Molecular Characterization of the Gastrula in the Turtle Emys orbicularis: An Evolutionary Perspective on Gastrulation

Due to the presence of a blastopore as in amphibians, the turtle has been suggested to exemplify a transition form from an amphibian- to an avian-type gastrulation pattern. In order to test this hypothesis and gain insight into the emergence of the unique characteristics of amniotes during gastrulation, we have performed the first molecular characterization of the gastrula in a reptile, the turtle Emys orbicularis. The study of Brachyury, Lim1, Otx2 and Otx5 expression patterns points to a highly conserved dynamic of expression with amniote model organisms and makes it possible to identify the site of mesoderm internalization, which is a long-standing issue in reptiles. Analysis of Brachyury expression also highlights the presence of two distinct phases, less easily recognizable in model organisms and respectively characterized by an early ring-shaped and a later bilateral symmetrical territory. Systematic comparisons with tetrapod model organisms lead to new insights into the relationships of the blastopore/blastoporal plate system shared by all reptiles, with the blastopore of amphibians and the primitive streak of birds and mammals. The biphasic Brachyury expression pattern is also consistent with recent models of emergence of bilateral symmetry, which raises the question of its evolutionary significance

Developmental toxicity and brain aromatase induction by high genistein concentrations in zebrafish embryos

Genistein is a phytoestrogen found at a high level in soybeans. In vitro and in vivo studies showed that high concentrations of genistein caused toxic effects. This study was designed to test the feasibility of zebrafish embryos for evaluating developmental toxicity and estrogenic potential of high genistein concentrations. The zebrafish embryos at 24 h post-fertilization were exposed to genistein (1 × 10−4 M, 0.5 × 10−4 M, 0.25 × 10−4 M) or vehicle (ethanol, 0.1%) for 60 h. Genistein-treated embryos showed decreased heart rates, retarded hatching times, decreased body length, and increased mortality in a dose-dependent manner. After 0.25 × 10−4 M genistein treatment, malformations of survived embryos such as pericardial edema, yolk sac edema, and spinal kyphosis were also observed. TUNEL assay results showed apoptotic DNA fragments in brain. This study also confirmed the estrogenic potential of genistein by EGFP expression in the brain of the mosaic reporter zebrafish embryos. This study first demonstrated that high concentrations of genistein caused a teratogenic effect on zebrafish embryos and confirmed the estrogenic potential of genistein in mosaic reporter zebrafish embryos

The expression of nuclear and membrane estrogen receptors in the European eel throughout spermatogenesis

[EN] Estradiol (E-2) can bind to nuclear estrogen receptors (ESR) or membrane estrogen receptors (GPER). While mammals possess two nuclear ESRs and one membrane GPER, the European eel, like most other teleosts, has three nuclear ESRs and two membrane GPERs, as the result of a teleost specific genome duplication. In the current study, the expression of the three nuclear ESRs (ESR1, ESR2a and ESR2b) and the two membrane GPERs (GPERa and GPERb) in the brain-pituitary-gonad (BPG) axis of the European eel was measured, throughout spermatogenesis. The eels were first transferred from freshwater (FW) to seawater (SW), inducing parallel increases in E2 plasma levels and the expression of ESRs. This indicates that salinity has a stimulatory effect on the E-2 signalling pathway along the BPG axis. Stimulation of sexual maturation by weekly injections of human chorionic gonadotropin (hCG) induced a progressive decrease in E-2 plasma levels, and different patterns of expression of ESRs and GPERs in the BPG axis. The expression of nuclear ESRs increased in some parts of the brain, suggesting a possible upregulation due to a local production of E-2. In the testis, the highest expression levels of the nuclear ESRs were observed at the beginning of spermatogenesis, possibly mediating the role of E2 as spermatogonia renewal factor, followed by a sharply decrease in the expression of ESRs. Conversely, there was a marked increase observed in the expression of both membrane GPERs throughout spermatogenesis, suggesting they play a major role in the final stages of spermatogenesis.Funded by the Spanish Ministry of Science and Innovation (REPRO-TEMP project; AGL2013-41646-R) and IMPRESS (Marie Sklodowska-Curie Actions; Grant agreement no: 642893). M.C. Vilchez has a predoctoral grant from UPV PAID Programme (2011-S2-02-6521), M. Morini has a predoctoral grant from Generalitat Valenciana (Programa Grisolia). D.S. Penaranda was supported by MICINN and UPV (PTA2011-4948-1).Morini, M.; Peñaranda, D.; Vilchez Olivencia, MC.; Tveiten, H.; Lafont, A.; Dufour, S.; Pérez Igualada, LM.... (2017). The expression of nuclear and membrane estrogen receptors in the European eel throughout spermatogenesis. Comparative Biochemistry and Physiology Part A Molecular & Integrative Physiology. 203:91-99. doi:10.1016/j.cbpa.2016.08.020S919920

