Chemical Contaminants from Plastics in the Animal Environment.

Accidental exposure of our mice to bisphenol A (BPA) from damaged polycarbonate cages 20 y ago provided some of the first evidence of the harmful effects of exposure to this common chemical. Recently we found that housing mice in damaged polysulfone cages resulted in similar harmful effects due to exposure to bisphenol S (BPS). This problem was unexpected for 2 reasons. First, polysulfone is a far more chemically resistant polymer than polycarbonate. Second, BPS is not a component in the manufacture of polysulfone. We report here our efforts to verify the source of the BPS and eliminate the exposure. Our analysis of new polysulfone caging materials confirmed that BPS is a breakdown product of damaged polysulfone plastic. Furthermore, we found that BPS can cross-contaminate new or undamaged cages in facilities that process damaged caging materials. Neither the use of disposable cages nor replacement of caging materials used solely for our colony was sufficient to eliminate exposure effects. Only the replacement of all cages and water bottles in the facility corrected the problem and allowed us to resume our studies. Taken together, our previous and current findings underscore the concern that chemicals from plastics are harmful environmental contaminants for both humans and animals. Furthermore, our results provide strong evidence that the presence of damaged plastic in a facility may be sufficient to affect research results and, by exten- sion, animal health.

Replacement Bisphenols Adversely Affect Mouse Gametogenesis with Consequences for Subsequent Generations.

20 years ago, accidental bisphenol A (BPA) exposure caused a sudden increase in chromosomally abnormal eggs from our control mice [1]. Subsequent rodent studies demonstrated developmental effects of exposure with repercussions on adult health and fertility (e.g., [2-9]; reviewed in [10-17]). Studies in monkeys, humans, fish, and worms suggest BPA effects extend across species (e.g., [18-30]; reviewed in [31-33]). Widespread use has resulted in ubiquitous environmental contamination and human BPA exposure. Consumer concern resulted in "BPA-free" products produced using structurally similar bisphenols that are now detectable environmental and human contaminants (e.g., [34-41]). We report here studies initiated by meiotic changes mirroring our previous BPA experience and implicating exposure to BPS (a common BPA replacement) from damaged polysulfone cages. Like with BPA [1, 2, 5], our data show that exposure to common replacement bisphenols induces germline effects in both sexes that may affect multiple generations. These findings add to growing evidence of the biological risks posed by this class of chemicals. Rapid production of structural variants of BPA and other EDCs circumvents efforts to eliminate dangerous chemicals, exacerbates the regulatory burden of safety assessment, and increases environmental contamination. Our experience suggests that these environmental contaminants pose a risk not only to reproductive health but also to the integrity of the research environment. EDCs, like endogenous hormones, can affect diverse processes. The sensitivity of the germline allows us to detect effects that, although not immediately apparent in other systems, may induce variability that undermines experimental reproducibility and impedes scientific advancement.

An old culprit but a new story: bisphenol A and "NextGen" bisphenols.

The concept that developmental events shape adult health and disease was sparked by the recognition of a link between maternal undernutrition and coronary disease in adults. From that beginning, a new field-the developmental origins of health and disease-emerged, and attention has focused on the effects of a wide array of developmental perturbations. Exposure to endocrine-disrupting chemicals has been of particular interest, and a ubiquitous environmental contaminant bisphenol A (BPA) has become the endocrine-disrupting chemical poster child. Bisphenol A has been the subject of intense investigation for nearly two decades, and exposure effects have been described in hundreds of experimental, epidemiological, and clinical studies. From the standpoint of reproductive health, the findings are particularly important, as they suggest that the ovary, testis, and reproductive tract in both sexes are targets of BPA action. The findings and the media and regulatory attention garnered by them have generated increasing public concern and resulted in legislative bans on BPA in some countries. The subsequent introduction of BPA-free products, although a masterful marketing strategy, is in reality only the beginning of a new and complex chapter of the BPA story. In this review we attempt to summarize what we have learned about the reproductive effects of BPA, present the reasons why studying the effects of this chemical in humans is no longer sufficient, and outline the challenges that the growing array of next generation bisphenols represents to clinicians, researchers, federal agencies, and the general public.

The Drosophila prage Gene, Required for Maternal Transcript Destabilization in Embryos, Encodes a Predicted RNA Exonuclease.

