Hydroxylated polychlorinated biphenyls may affect the thyroid hormone-induced brain development during metamorphosis of Xenopus laevis by disturbing the expression of matrix metalloproteinases.

BACKGROUND: Thyroid hormones are primarily responsible for the brain development in perinatal mammals. However, this process can be inhibited by external factors such as environmental chemicals. Perinatal mammals are viviparous, which makes direct fetal examination difficult. METHODS: We used metamorphic amphibians, which exhibit many similarities to perinatal mammals, as an experimental system. Therefore, using metamorphic amphibians, we characterized the gene expression of matrix metalloproteinases, which play an important role in brain development. RESULTS: The expression of many matrix metalloproteinases (mmps) was characteristically induced during metamorphosis. We also found that the expression of many mmps was induced by T3 and markedly inhibited by hydroxylated polychlorinated biphenyls (PCBs). CONCLUSION: Overall, our findings suggest that hydroxylated PCBs disrupt normal brain development by disturbing the gene expression of mmps.

(-)-Epicatechin gallate ameliorates cyprodinil-induced cardiac developmental defects through inhibiting aryl hydrocarbon receptor in zebrafish.

BACKGROUND: Cyprodinil is a widely used fungicide with broad-spectrum activity, but it has been associated with cardiac abnormalities. (-)-Epicatechin gallate (ECG), a natural polyphenolic compound, has been shown to possess protective properties in cardiac development. METHODS: In this study, we investigated whether ECG could mitigate cyprodinil-induced heart defects using zebrafish embryos as a model. Zebrafish embryos were exposed to cyprodinil with or without ECG. RESULTS: Our results demonstrated that ECG significantly improved the survival rate, embryo movement, and hatching delay induced by cyprodinil. Furthermore, ECG effectively ameliorated cyprodinil-induced cardiac developmental toxicity, including pericardial anomaly and impairment of cardiac function. Mechanistically, ECG attenuated the cyprodinil-induced alterations in mRNA expression related to cardiac development, such as amhc, vmhc, tbx5, and gata4, as well as calcium ion channels, such as ncx1h, atp2a2a, and cdh2. Additionally, ECG was found to inhibit the activity of the aryl hydrocarbon receptor (AhR) signaling pathways induced by cyprodinil. CONCLUSIONS: In conclusion, our findings provide evidence for the protective effects of ECG against cyprodinil-induced cardiac developmental toxicity, mediated through the inhibition of AhR activity. These findings contribute to a better understanding of the regulatory mechanisms and safe utilization of pesticide, such as cyprodinil.

Expression profile of viability and stress response genes as a result of resveratrol supplementation in vitrified and in vitro produced cattle embryos.

BACKGROUND: Resveratrol, a potent antioxidant, is known to induce the up-regulation of the internal antioxidant system. Therefore, it holds promise as a method to mitigate cryopreservation-induced injuries in bovine oocytes and embryos. This study aimed to (i) assess the enhancement in the quality of in vitro produced bovine embryos following resveratrol supplementation and (ii) monitor changes in the expression of genes associated with oxidative stress (GPX4, SOD, CPT2, NFE2L2), mitochondrial function (ATP5ME), endoplasmic reticulum function (ATF6), and embryo quality (OCT4, DNMT1, CASP3, ELOVL5). METHODS AND RESULTS: Three groups of in vitro bovine embryos were cultured with varying concentrations of resveratrol (0.01, 0.001, and 0.0001 µM), with a fourth group serving as a control. Following the vitrification process, embryos were categorized as either good or poor quality. Blastocysts were then preserved at - 80 °C for RNA isolation, followed by qRT-PCR analysis of selected genes. The low concentrations of resveratrol (0.001 µM, P < 0.05 and 0.0001 µM, P < 0.01) significantly improved the blastocyst rate compared to the control group. Moreover, the proportion of good quality vitrified embryos increased significantly (P < 0.05) in the groups treated with 0.001 and 0.0001 µM resveratrol compared to the control group. Analysis of gene expression showed a significant increase in OCT4 and DNMT1 transcripts in both good and poor-quality embryos treated with resveratrol compared to untreated embryos. Additionally, CASP3 expression was decreased in treated good embryos compared to control embryos. Furthermore, ELOVL5 and ATF6 transcripts were down-regulated in treated good embryos compared to the control group. Regarding antioxidant-related genes, GPX4, SOD, and CPT2 transcripts increased in the treated embryos, while NFE2L2 mRNA decreased in treated good embryos compared to the control group. CONCLUSIONS: Resveratrol supplementation at low concentrations effectively mitigated oxidative stress and enhanced the cryotolerance of embryos by modulating the expression of genes involved in oxidative stress response.

