational noise exposure and 50% of the sample reported having experienced hearing loss; of these, 57.9% (n = 32) associated it with their practice. Conclusions: A very low percentage of participants knew that there are regulations regarding occupational noise exposure. For this reason, it is important that, both during their training and their professional practice, dentists in Chile have greater access to information about these regulations and hearing protection measures.

Resumen Introducción. Los odontólogos son una población con un alto riesgo de desarrollar pérdida auditiva debido a la constante exposición a instrumentales que deben usar en su práctica profesional y que pueden generar ruidos de hasta 100 dB. Objetivo. Determinar el nivel de conocimiento de odontólogos laboralmente activos en Chile respecto a la pérdida auditiva causada por la exposición al ruido generado por maquinarias dentales. Materiales y métodos. Estudio transversal realizado en 114 odontólogos, quienes diligenciaron una encuesta virtual de 22 preguntas relativas a la percepción y el nivel de conocimiento sobre pérdida auditiva por exposición a ruidos fuertes y sobre la normativa nacional respecto a exposición ocupacional al ruido. Se evaluaron las diferencias entre percepción y niveles de conocimiento según los años de ejercicio profesional y la carga promedio de trabajo semanal en boxes de atención. Para el análisis de los datos se utilizó estadística descriptiva e inferencial (prueba de chi-cuadrado). Resultados. La mayoría de participantes eran chilenos (99.1%); el 58.8% fueron mujeres, y el 72.8% tenía menos de 10 años de ejercicio profesional. Además, el 97.4% desconocía las regulaciones nacionales sobre exposición ocupacional al ruido y el 50% reportó haber experimentado pérdida auditiva; de estos, 57.9% (n=32) lo asoció a su profesión. Conclusiones. Un muy bajo porcentaje de los participantes sabe que hay disposiciones sobre exposición ocupacional al ruido, por lo que es importante que, tanto en su formación, como durante su ejercicio profesional, los odontólogos en Chile tengan un mayor acceso a información relativa a estas normativas y a medidas ocupacionales de protección auditiva.

A Combined Morphometric and Statistical Approach to Assess Nonmonotonicity in the Developing Mammary Gland of Rats in the CLARITY-BPA Study.

BACKGROUND: The Consortium Linking Academic and Regulatory Insights on Bisphenol-A (CLARITY-BPA) is a rare collaboration of guideline-compliant (core) studies and academic hypothesis-based studies to assess the effects of bisphenol A (BPA). OBJECTIVES: We aimed to a) determine whether BPA showed effects on the developing rat mammary gland using new quantitative and established semiquantitative methods in two laboratories, b) develop a software tool for automatic evaluation of quantifiable aspects of the mammary ductal tree, and c) compare those methods. METHODS: Sprague-Dawley rats were exposed to BPA, vehicle, or positive control [ethinyl estradiol (EE2)] by oral gavage beginning on gestational day (GD)6 and continuing with direct dosing of the pups after birth. There were two studies: subchronic and chronic. The latter used two exposure regimes, one stopping at postnatal day (PND)21 (stop-dose) the other continuing until tissue harvest (continuous). Glands were harvested at multiple time points; whole mounts and histological specimens were analyzed blinded to treatment. RESULTS: The subchronic study's semiquantitative analysis revealed no significant differences between control and BPA dose groups at PND21, whereas at PND90 there were significant differences between control and the lowest BPA dose and between control and the lowest EE2 dose in animals in estrus. Quantitative, automatized analysis of the chronic PND21 specimens displayed nonmonotonic BPA effects, with a breaking point between the 25 and 250µg/kg body weight (BW) per day doses. This breaking point was confirmed by a global statistical analysis of chronic study animals at PND90 and 6 months analyzed by the quantitative method. The BPA response was different from the EE2 effect for many features. CONCLUSIONS: Both the semiquantitative and the quantitative methods revealed nonmonotonic effects of BPA. The quantitative unsupervised analysis used 91 measurements and produced the most striking nonmonotonic dose-response curves. At all time points, lower doses resulted in larger effects, consistent with the core study, which revealed a significant increase of mammary adenocarcinoma incidence in the stop-dose animals at the lowest BPA dose tested. https://doi.org/10.1289/EHP6301.

