Identification of Gene Expression Changes Associated With Uterine Receptivity in Mice.

Bisphenol A (BPA) is an endocrine disruptor widely used in polycarbonate plastics and epoxy resins. It has been reported that BPA has adverse effects on uterine development: it alters uterine expression of progesterone and estrogen receptors; and BPA at ~400 mg/kg is detrimental to embryo implantation. Embryo implantation is a synchronized process between a competent embryo and a receptive uterus. Uterine receptivity is a hormone-controlled transient state in which the uterus can accept an embryo to implant. The mechanism of BPA in disrupting embryo implantation is currently unknown. To investigate the potential effects of BPA on preimplantation embryos and uterine receptivity, timed pregnant wild type C57BL6 young adult females were treated with 0, 10, 40, and 100 mg/kg BPA subcutaneously daily from gestation day 0.5 to day 3.5, and detected for implantation sites on gestation day 4.5. No implantation sites were detected on gestation day 4.5 but retention of embryos in the oviduct and delayed embryo development were detected on day 3.5 in the 100 mg/kg/day BPA-treated females, indicating the detrimental effects of 100 mg/kg/day BPA on embryo transport and preimplantation embryo development. When untreated healthy embryos were transferred to pseudopregnant females treated with 100 mg/kg/day BPA from gestation day 0.5 to 3.5, no implantation sites were detected on day 4.5, indicating that 100 mg/kg/day BPA can also adversely affect uterine receptivity. Delayed implantation and prolonged gestation period, and increased perinatal lethality were detected in the 40 mg/kg/day BPA-treated females and their offspring, respectively. Progesterone receptor (PR), which disappears from uterine luminal epithelium (LE) upon embryo implantation, remained highly expressed in the LE of pregnant day 4.5 uteri from 40 and 100 mg/kg BPA-treated mice, further demonstrating defective embryo implantation in these BPA-treated mice. Normal implantation was observed in the 10 mg/kg/day BPA-treated females or the female offspring from 40 mg/kg/day BPA-treated group. These data demonstrate that preimplantation BPA exposure at 40 and 100 mg/kg/day can adversely affect embryo implantation. (Supported by University of Georgia and NIH R15HD066301) (poster)

Protein kinase C (PKC) isozymes are key signal transducers involved in normal physiology and disease and have been widely implicated in cancer progression. Despite our extensive knowledge of the signaling pathways regulated by PKC isozymes and their effectors, there is essentially no information on how individual members of the PKC family regulate gene transcription. Here, we report the first PKC isozyme-specific analysis of global gene expression by microarray using RNAi depletion of diacylglycerol/phorbol ester-regulated PKCs. A thorough analysis of this microarray data revealed unique patterns of gene expression controlled by PKCα, PKCδ, and PKCε, which are remarkably different in cells growing in serum or in response to phorbol ester stimulation. PKCδ is the most relevant isoform in controlling the induction of genes by phorbol ester stimulation, whereas PKCε predominantly regulates gene expression in serum. We also established that two PKCδ-regulated genes, FOSL1 and BCL2A1, mediate the apoptotic effect of phorbol esters or the chemotherapeutic agent etoposide in prostate cancer cells. Our studies offer a unique opportunity for establishing novel transcriptional effectors for PKC isozymes and may have significant functional and therapeutic implications.

In environmental risk assessment of substances, the 14-day growth inhibition test following OECD test guideline 239 is employed to assess toxicity in the macrophyte Myriophyllum spicatum. Currently, this test evaluates physiological parameters and does not allow the identification of the mode of action (MoA) by which adverse effects are induced. However, for an improved ecotoxicity assessment of substances, knowledge about their ecotoxic MoA in non-target organisms is required. It has previously been suggested that the identification of gene expression changes can contribute to MoA identification. Therefore, we developed a shortened three-day assay for M. spicatum including the transcriptomic assessment of global gene expression changes and applied this assay to two model substances, the herbicide and photosynthesis inhibitor bentazone and the pharmaceutical and HMG-CoA reductase inhibitor atorvastatin. Due to the lack of a reference genome for M. spicatum we performed a de novo transcriptome assembly followed by a functional annotation to use the toxicogenomic results for MoA discrimination. The gene expression changes induced by low effect concentrations of these substances were used to identify differentially expressed genes (DEGs) and impaired biological functions for the respective MoA. We observed both concentration-dependent numbers and differentiated patterns of DEGs for both substances. While bentazone impaired genes involved in the response to reactive oxygen species as well as light response, and also genes involved in developmental processes, atorvastatin exposure led to a differential regulation of genes related to brassinosteroid response as well as potential metabolic shifts between the mevalonate and methyl erythritol 4-phosphate pathway. Based on these responses, we identified biomarker candidates for the assessment of MoA in M. spicatum. Utilizing the shortened assay developed in this study, the investigation of the identified biomarker candidates may contribute to the development of future MoA-specific screening approaches in the ecotoxicological hazard prediction using aquatic non-standard model organisms.