Effect of low ethanol concentration in maturation medium on developmental ability, mitochondria, and gene expression profile in mouse oocytes.

Ethanol affects pre-conceptional oocyte quality in women. In this study, we examined the effect of low ethanol concentrations on mouse oocytes. Oocytes were collected from the ovaries of 9-10 week old mice and allowed to mature in vitro in the presence of low concentrations of ethanol (0.1% and 0.2% v/v) for 24 h. Treatment of oocytes with ethanol (0.2%) during maturation decreased the mitochondrial DNA content and membrane potential compared to that in untreated ones, whereas the ATP content did not differ between the groups. Both 0.1% and 0.2% ethanol reduced the lipid content in the oocytes. In addition, immunostaining revealed that oocytes cultured in maturation medium containing ethanol (0.2%) had reduced levels of global DNA methylation and DNMT3A compared with untreated oocytes, and decreased rate of blastocyst development with low mitochondrial protein levels (TOMM40) in embryo. RNA-sequencing of the ethanol-treated (0.2%) and untreated oocytes revealed that mitochondria were a major target of ethanol. In conclusion, treatment of oocytes with low concentration of ethanol reduces the developmental rate to the blastocyst stage, with a lower total cell number and global DNA methylation. In addition, ethanol affected mitochondrial function and mitochondria-related gene expression.

Toxicology of milrinone by zebrafish embryotoxicity test.

Milrinone, a phosphodiesterase III inhibitor with contractile and vasodilatory effects, is widely used in acute decompensated heart failure and medically refractory end-stage heart failure (HF). The adverse reactions of milrinone have been extensively explored clinically, but its possible toxicities and underlying molecular mechanisms in embryo development need further understanding as its clinical applications increase. Herein, we assessed the milrinone toxicity using the zebrafish embryotoxicity test (ZET), with a view of providing evidence and guidance for gravidas medicine. We carried out ZET by exposing embryos to a milrinone culture with a series concentration gradients since 1.5 hours post fertilization (hpf) and observed and assessed mortality and hatching rates of drug-treated zebrafishes at 24, 48, 72, and 96 hpf. No significant lethal effect was found in milrinone-treated zebrafish, but hatching rate of eggs at 48 hpf was up-regulated with the increase of milrinone concentration. The impact of milrinone on embryogenesis was assessed through body length, eye area, yolk sac area, swim bladder inflation area, pericardial area and venous congestion area at 96hpf. 150 µg/mL or higher milrinone treatment showed significant effects in the indicators. Organ disorders including enlarged pericardium, liver atrophy and decreased blood vessels were observed in dysplasia individuals versus controls. TUNEL assay suggested the ability of milrinone to induce apoptosis in malformation embryos. Quantitative real-time PCR showed aberrant expressions of transcription factors associated with heart development and genes related to liver development and apoptosis regulation. Therefore, ZET is feasible for the milrinone toxicity test, and high-dose milrinone causes harm to the embryonic development of zebrafish, especially in embryonic carcinogenesis, vasculogenesis, and hepatogenesis.

Maternal Diet High in Linoleic Acid Alters Renal Branching Morphogenesis and mTOR/AKT Signalling Genes in Rat Fetal Kidneys.

Linoleic acid (LA), an n-6 polyunsaturated fatty acid (PUFA), is obtained from the maternal diet during pregnancy, and is essential for normal fetal growth and development. A maternal high-LA (HLA) diet alters maternal and offspring fatty acids, maternal leptin and male/female ratio at embryonic (E) day 20 (E20). We investigated the effects of an HLA diet on embryonic offspring renal branching morphogenesis, leptin signalling, megalin signalling and angiogenesis gene expression. Female Wistar Kyoto rats were fed low-LA (LLA; 1.44% energy from LA) or high-LA (HLA; 6.21% energy from LA) diets during pregnancy and gestation/lactation. Offspring were sacrificed and mRNA from kidneys was analysed by real-time PCR. Maternal HLA decreased the targets involved in branching morphogenesis Ret and Gdnf in offspring, independent of sex. Furthermore, downstream targets of megalin, namely mTOR, Akt3 and Prkab2, were reduced in offspring from mothers consuming an HLA diet, independent of sex. There was a trend of an increase in the branching morphogenesis target Gfra1 in females (p = 0.0517). These findings suggest that an HLA diet during pregnancy may lead to altered renal function in offspring. Future research should investigate the effects an HLA diet has on offspring kidney function in adolescence and adulthood.

