In vivo in vitro comparison to retinoic acid in whole embryo culture and rat embryos
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ORGANISM(S): Rattus norvegicus
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PROVIDER: S-DIXA-072 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:The whole embryo culture (WEC) model serves as a potential replacement for classical in vivo developmental toxicity testing. In this alternative test, cultured rat embryos are exposed during neurulation and early organogenesis and evaluated for morphological adverse outcomes. Toxicogenomic-based approaches may improve the predictive ability of WEC by providing molecular-based markers associated with chemical exposure which can be compared across multiple parameters (e.g. time, dose, model). Additionally, comparisons between in vitro and in vivo models may identify objective, relevant molecular responses linked with developmental toxicity endpoints in vivo. In this study, using a transcriptomic approach, we compared all-trans-retinoic acid (RA)-exposed and non-exposed Wistar rat embryos derived using WEC (RA, 0.5µg/mL) or in vivo (RA, 50mg/kg, oral gavage) to identify overlapping and non-overlapping effects of RA on RNA expression in parallel with morphological changes. Across six time points (GD 10 + 2-48h), we observed strong similarities in RA-response at the gene (directionality, significance) and functional (e.g. embryonic development, cell differentiation) level which associated with RA-induced adverse morphological effects, including growth reduction as well as alterations of neural tube, limb, branchial, and mandible development. Differences between models in the timing of RA-induced impacts on the expression of select genes related to embryonic development and RA-metabolism associated with specific differential morphological outcomes. This study supports the use of WEC to examine compound-induced molecular responses relative to in vivo, and furthermore, assists in defining the applicability domain of the WEC in determining complementary windows of sensitivity for developmental toxicological investigations. Cultured embryos mimic the morphological developmental progression of embryos (in vivo) undergoing neurulation and early organogenesis. Using available genomics technologies, comparative molecular-based assessments between cultured embryos and in vivo models may further clarify commonalities and dissimilarities which contribute to differences between systems. Therefore, in this study, using a transcriptomic approach, we compared cultured whole rat embryos and embryos in vivo at comparable time points in development (gestational day (GD) 10 + 2-48h, GD 0 = copulatory plug) to assess for commonalities and differences in gene expression in relation to morphology. We reveal strong parallels in time-dependent expression of genes in terms of magnitude, directionality and functionality between whole embryo culture (WEC) and in vivo (rat). Genes changing in expression over time resemble previously hypothesized mechanisms underlying early development in mammalian systems. Furthermore, at the gene and functional level, we identify genes which differ in expression between models, including genes related to development, oxygen transport and metabolism. In summary, our results support the use of WEC for toxicological studies aimed at representing in vivo development during this time-window at the molecular level. Additionally, we indicate genes which differ in expression between models, providing possible insights for improvement of culture conditions.
2012-03-12 | GSE33195 | GEO
Project description:BackgroundWhole embryo culture is a valuable research method in mammalian developmental biology and birth defects research, enabling longitudinal studies of explanted organogenesis-stage rodent embryos. Rat serum is the primary culture medium, and can sustain growth and development over limited periods as in utero. However, the cost, labor, and time to produce culture serum are factors limiting the uptake of the methodology. The goal of replacing or at least reducing rat serum usage in culture would be in accordance with the principles of "replacement, reduction, and refinement" of animals in research (the 3Rs).MethodsWe performed cultures of mouse embryos for 24 hr from embryonic day 8.5 in serum-free media or in rat serum diluted with defined media, compared with 100% rat serum. Developmental parameters scored after culture included yolk sac circulation, dorsal axial length, somite number, protein content, and completion of cranial neural tube closure.ResultsA literature review revealed use of both serum-free and diluted rat serum-based media in whole embryo culture studies, but with almost no formal comparisons of culture success against 100% rat serum. Two serum-free media were tested, but neither could sustain development as in 100% rat serum. Dilution of rat serum 1:1 with Glasgow Minimum Essential Medium plus defined supplements supported growth and development as well as whole rat serum, whereas other diluent media yielded substandard outcomes.ConclusionRat serum usage cannot be avoided, to achieve high quality mouse embryo cultures, but rat usage can be reduced using medium containing diluted serum.
| S-EPMC6778057 | biostudies-literature
Project description:Analysis of gene expression and alternate splicing effects of retinoic acid treatment on gestational day 15 rat fetal testes in whole testis culture Retinoic acid exposure in cultured fetal testis has previously been demonstrated to have significant effects on the histology of the fetal testis in multiple species, as well as to alter the meiotic states of germ cells. However, previous experiments have not analyzed the mechanisms by which retinoic acid exposure leads to altered tubulogenesis and loss of seminiferous cord structure. This experiment demonstrated that retinoic acid exposure activated signaling pathways that promote the ovary development program and oppose normal testis development in mid-gestational rat fetal testes.