BackgroundRhodospirillum centenum is a photosynthetic member of the Gram-negative Azospirillum clade members of which exhibit a complex developmental life-cycle featuring morphologically distinct cell types. Under periods of nutrient deprivation, replicative vegetative cells differentiate into metabolically dormant cysts that survive harsh environmental stresses such as desiccation. Encystment involves a multi-stage developmental process that includes the rounding of cells, production of large intracellular storage granules of poly-hydroxybutyrate (PHB) and the excretion of a protective exopolysaccharide coating that envelops dormant cysts.ResultsTo study the process of cyst development, we performed RNA-seq studies on cells that were induced to undergo cyst development. To assay for temporal changes in gene expression, RNA was extracted at 4, 24, 48, 72, 96 hours during development and subjected to deep sequence analysis. These results show that 812 genes exhibit log2 ≥ 1.5-fold changes in expression over a 96 hour cyst induction period demonstrating large global changes in gene expression during cyst development.ConclusionsNotable changes in expression occurred in numerous genes involved in cell wall and lipid biosynthesis, metabolic enzymes, and numerous regulatory genes such as histidine kinases and transcription factors. Many genes involved in protein synthesis and DNA replication were also significantly reduced during late stages of cyst development. Genes previously identified by genetic screens as being critical for cyst development also exhibited changes of expression during cyst induction. This study provides the first transcriptome profile of global changes in gene expression that occur during development of cysts in a Gram-negative species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1250-9) contains supplementary material, which is available to authorized users.

Estrogen induction of uterine wet weight provides an excellent model to investigate relationships between changes in global gene expression and well-characterized physiological responses. In this study, time course microarray GeneChip data were analyzed using a novel approach to identify temporal changes in uterine gene expression following treatment of immature ovariectomized C57BL/6 mice with 0.1 mg/kg 17alpha-ethynylestradiol. Functional gene annotation information from public databases facilitated the association of changes in gene expression with physiological outcomes, which allowed detailed mechanistic inferences to be drawn regarding cell cycle control and proliferation, transcription and translation, structural tissue remodeling, and immunologic responses. These systematic approaches confirm previously established responses, identify novel estrogen-regulated transcriptional effects, and disclose the coordinated activation of multiple modes of action that support the uterotrophic response elicited by estrogen. In particular, it was possible to elucidate the physiological significance of the dramatic induction of arginase, a classic estrogenic response, by elucidating its mechanistic relevance and delineating the role of arginine and ornithine utilization in the estrogen-stimulated induction of uterine wet weight.

Caffeine consumption has been widely used as a central nervous system stimulant. Epidemiological studies, however, have suggested that maternal caffeine exposure during pregnancy is associated with increased abnormalities, including decreased fertility, delayed conception, early spontaneous abortions, and low birth weight. The mechanisms underlying the negative outcomes of caffeine consumption, particularly during early pregnancy, remain unclear. In present study, we found that pregnant mice treated with moderate (5 mg/kg) or high (30 mg/kg) dosage of caffeine (intraperitoneally or orally) during preimplantation resulted in retention of early embryos in the oviduct, defective embryonic development, and impaired embryo implantation. Transferring normal blastocysts into the uteri of caffeine-treated pseudopregnant females also showed abnormal embryo implantation, thus indicating impaired uterine receptivity by caffeine administration. The remaining embryos that managed to implant after caffeine treatment also showed increased embryo resorption rate and abnormal development at mid-term stage, and decreased weight at birth. In addition to a dose-dependent effect, significant variations between individual mice under the same caffeine dosage were also observed, suggesting different sensitivities to caffeine, similar to that observed in human populations. Collectively, our data revealed that caffeine exposure during early pregnancy impaired oviductal embryo transport, embryonic development, and uterine receptivity, which are responsible for abnormal implantation and pregnancy loss. The study raises the concern of caffeine consumption during early stages of pregnancy.

