ATP Binding Cassette Sub-family Member 2 (ABCG2) and Xenobiotic Exposure During Early Mouse Embryonic Stem Cell Differentiation.

BACKGROUND: ATP binding cassette sub-family member 2 (ABCG2) is a well-defined efflux transporter found in a variety of tissues. The role of ABCG2 during early embryonic development, however, is not established. Previous work which compared data from the ToxCast screening program with that from in-house studies suggested an association exists between exposure to xenobiotics that regulate Abcg2 transcription and differentiation of mouse embryonic stem cells (mESC), a relationship potentially related to redox homeostasis. METHODS: mESC were grown for up to 9 days. Pharmacological inhibitors were used to assess transporter function with and without xenobiotic exposure. Proliferation and differentiation were evaluated using RedDot1 and quantiative reverse transcriptase-polymerase chain reaction, respectively. ABCG2 activity was assessed using a Pheophorbide a-based fluorescent assay. Protein expression was measured by capillary-based immunoassay. RESULTS: ABCG2 activity increased in differentiating mESC. Treatment with K0143, an inhibitor of ABCG2, had no effect on proliferation or differentiation. As expected, mitoxantrone and topotecan, two chemotherapeutics, displayed increased toxicity in the presence of K0143. Exposure to K0143 in combination with chemicals predicted by ToxCast to regulate ABCG2 expression did not alter xenobiotic-induced toxicity. Moreover, inhibition of ABCG2 did not shift the toxicity of either tert-Butyl hydroperoxide or paraquat, two oxidative stressors. CONCLUSION: As previously reported, ABCG2 serves a protective role in mESC. The role of ABCG2 in regulating redox status, however, was unclear. The hypothesis that ABCG2 plays a fundamental role during mESC differentiation or that regulation of the receptor by xenobiotics may be associated with altered mESC differentiation could not be supported. Birth Defects Research, 110:35-47, 2018. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

The effects of triclosan on puberty and thyroid hormones in male Wistar rats.

Triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) is a potent antibacterial and antifungal compound that is widely used in personal care products, plastics, and fabrics. Recently triclosan has been shown to alter endocrine function in a variety of species. The purpose of this study was to determine effects of triclosan on pubertal development and thyroid hormone concentrations in the male rat. Weanling rats were exposed to 0, 3, 30, 100, 200, or 300 mg/kg of triclosan by oral gavage from postnatal day (PND) 23 to 53. Preputial separation (PPS) was examined beginning on PND 33. Rats were killed on PND 53, organ weights were recorded and serum was collected for subsequent analysis. Triclosan did not affect growth or the onset of PPS. Serum testosterone was significantly decreased at 200 mg/kg, however no effects were observed on androgen-dependent reproductive tissue weights. Triclosan significantly decreased total serum thyroxine (T4) in a dose-dependent manner at 30 mg/kg and higher (no observed effect level of 3 mg/kg). Triiodothyronine (T3) was significantly decreased only at 200 mg/kg, but thyroid stimulating hormone was not statistically different at any dose. Liver weights were significantly increased at 100 mg/kg triclosan and above suggesting that the induction of hepatic enzymes may have contributed to the altered T4 and T3 concentrations, but it does not appear to correlate with the T4 dose-response. This study demonstrates that triclosan exposure does not alter androgen-dependent tissue weights or onset of PPS; however, triclosan exposure significantly impacts thyroid hormone concentrations in the male juvenile rat.

The healthy men study: design and recruitment considerations for environmental epidemiologic studies in male reproductive health.

OBJECTIVE: To describe study design, conduct and response, and participant characteristics. DESIGN: Prospective cohort study. SETTING: Participants were male partners of women who were enrolled in a community-based prospective cohort study of drinking water disinfection by-products and pregnancy health. PATIENT(S): Two hundred thirty presumed fertile men recruited from 3 study sites in the United States. INTERVENTION(S): Men completed a telephone interview about demographics, health history, and exposures and provided a semen sample that was express mailed to the study laboratory. MAIN OUTCOME MEASURE(S): Response and participation rates, participant demographics, and lifestyle exposures. RESULT(S): We obtained a high participation rate (84%) among men who were located, but a low overall response rate (25%). Participants were more likely to be white, more highly educated, be married, and have a higher household income than the underlying study cohort. CONCLUSION(S): Our multisite study design may be applicable to the study of community environmental factors and reproductive health of men. Our design was efficient in that men from geographically disparate sites could be recruited, a semen sample was collected at home, and a telephone interview was conducted from a central study site. Despite these design features, the low response rates may suggest selection bias that can be addressed partially in the analysis.

Confocal laser scanning microscopy of whole mouse ovaries: excellent morphology, apoptosis detection, and spectroscopy.

