Papers of the Month
By Robert Brown, Deacqunita Diggs, Samantha Hall, Emily Mesev, and Simone Otto
High-throughput screening method accurate, say NTP scientists
National Toxicology Program (NTP) researchers and their collaborators have determined that the BG1 quantitative high-throughput screening (BG1 qHTS) method is as accurate as the accepted method that it was based on, the BG1Luc4E2 estrogen receptor transactivation (BG1Luc ER TA) method. The finding validates the use of BG1 qHTS as a faster, cheaper way to identify the hormone-mimicking environmental chemicals that produce reproductive or developmental problems in humans.
The BG1Luc ER TA method, which is part of the U.S. Environmental Protection Agency’s Endocrine Disruptor Screening Program, detects substances with estrogen receptor (ER) agonist activity. Because one of the goals of the Tox21 program is to develop test methods that reduce cost and animal use, the scientists adapted BG1Luc ER TA for high-throughput screening and tested approximately 10,000 chemicals, three times each. They found that 97 percent of the time, the new method was as accurate as BG1Luc ER TA. When a larger set of chemicals was used for comparison, the team demonstrated that the two methods had 92 percent agreement.
Based on this data, the authors say that the BG1 qHTS method is as reliable as the standard technique for identifying endocrine disrupting chemicals in the environment and should be considered as an additional method of testing. (DD)
Ceger P, Allen D, Huang R, Xia M, Casey W. 2015. Performance of the BG1Luc ER TA method in a qHTS format. ALTEX 32(4):287-296.
Study links glucocorticoid signaling to female fertility
Scientists at NIEHS are the first to provide compelling evidence that glucocorticoid signaling plays a central role in regulating female fertility. Characterization of glucocorticoid signaling in the uterus has significant clinical relevance and may lead to strategies to improve pregnancy rates.
Using a uterine-specific GR knockout mouse, investigators found that glucocorticoid signaling is involved in establishing uterine receptivity for the fertilized egg. Researchers further demonstrated that the absence of uterine GR signaling causes critical deficiencies in the cellular programming required for a healthy pregnancy. The loss of uterine GR signaling resulted in an exaggerated inflammatory response and altered immune cell abundance in response to maternal hormones.
These findings provide direct evidence that GR signaling is required to establish the necessary cellular context for maintaining normal uterine biology and fertility, and may lead to strategies to improve pregnancy rates. (RB)
Whirledge SD, Oakley RH, Myers PH, Lydon JP, DeMayo F, Cidlowski JA. 2015. Uterine glucocorticoid receptors are critical for fertility in mice through control of embryo implantation and decidualization. Proc Natl Acad Sci U S A 112(49):15166-15171.
Carbon substrate reprograming offers an advantage to T-cells
T cells that undergo repeated osmotic stress (OS) and recovery develop a resistance to cell death, or apoptosis, according to research conducted by NIEHS scientists. The group also found that the resistant T cells are completely dependent on glucose for life. Metabolically, they can live on galactose in the absence of glucose, but this modification primes them for apoptosis. The finding has immense implications for health, as diseased cells commonly resist cell death in conditions, such as neurodegeneration, cancer, and autoimmune disorders.
After scientists generated OS-resistant T cells, microarray analysis showed that many mechanisms of apoptotic signaling were down-regulated, but protein analysis of apoptotic regulator Bcl-2 showed no change. Further analysis revealed the cells were resistant to intrinsic signals for apoptosis and had become dependent on fermenting glucose for energy.
Fermentation in the presence of oxygen is called the Warburg effect, which is often seen in cancer cells and was originally proposed as a possible driver of tumorgenesis. The researchers discovered this occurs through changes in the protein kinase B mechanistic target of rapamycin (AKT-mTOR) pathway. This research provides insights into apoptotic resistance. (SO)
Bortner CD, Scoltock AB, Cain DW, Cidlowski JA. 2015. T-cell development of resistance to apoptosis is driven by a metabolic shift in carbon source and altered activation of death pathways. Cell Death Differ; doi:10.1038/cdd.2015.156 [Online 11 December 2015].
Estrogen receptor alpha role in fertilization and early embryo development
NIEHS scientists have shown that a lack of the epithelial estrogen receptor alpha (ERalpha) signaling alters uterine and oviduct physiology, which leads to disrupted sperm migration, restricted fertilization of eggs, and death of all zygotes. The observation marks an important role of oviductal ERalpha in both fertilization and the development of fertilized eggs before implantation in the uterus.
Scientists monitored fertilization in mice lacking epithelial ERalpha and reported a 50 percent reduction in zygote numbers compared to wild type controls. All the zygotes died before cleaving. Conversely, fertilization and embryo development in mice lacking ERalpha only in their mesenchymal cells remained unimpaired. Microarray analysis helped to correlate these findings with alterations in inflammatory response genes and regulatory enzymes.
The results suggest that estrogen signaling through epithelial ERalpha is necessary to control the secretion of immune response molecules, such as proteases, that can degrade preimplantation embryos. The scientists reported that elevated protease activity disrupted the integrity of the protective material that surrounds developing embryos, resulting in the embryos’ death. This study connects estrogen signaling via epithelial ERalpha with the balanced environment required for successful fertilization, raising possible implications for human fertility. (EM)
Winuthayanon W, Bernhardt ML, Padilla-Banks E, Myers PH, Edin ML, Hewitt SC, Korach KS, Williams CJ. 2015. Oviductal estrogen receptor alpha signaling prevents protease-mediated embryo death. Elife; doi:10.7554/eLife.10453 [Online 1 December 2015].
Improving the strategy of recombinase-based labeling
Researchers at NIEHS have recently developed a new way to visualize cells, which will be especially useful in studying the mammalian brain. Beyond its use in neuroscience research, this strategy will open new avenues for answering even broader developmental biology questions.
Recombinase-based labeling uses recombinase driver alleles with overlapping gene expression patterns in the mouse brain and an indicator allele that labels cells with a fluorescent protein in response to recombinase activity. One of the new indicator alleles, RC::RFLTG, expresses a red fluorescent marker in response to two recombinases called Dre and Flp and a green fluorescent marker in response to three recombinases (Dre, Flp, and Cre).
The scientists used RC::RFLTG to label specific subpopulations of noradrenergic neurons and their axonal projections, which are the connections they make to other regions of the brain. The researchers were able to simultaneously label two different noradrenergic subpopulations that were previously unobservable, revealing their three-dimensional structure and permitting direct comparison of their projections throughout the mouse brain.
This paper establishes an important new tool for investigating heterogeneity in genetically defined cell populations, facilitating future studies of their developmental origins and functional properties. (SH)
Plummer NW, Evsyukova IY, Robertson SD, de Marchena J, Tucker CJ, Jensen P. 2015. Expanding the power of recombinase-based labeling to uncover cellular diversity. Development 142(24):4385-4393.