Two-dimensional gel electrophoresis (2-DE) is currently unequalled in its capacity to separate individual components of complex protein mixtures, making it the method of choice for monitoring global changes in gene expression in response to defined conditions (Guerreiro et al, 1997). Using 2-DE we have examined the global changes in Rhizobium gene expression which occur in response to specific Tn5-induced mutations that lead to defective symbiotic phenotypes. Exopolysaccharides (EPS) of Rhizobium leguminosarum have been implicated to be involved in a complex series of interactions which lead to the establishment of an effective Rhizobium -legume symbiosis, but the precise role of EPS in this interaction is not well defined. The mutant ANU437, a derivative of R. l. bv. trifolli strain ANU794, contains a Tn5 insertion in pssA (also called pss4), produces very low levels of acidic exopolysaccharide and induces delayed formation of small non-nitrogen-fixing nodules on various clover species (Rolfe et al, 1996). Using 2-DE we have shown ANU437 to exhibit multiple differences in gene expression when compared with its wild-type parent. Such pleiotrophies would have been difficult to show via other methodologies. Twenty-one proteins in ANU437 were either newly-induced or up-regulated in their level of synthesis. Six of these proteins have been characterised by N-terminal sequencing, all were novel proteins except for one which showed a high similarity to a phosphogluconate dehydrogenase. These results have shown the PSSA protein, in addition to being a glycosyltransferase, may be directly or indirectly involved in influencing the expression of a complex series of genes whose levels of expression may be the actual contributing factor to the ineffective nodulation observed with exo− mutants.KeywordsBiological Nitrogen FixationComplex SeriesRhizobium LeguminosarumPhosphogluconate DehydrogenaseMultiple DifferenceThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Magnocellular neurons (MCNs) in the hypothalamo-neurohypophysial system (HNS) are highly specialized to release large amounts of arginine vasopressin (Avp) or oxytocin (Oxt) into the blood stream and play critical roles in the regulation of body fluid homeostasis. The MCNs are osmosensory neurons and are excited by exposure to hypertonic solutions and inhibited by hypotonic solutions. The MCNs respond to systemic hypertonic and hypotonic stimulation with large changes in the expression of their Avp and Oxt genes, and microarray studies have shown that these osmotic perturbations also cause large changes in global gene expression in the HNS. In this paper, we examine gene expression in the rat supraoptic nucleus (SON) under normosmotic and chronic salt-loading SL) conditions by the first time using “new-generation”, RNA sequencing (RNA-Seq) methods. We reliably detect 9,709 genes as present in the SON by RNA-Seq, and 552 of these genes were changed in expression as a result of chronic SL. These genes reflect diverse functions, and 42 of these are involved in either transcriptional or translational processes. In addition, we compare the SON transcriptomes resolved by RNA-Seq methods with the SON transcriptomes determined by Affymetrix microarray methods in rats under the same osmotic conditions, and find that there are 6,466 genes present in the SON that are represented in both data sets, although 1,040 of the expressed genes were found only in the microarray data, and 2,762 of the expressed genes are selectively found in the RNA-Seq data and not the microarray data. These data provide the research community a comprehensive view of the transcriptome in the SON under normosmotic conditions and the changes in specific gene expression evoked by salt loading.

Microcystis blooms occur worldwide and threaten aquatic ecosystems and human health. Sublethal effects on early developmental stages of fish are largely unknown, and research has mainly focused on microcystin toxins (such as MC-LR) rather than Microcystis cells. We exposed (96 h) zebrafish larvae to purified MC-LR (0-1000 μg/L) or lyophilized Microcystis aeruginosa containing 4.5 μg/L MC-LR and evaluated changes in global gene expression (Affymetrix GeneChip zebrafish genome arrays). Significant changes in gene expression (≥ 1.7-fold change, p < 0.0001) were determined with Rosetta Resolver 7.0, and ontology analysis was conducted with the DAVID bioinformatics tool. The number of differentially expressed genes relative to control increased with MC-LR concentration and included genes related to known mechanisms of action for MC-LR in mammals and older life stages of fish, as well as genes unique to larval zebrafish. Up-regulation of vitellogenin genes (vtg) (19.2-fold to >100-fold on arrays; 619.3-fold confirmed by quantitative PCR) was observed in Microcystis-exposed larvae but not in larvae exposed to MC-LR. Up-regulation of vtg indicates exposure to estrogenic substance(s) and suggests that Microcystis may be a natural source of environmental estrogens. Concerns about effects of Microcystis blooms may extend beyond those associated with the microcystin toxin.

Human angiotensin-converting enzyme 2 (hACE2) has recently received a great attention due to it play a critical role as SARS-CoV receptor in the infection of human body. However, no further analysis for gene regulation has been performed in target tissues of model mice during hACE2 overproduction. To characterize changes in global gene expression in the hearts and kidneys of rtTA/hACE2 double transgenic (dTg) mice in response to hACE2 overexpression, total RNA extracted from these tissues from dTg mice after doxycycline (Dox) treatment was hybridized to oligonucleotide microarrays. Briefly, dTg mice were generated by cross-mating pα-MHC/rtTA Tg mice with pTRE/hACE2 Tg mice. The expression level of hACE2 protein was determined to be high in hearts, kidneys, and brains of dTg mice, whereas lung, liver, and testis tissues expressed low levels. The level of hACE2 was significantly enhanced in hearts and kidneys of the Dox+dTg group compared to that in Vehicle+dTg mice although consistent levels of mouse ACE2 (mACE2) remained in the same tissues. Based on the microarray analysis of heart tissue, 385 genes were differentially expressed, including 168 upregulated and 217 downregulated, when comparing non-Tg and Vehicle+dTg mice, whereas 216 genes were differentially expressed, including 136 upregulated and 80 downregulated, between Vehicle+dTg and Dox+dTg mice. In the kidneys, 402 genes were differentially expressed, including 159 upregulated and 243 downregulated, between non-Tg and Vehicle+dTg mice. Dox-treated dTg mice exhibited the differential expression of 4735 genes including 1636 upregulated and 3109 downregulated. Taken together, these findings suggested that several functional groups and individual genes can be considered biomarkers that respond to hACE2 overexpression in dTg mice. Moreover, our results provided a lot of useful information to predict physiological responses when these dTg mice are applied as a susceptible model for novel coronavirus (SARS-CoV, COVID-19) in both vaccine and drug development.