Pre-natal exposure to glucocorticoids causes changes in developmental circadian clock gene expression and post-natal behaviour in the Japanese quail.

The embryonic environment is critical in shaping developmental trajectories and consequently post-natal phenotypes. Exposure to elevated stress hormones during this developmental stage is known to alter a variety of post-natal phenotypic traits, and it has been suggested that pre-natal stress can have long term effects on the circadian rhythm of glucocorticoid hormone production. Despite the importance of the circadian system, the potential impact of developmental glucocorticoid exposure on circadian clock genes, has not yet been fully explored. Here, we showed that pre-natal exposure to corticosterone (CORT, a key glucocorticoid) resulted in a significant upregulation of two key hypothalamic circadian clock genes during the embryonic period in the Japanese quail (Coturnix japonica). Altered expression was still present 10 days into post-natal life for both genes, but then disappeared by post-natal day 28. At post-natal day 28, however, diel rhythms of eating and resting were influenced by exposure to pre-natal CORT. Males exposed to pre-natal CORT featured an earlier acrophase, alongside spending a higher proportion of time feeding. Females exposed to pre-natal CORT featured a less pronounced shift in acrophase and spent less time eating. Both males and females exposed to pre-natal CORT spent less time inactive during the day. Pre-natal CORT males appeared to feature a delay in peak activity levels. Our novel data suggest that these circadian clock genes and aspects of diurnal behaviours are highly susceptible to glucocorticoid disruption during embryonic development, and these effects are persistent across developmental stages, at least into early post-natal life.

FGF18 impairs blastocyst viability, DNA double-strand breaks and maternal recognition of pregnancy genes.

Embryonic mortality in cattle is high, reaching 10-40 % in vivo and 60-70 % in vitro. Death of embryos involves reduced expression of genes related to embryonic viability, inhibition of DNA repair and increased DNA damage. In follicular granulosa cells, FGF18 from the theca layer increases apoptosis and DNA damage, so we hypothesized that FGF18 may also affect the oocyte and contribute to early embryonic death. The aims of this study were to identify the effects of FGF18 on cumulus expansion, oocyte maturation and embryo development from cleavage to blastocyst stage using a conventional bovine in vitro embryo production system using ovaries of abattoir origin. Addition of FGF18 during in-vitro maturation did not affect FSH-induced cumulus expansion or rates of nuclear maturation. When FGF18 was present in the culture system, rates of cleavage were not affected however, blastocyst and expanded blastocyst development was substantially inhibited (P < 0.05), indicating a delay of blastulation. The number of phosphorylated histone H2AFX foci per nucleus, a marker of DNA damage, was higher in cleavage-stage embryos cultured with FGF18 than in those from control group (P < 0.05). Furthermore, FGF18 decreased accumulation of PTGS2 and IFNT2 mRNA in blastocysts. In conclusion, these novel findings suggest that FGF18 plays a role in the regulation of embryonic death during the early stages of development by impairing DNA double-strand break repair and expression of genes associated with embryo viability and maternal recognition of pregnancy during the progression from oocyte to expanded blastocysts.

Developmental toxicity and mechanism of dibutyl phthalate and alternative diisobutyl phthalate in the early life stages of zebrafish (Danio rerio).

Diisobutyl phthalate (DiBP), is widely chemical replacement for Dibutyl phthalate (DBP). Although DBP and DiBP have been detected in surface water worldwide, few studies to date have systematically assessed the risks of DBP and its alternatives to aquatic organisms. The present study compared DBP and DiBP for their individual and joint toxicity as well as thyroid hormone levels in zebrafish embryo. Transcripts of key genes related to the hypothalamic-pituitary-thyroid (HPT) axis were investigated in developing zebrafish larvae by application of real time polymerase chain reaction. The median half-lethal concentrations of DBP and DiBP to zebrafish at 96 h were 0.545 mg L-1 and 1.149 mg L-1, respectively. The joint toxic effect of DBP-DiBP (0.25-0.53 mg L-1) with the same ratio showed a synergistic effect. Thyroid hormones levels increased with exposure to 10 µg L-1 of DBP or 50 µg L-1 of DiBP, and exposure to both compounds significantly increased thyroid gland-specific transcription of thyroglobulin gene (tg), hyronine deiodinase (dio2), and transthyretin (ttr), indicating an adverse effect associated with the HPT axis. Molecular docking results indicated that DBP (-7.10 kcal/M and -7.53 kcal/M) and DiBP (-6.63 kcal/M and -7.42 kcal/M) had the same docking energy with thyroid hormone receptors. Our data facilities an understand of potential harmful effects of DBP and its alternative (DiBP).

