comprehensive-assessment-germline-chemical-toxicity-using-nematode

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode <em>Caenorhabditis elegans</em>

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

University of California, Los Angeles

The stem-loop binding protein (SLBP) binds to the 3' end of histone mRNA and participates in 3'-processing of the newly synthesized transcripts, which protects them from degradation, and probably also promotes their translation. In proliferating cells, translation of SLBP mRNA begins at G1/S and the protein is degraded following DNA replication. These post-transcriptional mechanisms closely couple SLBP expression to S-phase of the cell cycle, and play a key role in restricting synthesis of replication-dependent histones to S-phase. In contrast to somatic cells, replication-dependent histone mRNAs accumulate and are translated independently of DNA replication in oocytes and early embryos. We report here that SLBP expression and activity also differ in mouse oocytes and early embryos compared with somatic cells. SLBP is present in oocytes that are arrested at prophase of G2/M, where it is concentrated in the nucleus. Upon entry into M-phase of meiotic maturation, SLBP begins to accumulate rapidly, reaching a very high level in mature oocytes arrested at metaphase II. Following fertilization, SLBP remains abundant in the nucleus and the cytoplasm throughout the first cell cycle, including both G1 and G2 phases. It declines during the second and third cell cycles, reaching a relatively low level by the late 4-cell stage. SLBP can bind the histone mRNA-stem-loop at all stages of the cell cycle in oocytes and early embryos, and it is the only stem-loop binding activity detectable in these cells. We also report that SLBP becomes phosphorylated rapidly following entry into M-phase of meiotic maturation through a mechanism that is sensitive to roscovitine, an inhibitor of cyclin-dependent kinases. SLBP is rapidly dephosphorylated following fertilization or parthenogenetic activation, and becomes newly phosphorylated at M-phase of mitosis. Phosphorylation does not affect its stem-loop binding activity. These results establish that, in contrast to Xenopus, mouse oocytes and embryos contain a single SLBP. Expression of SLBP is uncoupled from S-phase in oocytes and early embryos, which indicates that the mechanisms that impose cell-cycle-regulated expression of SLBP in somatic cells do not operate in oocytes or during the first embryonic cell cycle. This distinctive pattern of SLBP expression may be required for accumulation of histone proteins required for sperm chromatin remodelling and assembly of newly synthesized embryonic DNA into chromatin.

Stem-loop binding protein accumulates during oocyte maturation and is not cell-cycle-regulated in the early mouse embryo.

Lava fountains are spectacular continuous gas jets, propelling lava fragments to heights of several hundred metres, which occasionally occur during eruptions of low-viscosity magmas. Whether they are generated by the effervescent disruption of fast-rising bubbly melt or by the separate ascent of a bubble foam layer accumulated at depth still remains a matter of debate. No field measurement has yet allowed firm discrimination between these two models. A key insight into the origin of lava fountains may be gained by measuring the chemical composition of the driving gas phase. This composition should differ markedly depending on whether the magma degassing occurs before or during eruption. Here we report the analysis of magmatic gas during a powerful (250-600 m high) lava fountain, measured with Fourier transform infrared spectroscopy on Mount Etna, Sicily. The abundances of volcanic gas species, determined from absorption spectra of lava radiation, reveal a fountain gas having higher CO2/S and S/Cl ratios than other etnean emissions, and which cannot derive from syn-eruptive bulk degassing of Etna basalt. Instead, its composition suggests violent emptying of a gas bubble layer previously accumulated at about 1.5 km depth below the erupting crater.

Spectroscopic evidence for a lava fountain driven by previously accumulated magmatic gas.

Although messenger RNAs encoding the histone proteins are among the most abundant in mammalian oocytes, the mechanism regulating their translation has not been identified. The stem-loop binding protein (SLBP) binds to a highly conserved sequence in the 3'-untranslated region (utr) of the non-polyadenylated histone mRNAs in somatic cells and mediates their stabilization and translation. We previously showed that SLBP, which is expressed only during S-phase of proliferating cells, is expressed in growing oocytes at G2 of the cell cycle and accumulates substantially during meiotic maturation. We report here that elevating the amount of SLBP in immature (G2) oocytes is sufficient to increase translation of a reporter mRNA bearing the histone 3'-utr and endogenous histone synthesis and that this effect is not mediated through increased stability of the encoding mRNAs. We further report that translation of the reporter mRNA increases dramatically during meiotic maturation coincident with the accumulation of SLBP. Conversely, when SLBP accumulation during maturation is prevented using RNA interference, both translation of the reporter mRNA and synthesis of endogenous histones are significantly reduced. This effect is not mediated by a loss of the encoding mRNAs. Moreover, following fertilization, SLBP-depleted oocytes also show a significant decrease in pronuclear size and in the amount of acetylated histone detectable on the chromatin. These results demonstrate that histone synthesis in immature and maturing oocytes is governed by a translational control mechanism that is directly regulated by changes in the amount of SLBP.