Perinatal BPA exposure and reproductive axis function in CD-1 mice.

Perinatal Bisphenol-A (BPA) exposure reduces fertility and fecundity in mice. This study examined effects of early BPA exposure on activation of gonadotropin releasing hormone (GnRH) neurons in conjunction with a steroid-induced luteinizing hormone (LH) surge, characterized patterns of estrous cyclicity and fertility over time, and assessed the ovarian follicular reserve to further explore factors responsible for the reduced fertility we previously described in this model. The percent activated GnRH neurons was reduced in BPA-exposed females at 3-6 months, and periods of persistent proestrus were increased. These data suggest that perinatal exposure to BPA reduces GnRH neuronal activation required for the generation of the LH surge and estrous cyclicity. Assessments of anti-Müllerian hormone (AMH) levels failed to suggest a decline in the follicular reserve at the BPA exposure levels examined.

Perinatally administered bisphenol a as a potential mammary gland carcinogen in rats.

BACKGROUND: Environmental exposure to bisphenol A (BPA) affects mammary gland development in rodents and primates. Prenatal exposure to environmentally relevant doses of BPA increased the number of intraductal hyperplasias and ductal carcinomas in situ by 50 days of age in Wistar-Furth rats. OBJECTIVE: We aimed to determine whether BPA exposure of dams during gestation only or throughout lactation affects the incidence of mammary gland neoplasia in female offspring. METHODS: We treated pregnant Sprague-Dawley rats with BPA at 0, 0.25, 2.5, 25, or 250 µg BPA/kg BW/day from gestational day (GD) 9 to birth and from GD9 to postnatal day (PND) 21. Mammary glands from BPA-exposed offspring were examined at four time points for preneoplastic and neoplastic lesions. To assess circulating BPA levels, we exposed pregnant rats to vehicle or 250 µg BPA/kg BW/day during gestation only or during gestation/lactation and analyzed sera from dams, fetuses, and nursing pups for total and unconjugated BPA. RESULTS: Total and unconjugated BPA were detected in sera from 100% of dams and fetuses and 33% of pups exposed to 250 µg BPA/kg BW/day. Unconjugated BPA levels in exposed dams and fetuses (gestational) and in exposed dams and pups (gestational/lactational) were within levels found in humans. Preneoplastic lesions developed in BPA-exposed female offspring across all doses as early as PND50. Unexpectedly, mammary gland adenocarcinomas developed in BPA-exposed offspring by PND90. CONCLUSIONS: Our findings suggest that developmental exposure to environmentally relevant levels of BPA during gestation and lactation induces mammary gland neoplasms in the absence of any additional carcinogenic treatment. Thus, BPA may act as a complete mammary gland carcinogen.

Reprogramming within hours following nuclear transfer into mouse but not human zygotes.

Fertilized mouse zygotes can reprogram somatic cells to a pluripotent state. Human zygotes might therefore be useful for producing patient-derived pluripotent stem cells. However, logistical, legal and social considerations have limited the availability of human eggs for research. Here we show that a significant number of normal fertilized eggs (zygotes) can be obtained for reprogramming studies. Using these zygotes, we found that when the zygotic genome was replaced with that of a somatic cell, development progressed normally throughout the cleavage stages, but then arrested before the morula stage. This arrest was associated with a failure to activate transcription in the transferred somatic genome. In contrast to human zygotes, mouse zygotes reprogrammed the somatic cell genome to a pluripotent state within hours after transfer. Our results suggest that there may be a previously unappreciated barrier to successful human nuclear transfer, and that future studies could focus on the requirements for genome activation.

Receiver operating characteristic (ROC) analysis of day 5 morphology grading and metabolomic Viability Score on predicting implantation outcome.