Mangiferin improves early porcine embryonic development by reducing oxidative stress.

Mangiferin (MGN) is primarily found in the fruits, leaves, and bark of plants of the Anacardiaceae family, including mangoes. MGN exhibits various pharmacological effects, such as protection of the liver and gallbladder, anti-lipid peroxidation, and cancer prevention. This study aimed to investigate the effects of MGN supplementation during in vitro culture (IVC) on the antioxidant capacity of early porcine embryos and the underlying mechanisms involved. Porcine parthenotes in the IVC medium were exposed to different concentrations of MGN (0, 0.01, 0.1, and 1 µM). The addition of 0.1 µM MGN significantly increased the blastocyst formation rate of porcine embryos while reducing the apoptotic index and autophagy. Furthermore, the expression of antioxidation-related (SOD2, GPX1, NRF2, UCHL1), cell pluripotency (SOX2, NANOG), and mitochondria-related (TFAM, PGC1α) genes was upregulated. In contrast, the expression of apoptosis-related (CAS3, BAX) and autophagy-related (LC3B, ATG5) genes decreased after MGN supplementation. These findings suggest that MGN improves early porcine embryonic development by reducing oxidative stress-related genes.

Estradiol-17ß and bisphenol A affect growth and mineralization in early life stages of seabass.

Natural and synthetic estrogens are contaminants present in aquatic ecosystems. They can have significant consequences on the estrogen-sensitive functions of organisms, including skeletal development and growth of vertebrate larvae. Synthetic polyphenols represent a group of environmental xenoestrogens capable of binding the receptors for the natural hormone estradiol-17ß (E2). To better understand how (xeno-)estrogens can affect the skeleton in fish species with high ecological and commercial interest, 16 days post-hatch larvae of the seabass were experimentally exposed for 7 days to E2 and Bisphenol A (BPA), both used at the regulatory concentration of surface water quality (E2: 0.4 ng.L-1, BPA: 1.6 µg.L-1) or at a concentration 100 times higher. Skeletal mineralization levels were evaluated using Alizarin red staining, and expression of several genes playing key roles in growth, skeletogenesis and estrogen signaling pathways was assessed by qPCR. Our results show that E2 exerts an overall negative effect on skeletal mineralization at the environmental concentration of 0.4 ng.L-1, correlated with an increase in the expression of genes associated only with osteoblast bone cells. Both BPA exposures inhibited mineralization with less severe effects and modified bone homeostasis by regulating the expression of gene encoding osteoblasts and osteoclasts markers. Our results demonstrate that environmental E2 exposure inhibits larval growth and has an additional inhibitory effect on skeleton mineralization while both BPA exposures have marginal inhibitory effect on skeletal mineralization. All exposures have significant effects on transcriptional levels of genes involved in the skeletal development of seabass larvae.

Comparison of developmental toxicity of graphene oxide and graphdiyne to zebrafish larvae.

Graphdiyne (GDY) is a new member of family of carbon-based 2D nanomaterials (NMs), but the environmental toxicity is less investigated compared with other 2D NMs, such as graphene oxide (GO). In this study, we compared with developmental toxicity of GO and GDY to zebrafish larvae. It was shown that exposure of zebrafish embryos from 5 h post fertilization to GO and GDY for up to 5 days decreased hatching rate and induced morphological deformity. Behavioral tests indicated that GO and GDY treatment led to hyperactivity of larvae. However, blood flow velocity was not significantly affected by GO or GDY. RNA-sequencing data revealed that both types of NMs altered gene expression profiles as well as gene ontology terms and KEGG pathways related with metabolism. We further confirmed that the NMs altered the expression of genes related with lipid droplets and autophagy, which may be account for the delayed development of zebrafish larvae. At the same mass concentrations, GO induced comparable or even larger toxic effects compared with GDY, indicating that GDY might be more biocompatible compared with GO. These results may provide novel understanding about the environmental toxicity of GO and GDY in vivo.