The stem-loop binding protein regulates translation of histone mRNA during mammalian oogenesis.

In metazoans, translational regulation of a set of maternal mRNAs directs oocyte maturation and early embryogenesis. These transcripts are often kept dormant until their products are spatially and temporally required in development. The interaction between general translation factors (i.e. eIF4E) and their specific interactors influences translation initiation. A search of the protein database for a mouse homologue of the Drosophila Cup protein, a translational repressor during female germ-line development, identified the product of the Clast4 gene. In this report, we show that Clast4 mRNA and protein are highly expressed within the cytoplasm of growing oocytes. The Clast4 protein is stable during this developmental window and post-translationally modified by phosphorylation upon oocyte meiotic maturation. Additionally, we show that Clast4 and eIF4E directly interact by means of a canonical and functional eIF4E-binding motif. Our results suggest that Clast4, similar to Drosophila Cup, may act at the translational level during murine female germ-line development.

Clast4, the murine homologue of human eIF4E-Transporter, is highly expressed in developing oocytes and post-translationally modified at meiotic maturation.

Normal patterning of the developing limb requires a tight restriction of Sonic hedgehog (Shh) mRNA to the posterior margin of the limb bud. While several positive and negative regulatory factors have been identified which serve to position the Shh expression domain in the distal posterior limb, these factors cannot in themselves explain the tight restriction of Shh to the posterior margin, nor can they explain the similarly tight restriction of Shh to the anterior margin when the regulatory factors are disrupted or misexpressed. We suggest that the transcription factors Tbx2 and Tbx3 are excellent candidates for positively-acting factors responsible for limiting Shh expression to the margins of the limb bud. These closely related factors are indeed expressed at the anterior and posterior limb margins over a wide range of limb bud stages. Moreover, previous reports indicate that in addition, misexpression of Tbx2 beyond the limb margin is sufficient to anteriorly expand Shh, and conversely, antagonizing Tbx2 function leads to loss of Shh. In contrast to this idea, previous models have placed Tbx2 expression downstream of Shh and Bone Morphogenetic Protein (BMP) signaling. We find, however, that Tbx2 expression is neither affected by blocking Shh signaling with cyclopamine nor by genetic removal of several BMP activities in the limb bud. To understand the true source of the positional information responsible for limiting Tbx2, Tbx3 and Shh expression to the marginal mesenchyme of the limb bud, we undertook a series of grafting and extirpation experiments, which led to the identification of the dorsal-ventral (DV) border ectoderm exclusive of the apical ectodermal ridge (AER) as a new signaling center in the limb bud. We find that maintenance of Tbx2 expression in the limb mesoderm requires proximity to the non-AER D-V border. Using chick-quail graft chimeras, we find that a graft of the non-AER D-V border ectoderm to a location on the surface of the middle of the limb bud is sufficient to induce ectopic expression of Tbx2 in underlying mesoderm. These data demonstrate that the non-AER D-V border ectoderm is necessary and sufficient for Tbx2 expression at the anterior and posterior limb margins. Similarly, we find that a graft of the non-AER D-V border can expand the domain of Shh anteriorly when grafted just anterior to the ZPA. It is notable that Tbx2 expression does not extend distally to the mesoderm underlying the AER. Moreover, we find that grafts of the AER to more proximal locations result in downregulation of Tbx2 expression, suggesting that the AER produces a negatively-acting signal opposing the activity of the non-AER DV border ectoderm. Indeed, implantation of beads soaked in fibroblast growth factor 8 (Fgf8), expressed in the AER, downregulates Tbx2 expression. The data presented here identify the non-AER border of dorsal-ventral ectoderm as a new signaling center in limb development that localizes the ZPA to the limb margin. This finding explains the tight restriction of Shh expression to the posterior margin throughout limb outgrowth as well as the tight restriction of Shh expression to the anterior margin in many mutants exhibiting preaxial polydactyly.

Characterization of a novel ectodermal signaling center regulating Tbx2 and Shh in the vertebrate limb.

Strombolian-type eruptive activity, common at many volcanoes, consists of regular explosions driven by the bursting of gas slugs that rise faster than surrounding magma. Explosion quakes associated with this activity are usually localized at shallow depth; however, where and how slugs actually form remain poorly constrained. We used spectroscopic measurements performed during both quiescent degassing and explosions on Stromboli volcano (Italy) to demonstrate that gas slugs originate from as deep as the volcano-crust interface (approximately 3 kilometers), where both structural discontinuities and differential bubble-rise speed can promote slug coalescence. The observed decoupling between deep slug genesis and shallow (approximately 250-meter) explosion quakes may be a common feature of strombolian activity, determined by the geometry of plumbing systems.

