Gene Expression Patterns In Embryos Research Articles

P-156 cellular uptake of polystyrene nanoplastics by oocytes and their impact on subsequent embryo development: a preliminary insight using a bovine in vitro model

Abstract Study question Can polystyrene nanoplastics enter cumulus-enclosed oocytes (CEOs) and consequently alter oocyte quality and embryo development? Is this effect particle size-dependent? Summary answer After 24h in vitro exposure, polystyrene-nanoplastics entered the extracellular matrix and cytoplasm of peripheral cumulus cells, but not oocytes. Preimplantation embryo development was not altered. What is known already The ubiquitous use of plastic resulted in extreme global environmental plastic pollution. Chemical and physical processes contribute to the fragmentation of these macroplastic pollutants into heterogenous nanoplastics (NPs). Plastic intake is inevitable for humans by inhalation and ingestion of contaminated air, food (e.g. plastic ingested fish) and drinks (e.g. leakage of plastic bottles). Once imbibed, particles smaller than 150 µm can spread across the entire human body and reach the blood and lymphatic tissue. Due to the neovascularisation associated with an increased vascular permeability during follicular development, nanoparticles might reach the ovarian follicles, potentially affecting oocyte quality and embryo development. Study design, size, duration In a bottom-up approach, using a bovine in vitro oocyte maturation model, CEOs were exposed for 24h to 100µg/mL plain Nile Red-labelled polystyrene-nanoplastics (PS-NP) with average diameter of 50nm (PS-NP-50) or plain Flash Red-labelled PS-NP with average diameter of 200nm (PS-NP-200) (4 replicates using 714 CEOs). CEOs in NP-free control media (CONT) and in-vehicle control (SOLV) were included. Oocytes were in vitro fertilized (for 20h) and presumptive zygotes were cultured until 8 days post-insemination (p.i.). Participants/materials, setting, methods CEOs were fixed after IVM and examined for cumulus cell (CCs) expansion. The presence of NPs was assessed with laser-scanning Leica SP8 confocal microscope. Cleavage (48h p.i.) and blastocyst rates (8 days p.i.) were recorded together with differential staining of trophectoderm (TE, CDX2) and inner cell mass (ICM, Hoechst). The apoptotic-cell-index of the CCs and blastocysts was examined with Caspase-3 immunostaining. Images were quantified using ImageJ. Data were analysed using binary logistic regression and ANOVA. Main results and the role of chance At 24h of IVM, both PS-NP-50 and PS-NP-200 could be detected in the extracellular matrix surrounding the CCs and even within the cytoplasm of a few CCs in the outer layers of the CEOs. No NPs could be detected within the oocytes. Irrespective of the size of the NP’s, no significant differences in cumulus expansion rate or embryo cleavage rate were observed and no difference in apoptotic-cell-index of the CCs could be demonstrated due to a plastic effect (P > 0.1). However, the blastocyst/cleaved rate of PS-NP-50 (56%) was significantly higher than SOLV (36%, p < 0.05). On top, PS-NP-50 showed a lower apoptotic-cell-index (4.11±0.90) of the TE compared to CONT (7.08±0.99, p < 0.1), SOLV (9.09±1.49, p < 0.05) and PS-NP-200 (7.65±1.09, p < 0.05). There is a complete lack of univocal and sound scientific evidence of the actual amount of human plastic intake, making it difficult to determine a physiologically relevant concentration for testing possible effects. However, despite the high plastic concentration used in our model, no deteriorating effects on oocyte quality and embryo development were proven. Limitations, reasons for caution Exposure for only 24h during IVM resulted in a limited timeframe for uptake, consequently exerting its effects. However, long-term in vitro follicle culture models are not as straightforward. Limited reports give insight into the final destination of NPs inside the body, addressing the necessity for in vivo trials as confirmation. Wider implications of the findings While this study indicate that PS-NPs are not toxic for oocyte developmental competence upon direct exposure, this does not rule out the potential impact on e.g. embryo gene expression patterns and epigenetic programming. Future studies should also focus on indirect effects on the oocyte as PS-NP may alter follicular physiology. Trial registration number not applicable

Transcriptomes shed light on transgenerational and developmental effects of ocean warming on embryos of the sea urchin Strongylocentrotus intermedius