Retinoic Acid Upregulates METTL14 Expression and the m6A Modification Level to Inhibit the Proliferation of Embryonic Palate Mesenchymal Cells in Cleft Palate Mice.

Cleft palate only (CPO) is one of the most common craniofacial birth defects. Environmental factors can induce cleft palate by affecting epigenetic modifications such as DNA methylation, histone acetylation, and non-coding RNA. However, there are few reports focusing on the RNA modifications. In this study, all-trans retinoic acid (atRA) was used to simulate environmental factors to induce a C57BL/6J fetal mouse cleft palate model. Techniques such as dot blotting and immunofluorescence were used to find the changes in m6A modification when cleft palate occurs. RNA-seq and KEGG analysis were used to screen for significantly differentially expressed pathways downstream. Primary mouse embryonic palate mesenchymal (MEPM) cells were successfully isolated and used for in vitro experimental verification. We found that an increased m6A methylation level was correlated with suppressed cell proliferation in the palatine process mesenchyme of cleft palate mice. This change is due to the abnormally high expression of m6A methyltransferase METTL14. When using siRNAs and the m6A methyltransferase complex inhibitor SAH to interfere with the expression or function of METTL14, the teratogenic effect of atRA on primary cells was partially alleviated. In conclusion, METTL14 regulates palatal mesenchymal cell proliferation and cycle-related protein expression relies on m6A methylation modification, affecting the occurrence of cleft palate.

Nobiletin as a novel agent to enhance porcine in vitro embryo development and quality.

In vitro embryo production (IVP) is of great importance to the porcine industry, as well as for basic research and biomedical applications. Despite the large efforts made in laboratories worldwide to address suboptimal culture conditions, porcine IVP remains inefficient. Nobiletin (Nob, 5,6,7,8,3',4' hexamethoxyflavone) supplementation to in vitro culture (IVC) medium, enhances in vitro embryo development in various species. However, its impact on the quality and developmental capacity of in vitro-produced pig embryos is yet to be established. This study evaluated the effects of different concentrations (2.5 and 5 µM) of Nob during the early culture of in vitro-produced pig embryos on embryo developmental competence, mitochondrial activity, lipid content, intracellular Reactive Oxygen Species (ROS) and Glutathione (GSH) content, Total Cell Number (TCN) per blastocyst, and expression of genes related to embryo development, quality and oxidative stress. Embryos cultured in medium without Nob supplementation and in medium supplemented with 0.01 % dimethyl sulfoxide (DMSO-vehicle for Nob) constituted the Control and DMSO groups, respectively. Embryo development rates were evaluated on Days 2, 6 and 7 of IVC. Additionally, a representative group of embryos was selected to assess mitochondrial activity, lipid, ROS and GSH content (on Days 2 and 6 of IVC), TCN assessment and gene expression analyses (on Day 6 of IVC). No significant differences were observed in any of the parameters evaluated on Day 2 of IVC. In contrast, embryos cultured under the presence of Nob 2.5 showed higher developmental rates on Days 6 and 7 of IVC. In addition, Day 6 embryos showed increased mitochondrial activity, with decreased levels of ROS and GSH in the Nob 2.5 group compared to the other groups. Both Nob 2.5 and Nob 5 embryos showed higher TCN compared to the Control and DMSO groups. Furthermore, Nob 2.5 and Nob 5 upregulated the expression of Superoxide dismutase type 1 (SOD1) and Glucose-6-phosphate dehydrogenase (G6PDH) genes, which could help to counteract oxidative stress during IVC. In conclusion, the addition of Nob during the first 48 h of IVC increased porcine embryo development rates and enhanced their quality, including the upregulation of relevant genes that potentially improved the overall efficiency of the IVP system.

Amphetamine Exposure during Embryogenesis Alters Expression and Function of Tyrosine Hydroxylase and the Vesicular Monoamine Transporter in Adult C. elegans.