Egg activation, the transition of mature oocytes into developing embryos, is critical for the initiation of embryogenesis. This process is characterized by resumption of meiosis, changes in the egg's coverings and by alterations in the transcriptome and proteome of the egg; all of these occur in the absence of new transcription. Activation of the egg is prompted by ionic changes in the cytoplasm (usually a rise in cytosolic calcium levels) that are triggered by fertilization in some animals and by mechanosensitive cues in others. The egg's transcriptome is dramatically altered during the process, including by the removal of many maternal mRNAs that are not needed for embryogenesis. However, the mechanisms and regulators of this selective RNA degradation are not yet fully known. Forward genetic approaches in Drosophila have identified maternal-effect genes whose mutations prevent the transcriptome changes. One of these genes, prage (prg), was identified by Tadros et al. in a screen for mutants that fail to destabilize maternal transcripts. We identified the molecular nature of the prg gene through a combination of deficiency mapping, complementation analysis, and DNA sequencing of both extant prg mutant alleles. We find that prg encodes a ubiquitously expressed predicted exonuclease, consistent with its role in maternal mRNA destabilization during egg activation.

Calcium waves occur as Drosophila oocytes activate.

Egg activation is the process by which a mature oocyte becomes capable of supporting embryo development. In vertebrates and echinoderms, activation is induced by fertilization. Molecules introduced into the egg by the sperm trigger progressive release of intracellular calcium stores in the oocyte. Calcium wave(s) spread through the oocyte and induce completion of meiosis, new macromolecular synthesis, and modification of the vitelline envelope to prevent polyspermy. However, arthropod eggs activate without fertilization: in the insects examined, eggs activate as they move through the female's reproductive tract. Here, we show that a calcium wave is, nevertheless, characteristic of egg activation in Drosophila. This calcium rise requires influx of calcium from the external environment and is induced as the egg is ovulated. Pressure on the oocyte (or swelling by the oocyte) can induce a calcium rise through the action of mechanosensitive ion channels. Visualization of calcium fluxes in activating eggs in oviducts shows a wave of increased calcium initiating at one or both oocyte poles and spreading across the oocyte. In vitro, waves also spread inward from oocyte pole(s). Wave propagation requires the IP3 system. Thus, although a fertilizing sperm is not necessary for egg activation in Drosophila, the characteristic of increased cytosolic calcium levels spreading through the egg is conserved. Because many downstream signaling effectors are conserved in Drosophila, this system offers the unique perspective of egg activation events due solely to maternal components.

Cytoplasmic polyadenylation is a major mRNA regulator during oogenesis and egg activation in Drosophila.

The GLD-2 class of poly(A) polymerases regulate the timing of translation of stored transcripts by elongating the poly(A) tails of target mRNAs in the cytoplasm. WISPY is a GLD-2 enzyme that acts in the Drosophila female germline and is required for the completion of the egg-to-embryo transition. Though a handful of WISPY target mRNAs have been identified during both oogenesis and early embryogenesis, it was unknown whether WISP simply regulated a small pool of patterning or cell cycle genes, or whether, instead, cytoplasmic polyadenylation was widespread during this developmental transition. To identify the full range of WISPY targets, we carried out microarray analysis to look for maternal mRNAs whose poly(A) tails fail to elongate in the absence of WISP function. We examined the polyadenylated portion of the maternal transcriptome in both stage 14 (mature) oocytes and in early embryos that had completed egg activation. Our analysis shows that the poly(A) tails of thousands of maternal mRNAs fail to elongate in wisp-deficient oocytes and embryos. Furthermore, we have identified specific classes of genes that are highly regulated in this manner at each stage. Our study shows that cytoplasmic polyadenylation is a major regulatory mechanism during oocyte maturation and egg activation.

Calcium and egg activation in Drosophila.

In many animals, a rise in intracellular calcium levels is the trigger for egg activation, the process by which an arrested mature oocyte transitions to prepare for embryogenesis. In nearly all animals studied to date, this calcium rise, and thus egg activation, is triggered by the fertilizing sperm. However in the insects that have been examined, fertilization is not necessary to activate their oocytes. Rather, these insects' eggs activate as they transit through the female's reproductive tract, regardless of male contribution. Recent studies in Drosophila have shown that egg activation nevertheless requires calcium and that the downstream events and molecules of egg activation are also conserved, despite the difference in initial trigger. Genetic studies have uncovered essential roles for the calcium-dependent enzyme calcineurin and its regulator calcipressin, and have hinted at roles for calmodulin, in Drosophila egg activation. Physiological and in vitro studies have led to a model in which mechanical forces that impact the Drosophila oocyte as it moves through the reproductive tract triggers the influx of calcium from the external environment, thereby initiating egg activation. Future research will aim to test this model, as well as to determine the spatiotemporal dynamics of cytoplasmic calcium flux and mode of signal propagation in this unique system.

Pancreas-specific deletion of Prox1 affects development and disrupts homeostasis of the exocrine pancreas.