BACKGROUND: Ovaries consist of numerous follicles, oocytes, and granulosa cells in different stages of development. Many of these follicles will undergo an apoptotic process during the lifetime of the animal. By using proper tissue preparation methods, the events within the whole ovary can be observed by using 3D confocal microscopy. METHODS: Whole ovaries were stained with LysoTracker Red (LT), fixed with 4% paraformaldehyde (PF) and 1% glutaraldehyde (Glut), dehydrated with methanol (MEOH), and cleared with benzyl alcohol and benzyl benzoate (BABB). Using this tissue preparation technique, the ovary becomes relatively transparent, allowing its morphology to be observed with confocal microscopes. A spectral imaging system (PARISS) located on a conventional microscope was used to interpret the LT dye spectra and fixation products in the tissues with different excitation wavelengths. RESULTS: Apoptosis in the follicle was detected as clusters of intensely stained granulosa cells located in close proximity to the oocytes. The fixation with Glut and PF preserved morphological details, increased tissue fluorescence, thus increased the signal to noise of the background image. CONCLUSIONS: Thick tissues can be imaged after they are properly stained, aldehyde fixed, and BABB cleared. LT intensely stained single cells or clusters of apoptotic cells in the follicles and the nucleolus. Spectral differences between LT as an indicator of apoptosis and Glut-PF fixation was used to visualize ovarian morphology and apoptosis. The PARISS spectrophotometer revealed spectral peaks for LT at 609.6 nm and for Glut-PF at 471.3 nm. The proper use of the spectra from these fluorescence molecules is the foundation for high quality morphological images of apoptosis. By sequentially imaging the two probes with a 488 nm laser and a 543/568 nm laser, there was a reduction in fluorescent cross talk and an increase in image quality.

Glutathione (GSH) concentrations vary with the cell cycle in maturing hamster oocytes, zygotes, and pre-implantation stage embryos.

Glutathione (GSH) is thought to play critical roles in oocyte function including spindle maintenance and provision of reducing power needed to initiate sperm chromatin decondensation. Previous observations that GSH concentrations are higher in mature than immature oocytes and decline after fertilization, suggest that GSH synthesis may be associated with cell cycle events. To explore this possibility, we measured the concentrations of GSH in Golden Hamster oocytes and zygotes at specific stages of oocyte maturation and at intervals during the first complete embryonic cell cycle. Between 2 and 4 hr after the hormonal induction of oocyte maturation, GSH concentrations increased significantly (approximately doubling) in both oocytes and their associated cumulus cells. This increase was concurrent with germinal vesicle breakdown and the condensation of metaphase I chromosomes in the oocyte. GSH remained high in ovulated, metaphase II (MII) oocytes, but then declined significantly, by about 50%, shortly after fertilization, as the zygote progressed back into interphase (the pronucleus stage). GSH concentrations then plummeted by the two-cell embryo stage and remained at only 10% of those in MII oocytes throughout pre-implantation development. These results demonstrate that oocyte GSH concentrations fluctuate with the cell cycle, being highest during meiotic metaphase, the critical period for spindle growth and development and for sperm chromatin remodeling. These observations raise the possibility that GSH synthesis in maturing oocytes is regulated by gonadotropins, and suggest that GSH is more important during fertilization than during pre-implantation embryo development.

Abnormal fertilization is responsible for reduced fecundity following thiram-induced ovulatory delay in the rat.

Brief exposure to some pesticides, applied during a sensitive window for the neural regulation of ovulation, will block the preovulatory surge of LH and, thus, delay ovulation. Previously, we have shown that a single i.p. injection of 50 mg/kg of thiram, a dithiocarbamate fungicide that decreases norepinephrine synthesis, on proestrus (1300 h) suppresses the LH surge and delays ovulation for 24 h without altering the number of oocytes released. However, when bred, the treated dams had a decreased litter size and increased postimplantation loss. We hypothesized that the reduced litter size in thiram-delayed rats was a consequence of altered oocyte function arising from intrafollicular oocyte aging. To test this hypothesis, we examined delayed oocytes, zygotes, and 2-cell embryos for evidence of fertilization and polyspermy. In addition, we used confocal laser-scanning microscopy to evaluate and characterize cortical granule localization in oocytes and release in zygotes, because the cortical granule response is a major factor in the normal block to polyspermy. Our results demonstrate that a thiram-induced, 24-h delay in ovulation alters the fertilizability of the released oocyte. Although no apparent morphological differences were observed in the unfertilized mature oocytes released following the thiram-induced delay, the changes observed following breeding include a significant decrease in the percentage of fertilized oocytes, a significant increase in polyspermic zygotes (21%), and a 10-fold increase in the number of supernumerary sperm in the perivitelline space. Importantly, all the polyspermic zygotes exhibited an abnormal pattern of cortical granule exudate, suggestive of a relationship between abnormal cortical reaction and the polyspermy in the delayed zygotes. Because polyspermy is associated with polyploidy, abnormal development, and early embryonic death, the observed polyspermy could explain the abnormal development and decreased litter size that we observed previously following thiram-delayed ovulation.