Amphetamines (Amph) are psychostimulants broadly used as physical and cognitive enhancers. However, the long-term effects of prenatal exposure to Amph have been poorly investigated. Here, we show that continuous exposure to Amph during early development induces long-lasting changes in histone methylation at the C. elegans tyrosine hydroxylase (TH) homolog cat-2 and the vesicular monoamine transporter (VMAT) homologue cat-1 genes. These Amph-induced histone modifications are correlated with enhanced expression and function of CAT-2/TH and higher levels of dopamine, but decreased expression of CAT-1/VMAT in adult animals. Moreover, while adult animals pre-exposed to Amph do not show obvious behavioral defects, when challenged with Amph they exhibit Amph hypersensitivity, which is associated with a rapid increase in cat-2/TH mRNA. Because C. elegans has helped reveal neuronal and epigenetic mechanisms that are shared among animals as diverse as roundworms and humans, and because of the evolutionary conservation of the dopaminergic response to psychostimulants, data collected in this study could help us to identify the mechanisms through which Amph induces long-lasting physiological and behavioral changes in mammals.

Two thyroperoxidase-inhibiting chemicals induce shared transcriptional changes in hippocampus of developing rats.

Thyroid hormone (TH) system disrupting compounds can impair brain development by perturbing TH action during critical life stages. Human exposure to TH system disrupting chemicals is therefore of great concern. To better protect humans against such chemicals, sensitive test methods that can detect effects on the developing brain are critical. Worryingly, however, current test methods are not sensitive and specific towards TH-mediated effects. To address this shortcoming, we performed RNA-sequencing of rat brains developmentally exposed to two different thyroperoxidase (TPO) inhibiting compounds, the medical drug methimazole (MMI) or the pesticide amitrole. Pregnant and lactating rats were exposed to 8 and 16 mg/kg/day(d) MMI or 25 and 50 mg/kg/d amitrole from gestational day 7 until postnatal day 16. Bulk-RNA-seq was performed on hippocampus from the 16-day old male pups. MMI and amitrole caused pronounced changes to the transcriptomes; 816 genes were differentially expressed, and 425 gene transcripts were similarly affected by both chemicals. Functional terms indicate effects from key cellular functions to changes in cell development, migration and differentiation of several cell populations. Of the total number of DEGs, 106 appeared to form a consistent transcriptional fingerprint of developmental hypothyroidism as they were similarly and dose-dependently expressed across all treatment groups. Using a filtering system, we identified 20 genes that appeared to represent the most sensitive, robust and dose-dependent markers of altered TH-mediated brain development. These markers provide inputs to the adverse outcome pathway (AOP) framework where they, in the context of linking TPO inhibiting compounds to adverse cognitive function, can be used to assess altered gene expression in the hippocampus in rat toxicity studies.

Maternal PFOS exposure affects offspring development in Nrf2-dependent and independent ways in zebrafish (Danio rerio).

Perfluorooctanesulfonic acid (PFOS) is a ubiquitous legacy environmental contaminant detected broadly in human samples and water supplies. PFOS can cross the placenta and has been detected in cord blood and breastmilk samples, underscoring the importance of understanding the impacts of maternal PFOS exposure during early development. This study aimed to investigate the effects of a preconception exposure to PFOS on developmental endpoints in offspring, as well as examine the role of the transcription factor Nuclear factor erythroid-2-related factor (Nrf2a) in mediating these effects. This transcription factor regulates the expression of several genes that protect cells against oxidative stress including during embryonic development. Adult female zebrafish were exposed to 0.02, 0.08 or 0.14 mg/L PFOS for 1 week (duration of one cycle of oocyte maturation) and then paired with unexposed males from Nrf2a mutant or wildtype strains. Embryos were collected for two weeks or until completion of 5 breeding events. PFOS was maternally transferred to offspring independent of genotype throughout all breeding events in a dose-dependent manner, ranging from 2.77 to 23.72 ng/embryo in Nrf2a wildtype and 2.40 to 15.80 ng/embryo in Nrf2a mutants. Although embryo viability at collection was not impacted by maternal PFOS exposure, developmental effects related to nutrient uptake, growth and pancreatic ß-cell morphology were observed and differed based on genotype. Triglyceride levels were increased in Nrf2a wildtype eggs from the highest PFOS group. In Nrf2a wildtype larvae there was a decrease in yolk sac uptake while in Nrf2a mutants there was an increase. Additionally, there was a significant decrease in pancreatic ß-cell (islet) area in wildtype larvae from the 0.14 mg/L PFOS accompanied by an increase in the prevalence of abnormal islet morphologies compared to controls. Abnormal morphology was also observed in the 0.02 and 0.08 mg/L PFOS groups. Interestingly, in Nrf2a mutants there was a significant increase in the pancreatic ß-cell area in the 0.02 and 0.08 mg/L PFOS groups and no changes in the prevalence of abnormal islet morphologies. These results suggest that the regulation of processes like nutrient consumption, growth and pancreatic ß-cell development are at least partially modulated by the presence of a functional Nrf2a transcriptomic response. Overall, preconception exposure to environmental pollutants, such as PFOS, may impact the maturing oocyte and cause subtle changes that can ultimately impact offspring health and development.