The mineralocorticoid receptor: insights into its molecular and (patho)physiological biology

The last decade has witnessed tremendous progress in the understanding of the mineralocorticoid receptor (MR), its molecular mechanism of action, and its implications for physiology and pathophysiology. After the initial cloning of MR, and identification of its gene structure and promoters, it now appears as a major actor in protein-protein interaction networks. The role of transcriptional coregulators and the determinants of mineralocorticoid selectivity have been elucidated. Targeted oncogenesis and transgenic mouse models have identified unexpected sites of MR expression and novel roles for MR in non-epithelial tissues. These experimental approaches have contributed to the generation of new cell lines for the characterization of aldosterone signaling pathways, and have also facilitated a better understanding of MR physiology in the heart, vasculature, brain and adipose tissues. This review describes the structure, molecular mechanism of action and transcriptional regulation mediated by MR, emphasizing the most recent developments at the cellular and molecular level. Finally, through insights obtained from mouse models and human disease, its role in physiology and pathophysiology will be reviewed. Future investigations of MR biology should lead to new therapeutic strategies, modulating cell-specific actions in the management of cardiovascular disease, neuroprotection, mineralocorticoid resistance, and metabolic disorders

Screening Estrogenic Activities of Chemicals or Mixtures In Vivo Using Transgenic (cyp19a1b-GFP) Zebrafish Embryos

The tg(cyp19a1b-GFP) transgenic zebrafish expresses GFP (green fluorescent protein) under the control of the cyp19a1b gene, encoding brain aromatase. This gene has two major characteristics: (i) it is only expressed in radial glial progenitors in the brain of fish and (ii) it is exquisitely sensitive to estrogens. Based on these properties, we demonstrate that natural or synthetic hormones (alone or in binary mixture), including androgens or progestagens, and industrial chemicals induce a concentration-dependent GFP expression in radial glial progenitors. As GFP expression can be quantified by in vivo imaging, this model presents a very powerful tool to screen and characterize compounds potentially acting as estrogen mimics either directly or after metabolization by the zebrafish embryo. This study also shows that radial glial cells that act as stem cells are direct targets for a large panel of endocrine disruptors, calling for more attention regarding the impact of environmental estrogens and/or certain pharmaceuticals on brain development. Altogether these data identify this in vivo bioassay as an interesting alternative to detect estrogen mimics in hazard and risk assessment perspective

Nuclear Progesterone Receptors Are Up-Regulated by Estrogens in Neurons and Radial Glial Progenitors in the Brain of Zebrafish

In rodents, there is increasing evidence that nuclear progesterone receptors are transiently expressed in many regions of the developing brain, notably outside the hypothalamus. This suggests that progesterone and/or its metabolites could be involved in functions not related to reproduction, particularly in neurodevelopment. In this context, the adult fish brain is of particular interest, as it exhibits constant growth and high neurogenic activity that is supported by radial glia progenitors. However, although synthesis of neuroprogestagens has been documented recently in the brain of zebrafish, information on the presence of progesterone receptors is very limited. In zebrafish, a single nuclear progesterone receptor (pgr) has been cloned and characterized. Here, we demonstrate that this pgr is widely distributed in all regions of the zebrafish brain. Interestingly, we show that Pgr is strongly expressed in radial glial cells and more weakly in neurons. Finally, we present evidence, based on quantitative PCR and immunohistochemistry, that nuclear progesterone receptor mRNA and proteins are upregulated by estrogens in the brain of adult zebrafish. These data document for the first time the finding that radial glial cells are preferential targets for peripheral progestagens and/or neuroprogestagens. Given the crucial roles of radial glial cells in adult neurogenesis, the potential effects of progestagens on their activity and the fate of daughter cells require thorough investigation