2017-09-26 | GSE101634 | GEO
Project description:In vertebrates, 5'-Hoxd genes (Hoxd9), which are expressed in the hindlimb bud mesenchyme, participate in limb growth and patterning in early embryonic development. In the present study, We investigated the mechanisms by which ATRA regulates cultured E12.5 rat embryo hindlimb bud mesenchymal cells (rEHBMCs). Following exposure to ATRA over 24 h, mRNA and protein expression levels of HoxD9 were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time PCR (qPCR), and western blotting. Flow cytometry was used to detect apoptosis. ATRA inhibited the condensation and proliferation, and promoted the apoptosis rate of the rEHBMCs in a dose-dependent manner. Sox9 and Col2a1 in rEHBMCs were downregulated by ATRA in a dose-dependent manner at both mRNA and protein levels. Similarly, HoxD9 was downregulated by ATRA in a dose-dependent manner, in parallel with the cartilage-specific molecules Sox9 and Col2a1. Both qPCR and western blotting showed that both Shh and Gli3 were downregulated. Overexpression of HoxD9 reversed the effects of ATRA. These results demonstrate that ATRA suppresses chondrogenesis in rEHBMCs by inhibiting the expression of HoxD9 and its downstream protein targets, including Sox9 and Col2a1. This effect may also be correlated with inhibition of the Shh-Gli3 signaling pathway.
| S-EPMC8806522 | biostudies-literature
Project description: Not available
| S-EPMC7401760 | biostudies-literature
Project description:Preimplantation genetic screening (PGS) is widely used to select in vitro-fertilized embryos free of chromosomal abnormalities and to improve the clinical outcome of in vitro fertilization (IVF). A disadvantage of PGS is that it requires biopsy of the preimplantation human embryo, which can limit the clinical applicability of PGS due to the invasiveness and complexity of the process. Here, we present and validate a noninvasive chromosome screening (NICS) method based on sequencing the genomic DNA secreted into the culture medium from the human blastocyst. By using multiple annealing and looping-based amplification cycles (MALBAC) for whole-genome amplification (WGA), we performed next-generation sequencing (NGS) on the spent culture medium used to culture human blastocysts (n = 42) and obtained the ploidy information of all 24 chromosomes. We validated these results by comparing each with their corresponding whole donated embryo and obtained a high correlation for identification of chromosomal abnormalities (sensitivity, 0.882, and specificity, 0.840). With this validated NICS method, we performed chromosome screening on IVF embryos from seven couples with balanced translocation, azoospermia, or recurrent pregnancy loss. Six of them achieved successful clinical pregnancies, and five have already achieved healthy live births thus far. The NICS method avoids the need for embryo biopsy and therefore substantially increases the safety of its use. The method has the potential of much wider chromosome screening applicability in clinical IVF, due to its high accuracy and noninvasiveness.
| S-EPMC5081593 | biostudies-literature
Project description:Morphological phenotyping of the mouse embryo is described at neurulation stages, primarily as a guide to evaluating the outcome of whole embryo cultures between embryonic days 8.5 and 9.5. During this period, neural tube closure is initiated and progresses to completion in the cranial region. Spinal closure is still underway at the end of the culture period. The focus of this article is particularly on phenotyping that can be performed at the bench, using a stereomicroscope. This involves assessment of embryonic health, through observation and scoring of yolk sac blood circulation, measurement of developmental stage by somite counting, and determination of crown-rump length as a measure of growth. Axial rotation ("turning") can also be assessed using a simple scoring system. Neural tube closure assessment includes: 1) determining whether closure has been initiated at the Closure 1 site; 2) evaluating the complex steps of cranial neurulation including initiation at Closure sites 2 and 3, and completion of closure at the anterior and hindbrain neuropores; 3) assessment of spinal closure by measurement of posterior neuropore length. Interpretation of defects in neural tube closure requires an appreciation of, first, the stages that particular events are expected to be completed and, second, the correspondence between embryonic landmarks, for example, somite position, and the resulting adult axial levels. Detailed embryonic phenotyping, as described in this article, when combined with the versatile method of whole embryo culture, can form the basis for a wide range of experimental studies in early mouse neural development.