PURPOSE: Assessment of embryo viability is a key component of in vitro fertilization (IVF) and currently relies largely on embryo morphology and cleavage rate. In this study, we used receiver operating characteristic (ROC) analysis to compare the Viability Score (generated by metabolomic profiling of spent embryo culture media using near infrared (NIR) spectroscopy) to morphologic grading for predicting pregnancy in women undergoing single embryo transfer (SET) on day 5. METHODS: A total of 198 spent embryo culture media samples were collected in four IVF centers located in the USA, Europe and Australia. First, 137 samples (training set) were analyzed by NIR to develop an algorithm that generates a Viability Score predictive of pregnancy for each sample. Next, 61 samples (validation set) were analyzed by observers blinded to embryo morphology and IVF outcome, using the Day 5 algorithm generated with the training set. Pregnancy was defined as fetal cardiac activity (FCA) at 12 weeks of gestation. RESULTS: The Area Under the Curve (AUC) was greater for the metabolomic Viability Score compared to Morphology [Training set: 0.75 versus 0.55, p = 0.0011; Validation set: 0.68 versus 0.50, P = 0.021], and for a Composite score (obtained using a model combining Viability Score with morphologic grading), compared to morphology alone [0.74 versus 0.50, p = 0.004]. CONCLUSIONS: Our findings suggest that Viability Score alone or in combination with morphologic grading has the potential to be a better classifier for pregnancy outcome than morphology alone in women undergoing SET on day 5.

Influence of vitrification on mouse metaphase II oocyte spindle dynamics and chromatin alignment.

OBJECTIVE: To evaluate influences of vitrification and warming of metaphase II (MII) mouse oocytes on survival, spindle dynamics, spindle morphology, and chromatin alignment on metaphase plates. DESIGN: Experimental animal study. SETTING: University animal laboratory. ANIMAL(S): Eight-week-old B6D2F1 mice. INTERVENTION(S): Denuded MII oocytes were used fresh (control), exposed to vitrification/warming solutions (Sol Expos), or vitrified and warmed (Vitr). MAIN OUTCOME MEASURE(S): Oocyte recovery and survival after warming and the influence of solution exposure and cryopreservation on spindle dynamics and chromatin alignment. RESULT(S): Cryopreservation of two or 10 oocytes per straw resulted in recovery (100% +/- 0% and 95% +/- 4%, respectively; mean +/- SE) and survival (95% +/- 2% and 98% +/- 2%, respectively). Immediately after warming (Vitr), significantly fewer oocytes assessed with immunocytochemistry contained spindles, compared with control and Sol Expos. When oocytes were placed into a 37 degrees C environment for 2 hours after exposure or warming, the ability to recognize spindles by immunocytochemistry was not significantly different between groups. Using live-cell time-lapse imaging with LC-Polscope, similar time-dependent spindle formation dynamics were observed. At 2 hours after collection or treatment, spindle morphology and length were not significantly different between the groups, nor was the incidence of aberrant alignment of chromatin on metaphase plates. CONCLUSION(S): Immediately after warming of vitrified MII oocytes, beta-tubulin is depolymerized and chromatin remains condensed on the metaphase plate. Within a 2-hour period, beta-tubulin repolymerizes, forming morphologically normal metaphase spindles with properly aligned chromatin.

Insulin signaling in mouse oocytes.

Continuous exposure of follicles/oocytes to elevated levels of insulin compromises embryonic developmental competence, although the underlying cellular mechanisms are unknown. The objectives of the present study were to determine whether mouse oocytes have insulin receptors and a functional insulin signaling cascade, and whether insulin exposure during oocyte growth or maturation influences meiotic progression and chromatin remodeling. Immunoblot and immunocytochemical analyses of germinal vesicle-intact (GVI) oocytes demonstrated the presence of insulin receptor-beta. Insulin receptor expression in oocytes was increased by gonadotropin stimulation, and remained elevated throughout meiotic maturation. Fully grown GVI oocytes contained 3-phosphoinositide-dependent protein kinase-1 (PDPK1), thymoma viral proto-oncogene 1 (AKT1), and glycogen synthase kinase 3 (GSK3). In vitro maturation of GVI oocytes in 5 microg/ml insulin had no influence on meiotic progression or the incidence of normal metaphase II (MII) chromosome condensation. Treatment of oocytes during maturation had no effect on GSK3A/B protein expression or phosphorylation of S21/9. However, the culturing of preantral follicles for 10 days with 5 microg/ml insulin increased the phosphorylation of oocyte GSK3B, indicating GSK3 inactivation. The rates of development to metaphase I (MI) were similar for oocytes obtained from insulin-treated follicles and controls, whereas the incidence of abnormal MI chromatin condensation was significantly higher in oocytes obtained from follicles cultured with insulin compared to those cultured without insulin. These results demonstrate that oocytes contain a functional insulin signaling pathway, and that insulin exposure during oocyte growth results in chromatin remodeling aberrations. These findings begin to elucidate the mechanisms by which chronic elevated insulin influences oocyte meiosis, chromatin remodeling, and embryonic developmental competence.