Age-Related Impairment of Ultrasonic Vocalization in Tau.P301L Mice: Possible Implication for Progressive Language Disorders

Tauopathies, including Alzheimer's Disease, are the most frequent neurodegenerative diseases in elderly people and cause various cognitive, behavioural and motor defects, but also progressive language disorders. For communication and social interactions, mice produce ultrasonic vocalization (USV) via expiratory airflow through the larynx. We examined USV of Tau.P301L mice, a mouse model for tauopathy expressing human mutant tau protein and developing cognitive, motor and upper airway defects.At age 4-5 months, Tau.P301L mice had normal USV, normal expiratory airflow and no brainstem tauopathy. At age 8-10 months, Tau.P301L mice presented impaired USV, reduced expiratory airflow and severe tauopathy in the periaqueductal gray, Kolliker-Fuse and retroambiguus nuclei. Tauopathy in these nuclei that control upper airway function and vocalization correlates well with the USV impairment of old Tau.P301L mice.In a mouse model for tauopathy, we report for the first time an age-related impairment of USV that correlates with tauopathy in midbrain and brainstem areas controlling vocalization. The vocalization disorder of old Tau.P301L mice could be, at least in part, reminiscent of language disorders of elderly suffering tauopathy

Adult Male Mice Emit Context-Specific Ultrasonic Vocalizations That Are Modulated by Prior Isolation or Group Rearing Environment

Social interactions in mice are frequently analysed in genetically modified strains in order to get insight of disorders affecting social interactions such as autism spectrum disorders. Different types of social interactions have been described, mostly between females and pups, and between adult males and females. However, we recently showed that social interactions between adult males could also encompass cognitive and motivational features. During social interactions, rodents emit ultrasonic vocalizations (USVs), but it remains unknown if call types are differently used depending of the context and if they are correlated with motivational state. Here, we recorded the calls of adult C57BL/6J male mice in various behavioral conditions, such as social interaction, novelty exploration and restraint stress. We introduced a modulator for the motivational state by comparing males maintained in isolation and males maintained in groups before the experiments. Male mice uttered USVs in all social and non-social situations, and even in a stressful restraint context. They nevertheless emitted the most important number of calls with the largest diversity of call types in social interactions, particularly when showing a high motivation for social contact. For mice maintained in social isolation, the number of calls recorded was positively correlated with the duration of social contacts, and most calls were uttered during contacts between the two mice. This correlation was not observed in mice maintained in groups. These results open the way for a deeper understanding and characterization of acoustic signals associated with social interactions. They can also help evaluating the role of motivational states in the emission of acoustic signals

Interactions Between Estrogen- and Ah-Receptor Signalling Pathways in Primary Culture of Salmon Hepatocytes Exposed to Nonylphenol and 3,3',4,4'-Tetrachlorobiphenyl (Congener 77)