| S-EPMC10435082 | biostudies-literature
Project description:Marine foods can be contaminated with organochlorines and the risk to human beings who consume these foods needs to be evaluated. We examined the teratogenic effects of contaminants extracted from whale bacon on rat embryos using a whole-embryo culture system. Embryonic day 11.5 embryos were cultured for 48 h with organohalogens extracted from whale bacon at low (polychlorinated biphenyls (PCBs): 0.32 ppm, dichlorodiphenyltrichloroethanes (DDTs): 0.16 ppm, chlordanes (CHLs): 0.02 ppm) and high (PCBs: 2.15 ppm, DDTs: 1.99 ppm, CHLs: 0.20 ppm) doses. The levels of organohalogen compounds in cultured embryos were determined. The organochlorine contaminants extracted from whale products were readily transferred to the cultured rat embryos. The number of heartbeats, yolk sac circulation score, and embryonic body circulation score of embryos did not change during the culture period in either exposure group. Cultured embryos treated with the low-dose contaminated medium for 48 h showed abnormalities of the mandible, and craniofacial or forelimb hematomas with an incidence of 50%. All embryos treated with the high-dose medium showed craniofacial abnormalities and cleft lip, and limb abnormalities and hematomas. These results indicate that the organohalogen contaminants in whale bacon may be teratogenic in a dose-dependent manner. Further studies are necessary to determine the dose-effect relationship.
| S-EPMC9566164 | biostudies-literature
Project description:Using sex-sorted semen to produce offspring of desired sex is associated with reduced developmental competence in vitro and lower fertility rates in vivo. The objectives of the present study were to investigate the effects of exogenous follistatin supplementation on the developmental competence of bovine embryos produced with sex-sorted semen and possible link between TGF-β regulated pathways and embryotrophic actions of follistatin. Effects of follistatin on expression of cell lineage markers (CDX2 and Nanog) and downstream targets of SMAD signaling (CTGF, ID1, ID2 and ID3) and AKT phosphorylation were investigated. Follistatin was supplemented during the initial 72 h of embryo culture. Exogenous follistatin restored the in vitro developmental competence of embryos produced with sex-sorted semen to the levels of control embryos produced with unsorted semen, and comparable results were obtained using sorted semen from three different bulls. The mRNA abundance for SMAD signaling downstream target genes, CTGF (SMAD 2/3 pathway) and ID2 (SMAD 1/5 pathway), was lower in blastocysts produced using sex-sorted versus unsorted semen, but mRNA levels for CDX2, NANOG, ID1 and ID3 were similar in both groups. Follistatin supplementation restored CTGF and ID2 mRNA in blastocysts produced using sex-sorted semen to levels of control embryos. Moreover, levels of phosphorylated (p)AKT (Ser-473 and Thr-308) were similar in embryos derived from sex-sorted and unsorted semen, but follistatin treatment increased pAKT levels in both groups. Taken together, results demonstrated that follistatin improves in vitro development of embryos produced with sex-sorted semen and such effects are associated with enhanced indices of SMAD signaling.
| S-EPMC7747478 | biostudies-literature
Project description:Recent studies have shown that the metabolome of single embryo culture media is linked to successful pregnancy. In this study, the analysis was expanded to compare the metabolomes of viable and non-viable early-stage embryos and to examine metabolomic markers associated with hatching in viable embryos. The authors hypothesized that the metabolomic profiles of high-quality early blastocysts differ from those of non-viable embryos that reach the blastocyst stage but undergo developmental arrest at later stages. The metabolic profile of 43 spent bovine embryo culture medium samples were analyzed using liquid chromatography-mass spectrometry, covering 189 metabolites, including 40 acylcarnitines, 42 amino acids/biogenic amines, 91 phospholipids, 15 sphingolipids, and the sum of hexoses. Embryos were produced from abattoir-derived oocytes, and the culture medium samples were derived from Grade 1 early blastocysts that progressed to hatching (VBL; n = 10), non-viable early blastocysts that developed to the blastocyst stage but failed to hatch (DBL; n = 12), Grade 1 hatched blastocysts (HBL; n = 16), and plain growth media for control (CM; n = 5). It was observed that methionine sulfoxide (Met-SO) and lysophosphatidylcholine (lysoPC) C24:0 concentrations were significantly lower in the culture media from viable blastocysts compared to those from non-viable blastocysts (p < 0.001). Additionally, blastocysts that resulted in successful hatching had significantly lower levels of phospholipid, arginine (Arg), and methionine-related metabolites that significantly differentiated the control and viable blastocyst culture media from the media containing non-viable embryos. Building on previous studies, there appears to be an overlap in metabolites released during hatching that are also associated with successful pregnancy. The identified biomarkers can aid in assessing an embryo's developmental potential and enhance embryo selection for transfer or cryopreservation.
| S-EPMC11900063 | biostudies-literature