Glycogen synthase kinase-3 regulation of chromatin segregation and cytokinesis in mouse preimplantation embryos.

Glycogen synthase kinase-3 (GSK-3) is a highly conserved serine/threonine protein kinase implicated in diverse cellular processes. Activity of GSK-3 is essential for meiotic chromatin segregation in oocytes, yet expression and/or function of GSK-3 have not been reported in mammalian preimplantation embryos. Objectives of this study were to characterize GSK-3 protein expression/phosphorylation in mouse preimplantation embryos, to assess the effect of GSK-3 activity inhibition on early mitotic events, and to differentiate nuclear and cytoplasmic anomalies in GSK-3 inhibited embryos. Both GSK-3 isoforms were expressed during embryo development, with a differential expression of alpha versus beta. Phosphorylation of GSK-3alpha/beta at residues Y279/Y216 indicated constitutive activation throughout preimplantation development. Phosphorylation at N-terminal residues S21/S9 indicated inhibition of GSK-3alpha/beta activity that was differentially regulated during early development; both alpha and beta isoforms were phosphorylated during early divisions, whereas at the blastocyst stage, only beta was phosphorylated. Cytoplasmic microinjection of zygotes with anti-GSK-3alpha/beta antibody significantly compromised embryonic development past the two-cell stage compared to controls. Reversibility of developmental block was tested via pharmacological inhibitors of GSK-3, lithium chloride (LiCl) and alsterpaullone. Similar to immunoneutralization, significantly fewer zygotes cultured with either LiCl or alsterpaullone developed past the two-cell stage compared to controls and this mitotic block was not reversible. Inhibition of GSK-3 activity significantly compromised timing of pronuclear membrane breakdown and mitosis initiation, nuclear development, and cytokinesis. Inhibition of GSK-3 also resulted in abnormal chromatin segregation, evidenced by incomplete karyokinesis and micronuclei formation. These results suggest that GSK-3 activity is critical for early preimplantation embryonic development.

Oocyte-specific gene signaling and its regulation of mammalian reproductive potential.

Oocyte-specific genes play important roles in regulating ovarian development, principally through the proper and timely progression of oogenesis and folliculogenesis. Development of transgenic mouse models has been critical in revealing how oocyte-specific transcripts influence oocyte development and growth, integrity of the oocyte-granulosa cell complex, oocyte maturation, fertilization, and early embryonic development. Oocyte-derived genes that mediate recombination of homologous chromosomes and DNA mismatch repair include Spo11, Atm, Dmc1, Msh5, Mlh1, and Msh4. Transcripts such as Dazla and Fig-alpha regulate initial proliferation of the primordial germ cell and follicle. Transition from the primordial to primary follicle relies on the expression of growth factors bFGF, Gdf9 and Bmp15, as well as on the expression of various transcripts that mediate oocyte-granulosa cell interactions. Oocyte growth is predominantly under exogenous control, however resumption of meiotic progression is dictated by genes that influence proper chromatin and spindle regulation, such as Cdk, Histone H1oo, Fmn-2, Mad2, and Bub3. Maintenance of meiotic metaphase II arrest prior to fertilization is mediated primarily by c-mos, and successful fertilization requires the expression of zona pellucida glycoproteins (Zp1, Zp2, and Zp3) and Cd9. Following fertilization, maternal-effect and maternally expressed 'imprinting' genes are necessary for the completion of meiosis and for patterning early embryonic development. Recent utilization of suppressive subtractive hybridization (SSH), PCR amplification, and cDNA microarray analysis techniques alongside established transgenesis models are expanding the classification of novel oocyte-specific genes required for reproductive fitness in various species, including human.