Human cortical neurogenesis is altered via glucocorticoid-mediated regulation of ZBTB16 expression.

Glucocorticoids are important for proper organ maturation, and their levels are tightly regulated during development. Here, we use human cerebral organoids and mice to study the cell-type-specific effects of glucocorticoids on neurogenesis. We show that glucocorticoids increase a specific type of basal progenitors (co-expressing PAX6 and EOMES) that has been shown to contribute to cortical expansion in gyrified species. This effect is mediated via the transcription factor ZBTB16 and leads to increased production of neurons. A phenome-wide Mendelian randomization analysis of an enhancer variant that moderates glucocorticoid-induced ZBTB16 levels reveals causal relationships with higher educational attainment and altered brain structure. The relationship with postnatal cognition is also supported by data from a prospective pregnancy cohort study. This work provides a cellular and molecular pathway for the effects of glucocorticoids on human neurogenesis that relates to lasting postnatal phenotypes.

Ethynylestradiol feminizes gene expression partly in testis developing as ovotestis and disrupts asymmetric Müllerian duct development by eliminating asymmetric gene expression in Japanese quail embryos.

In avian embryos, xenoestrogens induce abnormalities in reproductive organs, particularly the testes and Müllerian ducts (MDs). However, the molecular mechanisms remain poorly understood. We investigated the effects of ethynylestradiol (EE2) exposure on gene expression associated with reproductive organ development in Japanese quail embryos. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis revealed that the left testis containing ovary-like tissues following EE2 exposure highly expressed the genes for steroidogenic enzymes (P450scc, P45017α, lyase, and 3ß-HSD) and estrogen receptor-ß, compared to the right testis. No asymmetry was found in these gene expression without EE2. EE2 induced hypertrophy in female MDs and suppressed atrophy in male MDs on both sides. RNA sequencing analysis of female MDs showed 1,366 differentially expressed genes between developing left MD and atrophied right MD in the absence of EE2, and these genes were enriched in Gene Ontology terms related to organogenesis, including cell proliferation, migration and differentiation, and angiogenesis. However, EE2 reduced asymmetrically expressed genes to 21. RT-qPCR analysis indicated that genes promoting cell cycle progression and oncogenesis were more highly expressed in the left MD than in the right MD, but EE2 eliminated such asymmetric gene expression by increasing levels on the right side. EE2-exposed males showed overexpression of these genes in both MDs. This study reveals part of the molecular basis of xenoestrogen-induced abnormalities in avian reproductive organs, where EE2 may partly feminize gene expression in the left testis, developing as the ovotestis, and induce bilateral MD malformation by canceling asymmetric gene expression underlying MD development.

Rewiring of the epigenome and chromatin architecture by exogenously induced retinoic acid signaling during zebrafish embryonic development.

Retinoic acid (RA) is the ligand of RA receptors (RARs), transcription factors that bind to RA response elements. RA signaling is required for multiple processes during embryonic development, including body axis extension, hindbrain antero-posterior patterning and forelimb bud initiation. Although some RA target genes have been identified, little is known about the genome-wide effects of RA signaling during in vivo embryonic development. Here, we stimulate the RA pathway by treating zebrafish embryos with all-trans-RA (atRA) and use a combination of RNA-seq, ATAC-seq, ChIP-seq and HiChIP to gain insight into the molecular mechanisms by which exogenously induced RA signaling controls gene expression. We find that RA signaling is involved in anterior/posterior patterning, central nervous system development, and the transition from pluripotency to differentiation. AtRA treatment also alters chromatin accessibility during early development and promotes chromatin binding of RARαa and the RA targets Hoxb1b, Meis2b and Sox3, which cooperate in central nervous system development. Finally, we show that exogenous RA induces a rewiring of chromatin architecture, with alterations in chromatin 3D interactions involving target genes. Altogether, our findings identify genome-wide targets of RA signaling and provide a molecular mechanism by which developmental signaling pathways regulate target gene expression by altering chromatin topology.