Magmatic gas composition reveals the source depth of slug-driven strombolian explosive activity.

While the various internal organs of vertebrates display many obvious left-right asymmetries in their location and/or morphology, external features exhibit a high degree of bilateral symmetry. How this external bilateral symmetry is established during development is largely unknown. In this review, we explore several mechanisms, in place during development, that regulate the final size of the limb. These mechanisms rely on the presence of positive signaling feedback loops during limb bud growth. Through the activity of these signaling loops and their eventual breakdown when the limb bud has reached a certain size, bilateral symmetry can be achieved.

Achieving bilateral symmetry during vertebrate limb development.

Toxicity reference values (TRVs) are essential in models used in the prediction of the potential for adverse impacts of environmental contaminants to avian and mammalian wildlife; however, issues in their derivation and application continue to result in inconsistent hazard and risk assessments that present a challenge to site managers and regulatory agencies. Currently, the available science does not support several common practices in TRV derivation and application. Key issues include inappropriate use of hazard quotients and the inability to define the probability of adverse outcomes. Other common problems include the continued use of no-observed- and lowest-observed-adverse-effect levels (NOAELs and LOAELs), the use of allometric scaling for interspecific extrapolation of chronic TRVs, inappropriate extrapolation across classes when data are limited, and extrapolation of chronic TRVs from acute data without scientific basis. Recommendations for future TRV derivation focus on using all available qualified toxicity data to include measures of variation associated with those data. This can be achieved by deriving effective dose (EDx)-based TRVs where x refers to an acceptable (as defined in a problem formulation) reduction in endpoint performance relative to the negative control instead of relying on NOAELs and LOAELs. Recommendations for moving past the use of hazard quotients and dealing with the uncertainty in the TRVs are also provided.

Recommendations for the development and application of wildlife toxicity reference values.

Developmentally regulated translation plays a key role in controlling gene expression during oogenesis. In particular, numerous mRNA species are translationally repressed in growing oocytes and become translationally activated during meiotic maturation. While many studies have focused on a U-rich sequence, termed the cytoplasmic polyadenylation element (CPE), located in the 3'-untranslated region (UTR) and the CPE-binding protein (CPEB) 1, multiple mechanisms likely contribute to translational control in oocytes. The stem-loop-binding protein (SLBP) is expressed in growing oocytes, where it is required for the accumulation of nonpolyadenylated histone mRNAs, and then accumulates substantially during meiotic maturation. We report that, in immature oocytes, Slbp mRNA carries a short poly(A) tail, and is weakly translated, and that a CPE-like sequence in the 3'-UTR is required to maintain this low activity. During maturation, Slbp mRNA becomes polyadenylated and translationally activated. Unexpectedly, proteasomal activity is required both to initiate and to sustain translational activation. This proteasomal activity is not required for the polyadenylation of Slbp mRNA during early maturation; however, it is required for a subsequent deadenylation of the mRNA that occurs during late maturation. Moreover, although CPEB1 is degraded during maturation, inhibiting its degradation by blocking mitogen-activated protein kinase 1/3 activity does not prevent the accumulation of SLBP, indicating that CPEB1 is not the protein whose degradation is required for translational activation of Slbp mRNA. These results identify a new role for proteasomal activity in initiating and sustaining translational activation during meiotic maturation.

Proteasomal activity is required to initiate and to sustain translational activation of messenger RNA encoding the stem-loop-binding protein during meiotic maturation in mice.

Bisphenol A (BPA) is a highly prevalent constituent of plastics that has been associated with diabetes, cardiovascular disease, and an increased risk of miscarriages in humans. In mice, BPA exposure disrupts the process of meiosis; however, analysis of the affected molecular pathways is lagging and has been particularly challenging. Here we show that exposure of the nematode Caenorhabditis elegans to BPA, at internal concentrations consistent with mammalian models, causes increased sterility and embryonic lethality. BPA exposure results in impaired chromosome synapsis and disruption of meiotic double-strand break repair (DSBR) progression. BPA carries an anti-estrogenic activity in the germline and results in germline-specific down-regulation of DSBR genes, thereby impairing maintenance of genomic integrity during meiosis. C. elegans therefore constitutes a model of remarkable relevance to mammals with which to assess how our chemical landscape affects germ cells and meiosis.

Bisphenol A impairs the double-strand break repair machinery in the germline and causes chromosome abnormalities.

Mechanistic insights into the action of Bisphenol A on the germline using C. elegans.