Ocean warming increasingly endangers the fitness of marine invertebrates. Transgenerational effects (TE) potentially mitigate the impacts of environmental stress on the embryos of marine invertebrates. The molecular mechanisms, however, remain largely unknown. Using high-throughput RNA sequencing technology, we investigated the gene expression patterns of embryos (the gastrula stage) of the sea urchin Strongylocentrotus intermedius at different developmental temperatures, whose parents were exposed to long-term (15 months) elevated temperature (A) or not (B). The temperatures at which adults were held for ~4 weeks prior to the start of the experiment (21 °C for A and 18 °C for B) were also used for the development of offspring (high: 21 °C and ambient (laboratory): 18 °C) resulting in four experimental groups (HA and HB at 21 °C, and LA and LB at 18 °C). The embryos were sampled ~24 h after fertilization. All samples were in the gastrula stage. Twelve mRNA libraries (groups HA, HB, LA, LB, 3 replicates for each group) were established for the following sequencing. Embryos whose parents were exposed to elevated temperatures or not showed 1891 significantly different DEGs (differentially expressed genes) at the ambient developmental temperature (LB vs LA, LB as control) and 2203 significantly different DEGs at the high developmental temperature (HB vs HA, HB as control), respectively. This result indicates complex molecular mechanisms of transgenerational effects of ocean warming, in which a large number of genes are involved. With the TE, we found 904 shared DEGs in both LB vs LA (LB as control) and HB vs HA (HB as control) changed in the same direction of expression (i.e., up- or down-regulated), indicating that parental exposed temperatures affect the expression of these genes in the same manner regardless of the development temperature. With developmental exposure, we found 198 shared DEGs in both HB vs LB (HB as control) and HA vs LA (HA as control) changed in the same direction of expression. Of the 198 DEGs, more genes were up-regulated at high developmental temperature. Interestingly, embryos whose parents were exposed to high temperature showed fewer differently expressed DEGs between high and low developmental temperatures than the individuals whose parents were exposed to ambient temperature. The results indicate that gene expressions are probably depressed by the transgenerational effect of ocean warming. The roles of hsp70 and hnf6 in thermal acclimation are highlighted for future studies. The present study provides new insights into the molecular mechanisms of the transgenerational and developmental effects of ocean warming on the embryos of sea urchins.

Single embryo-resolution quantitative analysis of reporters permits multiplex spatial cis-regulatory analysis

Cis-regulatory modules (CRMs) control spatiotemporal gene expression patterns in embryos. While measurement of quantitative CRM activities has become efficient, measurement of spatial CRM activities still relies on slow, one-by-one methods. To overcome this bottleneck, we have developed a high-throughput method that can simultaneously measure quantitative and spatial CRM activities. The new method builds profiles of quantitative CRM activities measured at single-embryo resolution in many mosaic embryos and uses these profiles as a ‘fingerprint’ of spatial patterns. We show in sea urchin embryos that the new method, Multiplex and Mosaic Observation of Spatial Information encoded in Cis-regulatory modules (MMOSAIC), can efficiently predict spatial activities of new CRMs and can detect spatial responses of CRMs to gene perturbations. We anticipate that MMOSAIC will facilitate systems-wide functional analyses of CRMs in embryos.

Suboptimal culture conditions induce more deviations in gene expression in male than female bovine blastocysts.

BackgroundSince the development of in vitro embryo production in cattle, different supplements have been added to culture media to support embryo development, with serum being the most popular. However, the addition of serum during embryo culture can induce high birthweights and low viability in calves (Large Offspring Syndrome). Analysis of global gene expression in bovine embryos produced under different conditions can provide valuable information to optimize culture media for in vitro embryo production.ResultsWe used RNA sequencing to examine the effect of in vitro embryo production, in either serum-containing or serum-free media, on the global gene expression pattern of individual bovine blastocysts. Compared to in vivo derived embryos, embryos produced in serum-containing medium had five times more differentially expressed genes than embryos produced in serum-free conditions (1109 vs. 207). Importantly, in vitro production in the presence of serum appeared to have a different impact on the embryos according to their sex, with male embryos having three times more genes differentially expressed than their female counterparts (1283 vs. 456). On the contrary, male and female embryos produced in serum-free conditions showed the same number (191 vs. 192) of genes expressed differentially; however, only 44 of those genes were common in both comparisons. The pathways affected by in vitro production differed depending on the type of supplementation. For example, embryos produced in serum-containing conditions had a lower expression of genes related to metabolism while embryos produced in serum-free conditions showed aberrations in genes involved in lipid metabolism.ConclusionsSerum supplementation had a major impact on the gene expression pattern of embryos, with male embryos being the most affected. The transcriptome of embryos produced in serum-free conditions showed a greater resemblance to that of in vivo derived embryos, although genes involved in lipid metabolism were altered. Male embryos appeared to be most affected by suboptimal in vitro culture, i.e. in the presence of serum.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2393-z) contains supplementary material, which is available to authorized users.