BACKGROUND & AIMS: The exocrine portion of the pancreas functions in digestion and preserves pancreatic homeostasis. Learning how this tissue forms during embryogenesis could improve our understanding of human pancreatic diseases. Expression of the homeobox gene Prox1 in the exocrine pancreas changes throughout development in mice. We investigated the role of Prox1 in development of the exocrine pancreas in mice. METHODS: Mice with pancreas-specific deletion of Prox1 (Prox1(ΔPanc)) were generated and their pancreatic tissues were analyzed using immunohistochemistry, transmission electron microscopy, histologic techniques, quantitative real-time polymerase chain reaction, immunoblotting, and morphometric analysis. RESULTS: Loss of Prox1 from the pancreas led to multiple exocrine alterations, most notably premature acinar cell differentiation, increased ductal cell proliferation, altered duct morphogenesis, and imbalanced expression of claudin proteins. Prox1(ΔPanc) mice also had some minor alterations in islet cells, but beta-cell development was not affected. The exocrine congenital defects of Prox1(ΔPanc) pancreata appeared to initiate a gradual process of deterioration that resulted in extensive loss of acinar cells, lipomatosis, and damage to ductal tissue in adult mice. CONCLUSIONS: Pancreas-specific deletion of Prox1 causes premature differentiation of acinar cells and poor elongation of epithelial branches; these defects indicate that Prox1 controls the expansion of tip progenitors in the early developing pancreas. During later stages of embryogenesis, Prox1 appears to regulate duct cell proliferation and morphogenesis. These findings identify Prox1 as an important regulator of pancreatic exocrine development.

The poly(A) polymerase GLD2 is required for spermatogenesis in Drosophila melanogaster.

The DNA of a developing sperm is normally inaccessible for transcription for part of spermatogenesis in many animals. In Drosophila melanogaster, many transcripts needed for late spermatid differentiation are synthesized in pre-meiotic spermatocytes, but are not translated until later stages. Thus, post-transcriptional control mechanisms are required to decouple transcription and translation during spermatogenesis. In the female germline, developing germ cells accomplish similar decoupling through poly(A) tail alterations to ensure that dormant transcripts are not prematurely translated: a transcript with a short poly(A) tail will remain untranslated, whereas elongating the poly(A) tail permits protein production. In Drosophila, the ovary-expressed cytoplasmic poly(A) polymerase WISPY is responsible for stage-specific poly(A) tail extension in the female germline. Here, we examine the possibility that a recently derived testis-expressed WISPY paralog, GLD2, plays a similar role in the Drosophila male germline. We show that knockdown of Gld2 transcripts causes male sterility, as GLD2-deficient males do not produce mature sperm. Spermatogenesis up to and including meiosis appears normal in the absence of GLD2, but post-meiotic spermatid development rapidly becomes abnormal. Nuclear bundling and F-actin assembly are defective in GLD2 knockdown testes and nuclei fail to undergo chromatin reorganization in elongated spermatids. GLD2 also affects the incorporation of protamines and the stability of dynamin and transition protein transcripts. Our results indicate that GLD2 is an important regulator of late spermatogenesis and is the first example of a Gld-2 family member that plays a significant role specifically in male gametogenesis.

Two GDP-mannose transporters contribute to hyphal form and cell wall integrity in Aspergillus nidulans.

In order to identify novel genes affecting cell wall integrity, we have generated mutant strains of the filamentous fungus Aspergillus nidulans that show hypersensitivity to the chitin-binding agent Calcofluor White (CFW). Affected loci are designated cal loci. The phenotype of one of these alleles, calI11, also includes shortened hyphal compartments and increased density of branching in the absence of CFW, as well as reduced staining of cell walls by the lectin FITC-Concanavalin A (ConA), which has strong binding affinity for mannosyl residues. We have identified two A. nidulans genes (AN8848.3 and AN9298.3, designated gmtA and gmtB, respectively) that complement all aspects of the phenotype. Both genes show strong sequence similarity to GDP-mannose transporters (GMTs) of Saccharomyces and other yeasts. Sequencing of gmtA from the calI11 mutant strain reveals a G to C mutation at position 943, resulting in a predicted alanine to proline substitution at amino acid position 315 within a region that is highly conserved among other fungi. No mutations were observed in the mutant strain's allele of gmtB. Meiotic mapping demonstrated a recombination frequency of under 1 % between the calI locus and the phenA locus (located approximately 9.5 kb from AN8848.3), confirming that gmtA and calI are identical. A GmtA-GFP chimera exhibits a punctate distribution pattern, consistent with that shown by putative Golgi markers in A. nidulans. However, this distribution did not overlap with that of the putative Golgi equivalent marker CopA-monomeric red fluorescent protein (mRFP), which may indicate that the physically separated Golgi-equivalent organelles of A. nidulans represent physiologically distinct counterparts of the stacked cisternae of plants and animals. These findings demonstrate that gmtA and gmtB play roles in cell wall metabolism in A. nidulans similar to those previously reported for GMTs in yeasts.