BACKGROUND: The estrogenic and xenobiotic biotransformation gene expressions are receptor-mediated processes that are ligand structure-dependent interactions with estrogen-receptor (ER) and aryl hydrocarbon receptor (AhR), probably involving all subtypes and other co-factors. The anti-estrogenic activities of AhR agonists have been reported. In teleost fish, exposure to AhR agonists has been associated with reduced Vtg synthesis or impaired gonadal development in both in vivo- and in vitro studies. Inhibitory AhR and ER cross-talk have also been demonstrated in breast cancer cells, rodent uterus and mammary tumors. Previous studies have shown that AhR-agonists potentiate xenoestrogen-induced responses in fish in vivo system. Recently, several studies have shown that AhR-agonists directly activate ERα and induce estrogenic responses in mammalian in vitro systems. In this study, two separate experiments were performed to study the molecular interactions between ER and AhR signalling pathways using different concentration of PCB-77 (an AhR-agonist) and time factor, respectively. Firstly, primary Atlantic salmon hepatocytes were exposed to nonylphenol (NP: 5 μM – an ER agonist) singly or in combination with 0.001, 0.01 and 1 μM PCB-77 and sampled at 48 h post-exposure. Secondly, hepatocytes were exposed to NP (5 μM) or PCB-77 (1 μM) singly or in combination for 12, 24, 48 and 72 h. Samples were analyzed using a validated real-time PCR for genes in the ER pathway or known to be NP-responsive and AhR pathway or known to be PCB-77 responsive. RESULTS: Our data showed a reciprocal inhibitory interaction between NP and PCB-77. PCB-77 produced anti-NP-mediated effect by decreasing the mRNA expression of ER-responsive genes. NP produced anti-AhR mediated effect or as inhibitor of AhRα, AhRR, ARNT, CYP1A1 and UDPGT expression. A novel aspect of the present study is that low (0.001 μM) and medium (0.01 μM) PCB-77 concentrations increased ERα mRNA expression above control and NP exposed levels, and at 12 h post-exposure, PCB-77 exposure alone produced significant elevation of ERα, ERβ and Zr-protein expressions above control levels. CONCLUSION: The findings in the present study demonstrate a complex mode of ER-AhR interactions that were dependent on time of exposure and concentration of individual chemicals (NP and PCB-77). This complex mode of interaction is further supported by the effect of PCB-77 on ERα and ERβ (shown as increase in transcription) with no concurrent activation of Vtg (but Zr-protein) response. These complex interactions between two different classes of ligand-activated receptors provide novel mechanistic insights on signalling pathways. Therefore, the degree of simultaneous interactions between the ER and AhR gene transcripts demonstrated in this study supports the concept of cross-talk between these signalling pathways

Perturbation of lipids and glucose metabolism associated with previous 2,4-D exposure: a cross-sectional study of NHANES III data, 1988-1994

<p>Abstract</p> <p>Background</p> <p>Results from previous population studies showed that mortality rates from acute myocardial infarction and type-2 diabetes during the 1980s and 1990s in rural, agricultural counties of Minnesota, Montana, North and South Dakota, were higher in counties with a higher level of spring wheat farming than in counties with lower levels of this crop. Spring wheat, one of the major field crops in these four states, was treated for 85% or more of its acreage with chlorophenoxy herbicides. In the current study NHANES III data were reviewed for associations of 2,4-dichlorophenoxy acetic acid (2,4-D) exposure, one of the most frequently used chlorophenoxy herbicides, with risk factors that are linked to the pathogenesis of acute myocardial infarction and type-2 diabetes, such as dyslipidemia and impaired glucose metabolism.</p> <p>Methods</p> <p>To investigate the toxicity pattern of chlorophenoxy herbicides, effects of a previous 2,4-D exposure were assessed by comparing levels of lipids, glucose metabolism, and thyroid stimulating hormone in healthy adult NHANES III subjects with urinary 2,4-D above and below the level of detection, using linear regression analysis. The analyses were conducted for all available subjects and for two susceptible subpopulations characterized by high glycosylated hemoglobin (upper 50^thpercentile) and low thyroxine (lower 50^thpercentile).</p> <p>Results</p> <p>Presence of urinary 2,4-D was associated with a decrease of HDL levels: 8.6% in the unadjusted data (p-value = 0.006), 4.8% in the adjusted data (p-value = 0.08), and 9% in the adjusted data for the susceptible subpopulation with low thyroxine (p-value = 0.02). An effect modification of the inverse triglycerides-HDL relation was observed in association with 2,4-D. Among subjects with low HDL, urinary 2,4-D was associated with increased levels of triglycerides, insulin, C-peptide, and thyroid stimulating hormone, especially in the susceptible subpopulations. In contrast, subjects with high HDL did not experience adverse 2,4-D associated effects.</p> <p>Conclusions</p> <p>The results indicate that exposure to 2,4-D was associated with changes in biomarkers that, based on the published literature, have been linked to risk factors for acute myocardial infarction and type-2 diabetes.</p