Unknown,Transcriptomics,Genomics,Proteomics

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Effects of egg enrichment with glucocorticoid hormone, antagonist and receptor messenger in the modulation of gene expression in zebrafish (Danio rerio) embryos

ABSTRACT: Several studies have shown the presence of large reservoirs of maternally contributed liposoluble hormones in the yolk of mature teleost oocytes, such as steroid, thyroid and retinoid hormones, as found in other oviparous vertebrates. This supports the idea that maternal hormones could play a major role in regulating developmental processes post-fertilization. The aims of this study were: A) to verify whether a cortisol treatment of zebrafish eggs affects the growth rate of the progenies as compared to controls in F1-F4 treated generations (ontogenetic programming). It was to be established whether: 1) this is due to a short-term effect of maternal cortisol, directly producing an initial growth retard that is not compensated later on in lifetime (short-term epigenetic effect); or 2) this is due to a long-term priming of growth rate by maternal cortisol that persists later on in lifetime (long-term epigenetic effect); B) to verify whether the ontogenetic programming is inheritable, being transmitted also to the untreated F4 generation. It was to be established whether the programmed genes are developmental and/or growth genes that are inhibited, or genes encoding hormones of the corticoid stress axis (hypothalamo-hypophyso-interrenal axis) that are amplified, or both gene clusters. Control embryos (treated with the vehicle ethanol only) were compared to cortisol-treated embryos at 5 hpf, 12 hpf and 24 hpf. Four replicates were performed for the 5 hpf samples, three biological replicates were carried out for the 12 hpf samples, and a single experiment was performed for the 24 hpf samples.

ORGANISM(S): Danio rerio

SUBMITTER: Andrea Celeghin

PROVIDER: E-GEOD-52398 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We have previously shown in sheep that 10 days of modest chronic increase in maternal cortisol result in fetal heart enlargement and Purkinje cell apoptosis. In subsequent studies in which we extended the duration of cortisol infusion (1mg/kg/d) to term, we found a dramatic incidence of stillbirth in the pregnancies with chronically increased cortisol and associated maternal hyperglycemia. To investigate the effects on the heart, transcriptomic analyses were performed on the septa using ovine microarrays and Webgestalt and Cytoscape programs for pathway inference. Analyses of the effects of 10 days of maternal cortisol infusion (130d-cortisol vs 130d control), ~25 days (term at ~140d-cortisol vs 140d control), normal maturation (140d-control vs 130d control) were performed. In all analyses gene ontology (GO) terms related to immune function and cytokine actions were significantly over-represented. After 10 days of cortisol, growth factor and muscle cell apoptosis pathways were significantly over-represented, consistent with our previous findings. We found significantly differentially regulated genes in the term fetuses (ie after ~25 days of cortisol) in pathways consistent with altered metabolism in the heart, particularly in mitochondria, associated with responses to hypoxia and to nutrient. Analysis of mitochondrial number by quantitative real-time PCR confirmed a significant decrease. These pathways were different from those modeled following the normal increase in cortisol in late gestation which contributes to normal maturation of the heart, and thus may be indicative of the fetal heart pathophysiologies seen in pregnancies complicated by diabetes, CushingM-bM-^@M-^Ys disease and chronic stress. 2 cohorts of singleton sheep fetuses at 129-131d gestation or 139-144d gestation (approximately term) were used. The first cohort received maternal cortisol infusion of 1mg/kg/day or vehicle for 10 days until approximately day 130 of gestation (n=6/group), the second cohort received the same dose of cortisol for approximately 25 days until near term (n=7, cortisol; n=7ewes, n=8 fetuses due to one set of twins, control)

Project description:In zebrafish, vitellogenic oocytes can incorporate significant amounts of 17β-estradiol released from nearby granulosa cells according to a first-order kinetics, since the steroid low polarity ensures high permeability and affinity for yolk lipids. Estrogen bioactivity is likely, because the maternal mRNA for the estrogen receptor-β2 (ers2a) is highly expressed in ovulated oocytes. This transcript is available for translation in the embryo until its sharp decline from 4 to 8 hours post-fertilization (hpf), being replaced by low levels of zygotic ers2a mRNA from 24 hpf to hatching at 48 hpf, as determined by qRT-PCR. Estrogen receptors-α and -β1 are only expressed zygotically at low levels from 24 hpf onwards. To test the functional role of maternal ers2a mRNA, 1- or 2-cell embryos were injected with 10.3 ng each of morpholino to knockdown translation (MO2-ers2a) of both maternal and zygotic ers2a transcripts, missplicing morpholino (MO3-ers2a) to block post-transcriptionally the zygotic transcript alone, and a nonspecific morpholino (MO-control) as a control. Treatment with MO2-ers2a caused severe malformations in 63% of 1-5 dpf larvae, as compared to 10-11% in those treated with MO3-ers2a and MO2-control. Defects included body growth delay and curved shape, abnormal brain and splanchnocranium development, enlarged and hemorrhagic pericardial cavity, uninflated swim bladder and rudimentary caudal fin with aberrant circular motion. Affected larvae could survive for only 12-14 days. Co-injection of an anti-p53 MO failed to rescue the MO2-ers2a-phenotypes, eliminating the possibility of off-target effects. Pangenomic microarray analysis revealed that 240 and 219 significantly expressed transcripts were up- and down-regulated, respectively, by maternal Ers2a protein deficiency in 8-hpf MO2-ers2a-embryos. Also at 48 hpf, 162 and 120 presumably zygotic transcripts were up- and down-regulated, respectively, but only 18 were in common with each of the 8-hpf sets. Whole-mount in situ hybridization revealed an intensified expression of the genes six3.1 and emx1 in MO2-ers2a-embryos at 24-48 hpf, as compared to controls. These findings suggest the involvement of maternal ers2a mRNA in the epigenetic programming of zebrafish development. MO2-ers2a morphants were compared with MO-control at 8 hpf and 48 hpf. MO2-ers2a is a morpholinos selected to knockdown translation of ers2a mRNA

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Effects of egg enrichment with glucocorticoid hormone, antagonist and receptor messenger in the modulation of gene expression in zebrafish (Danio rerio) embryos

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