[This corrects the article DOI: 10.1371/journal.pone.0221519.].

Plants respond to the proximity of neighboring vegetation by elongating to prevent shading. Red-depleted light reflected from neighboring vegetation triggers a shade avoidance response leading to a dramatic change in plant architecture. These changes in light quality are detected by the phytochrome family of photoreceptors. We analyzed global changes in gene expression over time in wild-type, phyB mutant, and phyA phyB double mutant seedlings of Arabidopsis in response to simulated shade. Using pattern fitting software, we identified 301 genes as shade responsive with patterns of expression corresponding to one of various physiological response modes. A requirement for a consistent pattern of expression across 12 chips in this way allowed more subtle changes in gene expression to be considered meaningful. A number of previously characterized genes involved in light and hormone signaling were identified as shade responsive, as well as several putative, novel shade-specific signal transduction factors. In addition, changes in expression of genes in a range of pathways associated with elongation growth and stress responses were observed. The majority of shade-responsive genes demonstrated antagonistic regulation by phyA and phyB in response to shade following the pattern of many physiological responses. An analysis of promoter elements of genes regulated in this way identified conserved promoter motifs potentially important in shade regulation.

Nitrogen (N) is an essential macronutrient for plant growth and development. Plants adapt to changes in N availability partly by changes in global gene expression. We integrated publicly available root microarray data under contrasting nitrate conditions to identify new genes and functions important for adaptive nitrate responses in Arabidopsis thaliana roots. Overall, more than 2000 genes exhibited changes in expression in response to nitrate treatments in Arabidopsis thaliana root organs. Global regulation of gene expression by nitrate depends largely on the experimental context. However, despite significant differences from experiment to experiment in the identity of regulated genes, there is a robust nitrate response of specific biological functions. Integrative gene network analysis uncovered relationships between nitrate-responsive genes and 11 highly co-expressed gene clusters (modules). Four of these gene network modules have robust nitrate responsive functions such as transport, signaling, and metabolism. Network analysis hypothesized G2-like transcription factors are key regulatory factors controlling transport and signaling functions. Our meta-analysis highlights the role of biological processes not studied before in the context of the nitrate response such as root hair development and provides testable hypothesis to advance our understanding of nitrate responses in plants.

The present study was designed to examine and analyze the global gene expression changes during the tumorigenesis of a human immortalized oral epithelial cell line, and search for the possible genes that may play a role in the carcinogenesis of oral cancer associated with benzo (a) pyrene. The human immortalized oral epithelial cells, which have been established through transfection of E6/E7 genes of human papillomavirus type 16 and proved to be non-tumorigenic in nude mice, were treated with benzo (a) pyrene. Tumorigenicity of the treated cells were examined through nude mice subcutaneous injection. The global gene expression profiles of immortalized cells and the tumorigenic cells were acquired through hybridization of a microarray of Affymetrix U133 plus 2.0. The data were analyzed using Spring 7.0 software and treated statistically using one-way analysis of variance (ANOVA). The differentially expressed genes were classified using a Venn diagram and annotated with gene ontology. Several highlighted genes were validated in cells using a real-time polymerase chain reaction. There were 883 differentially expressed genes during the tumorigenesis and most of them changed expression in the early stage of tumorigenesis. These genes mainly involved in macromolecule metabolism and signal transduction, possessed the molecular function of transition metal ion binding, nucleotide binding and kinase activity; their protein products were mainly integral to membranes or localized in the nucleus and cytoskeleton. The expression patterns of IGFBP3, S100A8, MAP2K, KRT6B, GDF15, MET were validated in cells using a real-time polymerase chain reaction; the expression of IGFBP3 was further validated in clinical oral cancer specimens. This study provides the global transcription profiling associated with the tumorigenesis of oral epithelial cells exposed to benzo (a) pyrene; IGFBP3 may play a potential role in the initiation of oral cancer related to benzo (a) pyrene exposure.

Global Gene Expression Changes Induced in the Human Placenta during Labor