The acquisition of essential metals, such as the metal cofactors (molybdenum (Mo) and iron (Fe)) of the nitrogenase, the enzyme responsible for the reduction of dinitrogen (N(2)) to ammonium, is critical to N(2) fixing bacteria in soil. The release of metal nanoparticles (MNPs) to the environment could be detrimental to N(2) fixing bacteria by introducing a new source of toxic metals and by interfering with the acquisition of essential metals such as Mo. Since Mo has been reported to limit nonsymbiotic N(2) fixation in many ecosystems from tropical to cold temperate, this question is particularly acute in the context of Mo limitation. Using a combination of microbiology and analytical chemistry techniques, we have evaluated the effect of titanium (Ti) and tungsten (W) oxide nanoparticles on the diazotrophic growth and metals acquisition in pure culture of the ubiquitous N(2) fixing bacterium Azotobacter vinelandii under Mo replete and Mo limiting conditions. We report that under our conditions (≤10 mg·L(-1)) TiO(2) NPs have no effects on the diazotrophic growth of A. vinelandii while WO(3) NPs are highly detrimental to the growth especially under Mo limiting conditions. Our results show that the toxicity of WO(3) NPs to A. vinelandii is due to an interference with the catechol-metalophores assisted uptake of Mo.

Effects of tungsten and titanium oxide nanoparticles on the diazotrophic growth and metals acquisition by Azotobacter vinelandii under molybdenum limiting condition.

Despite the developmental impact of chromosome segregation errors, we lack the tools to assess environmental effects on the integrity of the germline in animals.

A C. elegans screening platform for the rapid assessment of chemical disruption of germline function.

Hazard quotients based on a point-estimate comparison of exposure to a toxicity reference value (TRV) are commonly used to characterize risks for wildlife. Quotients may be appropriate for screening-level assessments but should be avoided in detailed assessments, because they provide little insight regarding the likely magnitude of effects and associated uncertainty. To better characterize risks to wildlife and support more informed decision making, practitioners should make full use of available dose-response data. First, relevant studies should be compiled and data extracted. Data extractions are not trivial--practitioners must evaluate the potential use of each study or its components, extract numerous variables, and in some cases, calculate variables of interest. Second, plots should be used to thoroughly explore the data, especially in the range of doses relevant to a given risk assessment. Plots should be used to understand variation in dose-response among studies, species, and other factors. Finally, quantitative dose-response models should be considered if they are likely to provide an improved basis for decision making. The most common dose-response models are simple models for data from a particular study for a particular species, using generalized linear models or other models appropriate for a given endpoint. Although simple models work well in some instances, they generally do not reflect the full breadth of information in a dose-response data set, because they apply only for particular studies, species, and endpoints. More advanced models are available that explicitly account for variation among studies and species, or that standardize multiple endpoints to a common response variable. Application of these models may be useful in some cases when data are abundant, but there are challenges to implementing and interpreting such models when data are sparse.

Article	Year
Stem-loop binding protein accumulates during oocyte maturation and is not cell-cycle-regulated in the early mouse embryo. Journal of cell science\| PubMed ID: 12415002	2002
Spectroscopic evidence for a lava fountain driven by previously accumulated magmatic gas. Nature\| PubMed ID: 15674289	2005
The stem-loop binding protein regulates translation of histone mRNA during mammalian oogenesis. Developmental biology\| PubMed ID: 16125165	2005
Clast4, the murine homologue of human eIF4E-Transporter, is highly expressed in developing oocytes and post-translationally modified at meiotic maturation. Gene\| PubMed ID: 16343815	2006
Characterization of a novel ectodermal signaling center regulating Tbx2 and Shh in the vertebrate limb. Developmental biology\| PubMed ID: 17300775	2007
Magmatic gas composition reveals the source depth of slug-driven strombolian explosive activity. Science (New York, N.Y.)\| PubMed ID: 17626881	2007
Achieving bilateral symmetry during vertebrate limb development. Seminars in cell & developmental biology\| PubMed ID: 19027866	2009
Recommendations for the development and application of wildlife toxicity reference values. Integrated environmental assessment and management\| PubMed ID: 19558201	2010
Proteasomal activity is required to initiate and to sustain translational activation of messenger RNA encoding the stem-loop-binding protein during meiotic maturation in mice. Biology of reproduction\| PubMed ID: 19759367	2010
Bisphenol A impairs the double-strand break repair machinery in the germline and causes chromosome abnormalities. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 21059909	2010
Mechanistic insights into the action of Bisphenol A on the germline using C. elegans. Cell cycle (Georgetown, Tex.)\| PubMed ID: 21228622	2011
Effects of tungsten and titanium oxide nanoparticles on the diazotrophic growth and metals acquisition by Azotobacter vinelandii under molybdenum limiting condition. Environmental science & technology\| PubMed ID: 23339336	2013
A C. elegans screening platform for the rapid assessment of chemical disruption of germline function. Environmental health perspectives\| PubMed ID: 23603051	2013
Using sparse dose-response data for wildlife risk assessment. Integrated environmental assessment and management\| PubMed ID: 23913468	2014

Patrick Allard

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