Ovarian response and embryo gene expression patterns after nonsuperovulatory gonadotropin stimulation in primiparous rabbits does

Ovarian stimulation with equine chorionic gonadotropin (eCG) is largely used in animal reproductive technologies to provide a larger number of oocytes and embryos and to improve the reproductive outcome. However, the consequences of maternal treatment with eCG on embryo gene expression patterns are not widely studied. The aim of this work was to assess the ovarian response (preovulatory follicular population, oocyte maturation, ovulation rate, and serum steroid concentrations), the early embryo survival and gene expression patterns of a panel of quality-genes involved in glucose intake, oxidative stress, apoptosis, proliferation, implantation, and fetal growth in embryos of lactating rabbits treated with eCG. A total of 34 primiparous rabbit does (Oryctolagus cuniculus) were randomly distributed at Day 23 postpartum into a treatment group receiving a unique nonsuperovulatory dose (25 IU) of eCG (eCG group; N = 17 does); or a control group without eCG treatment previously to artificial insemination (control group; N = 17 does). After 48 hours, 8 does of each group were euthanized and their ovarian response was studied. The rest of animals were artificially inseminated and their ovulation was induced with a GnRH analogue. Embryos were recovered 3.5 days later. The oocytes retrieved for in vitro maturation showed no differences in metaphase II rate in both experimental groups, although oocyte cytoplasmic maturation, in terms of cortical granule migration rate, was improved in eCG-treated does (P < 0.05). The mean number of preovulatory follicles was similar between groups but the ovulation rate was significantly higher in eCG-treated does compared with does not stimulated (P < 0.05). No differences were found in serum estradiol and progesterone concentrations of does the day of oocyte and embryo recovery, respectively. However, progesterone:estradiol ratio was slightly increased in eCG group on embryo recovery day (P = 0.1). The percentage of embryos recovered at the blastocyst stage was also increased in eCG-treated does (P < 0.05), nevertheless, there were no differences in the gene expression patterns of candidate genes SLC2A4, IGF1R, IGF2R, SHC1-SHC, TP53, PTGS2, and PLAC8; except for the transcripts of SOD1 mRNA which were downregulated in eCG-derived embryos (P < 0.05). In conclusion, the administration of eCG improves ovulation rate, oocyte cytoplasmic maturation, and blastocyst formation in primiparous rabbit does inseminated on Day 25 postpartum. Although it seems not to influence the gene expression patterns studied, a lower antioxidant defense of embryos developed after the maternal eCG treatment is suggested.

Whole Mount &lt;em&gt;in Situ&lt;/em&gt; Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount &lt;em&gt;in Situ&lt;/em&gt; Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Automatically identifying and annotating mouse embryo gene expression patterns

Motivation: Deciphering the regulatory and developmental mechanisms for multicellular organisms requires detailed knowledge of gene interactions and gene expressions. The availability of large datasets with both spatial and ontological annotation of the spatio-temporal patterns of gene expression in mouse embryo provides a powerful resource to discover the biological function of embryo organization. Ontological annotation of gene expressions consists of labelling images with terms from the anatomy ontology for mouse development. If the spatial genes of an anatomical component are expressed in an image, the image is then tagged with a term of that anatomical component. The current annotation is done manually by domain experts, which is both time consuming and costly. In addition, the level of detail is variable, and inevitably errors arise from the tedious nature of the task. In this article, we present a new method to automatically identify and annotate gene expression patterns in the mouse embryo with anatomical terms.Results: The method takes images from in situ hybridization studies and the ontology for the developing mouse embryo, it then combines machine learning and image processing techniques to produce classifiers that automatically identify and annotate gene expression patterns in these images. We evaluate our method on image data from the EURExpress study, where we use it to automatically classify nine anatomical terms: humerus, handplate, fibula, tibia, femur, ribs, petrous part, scapula and head mesenchyme. The accuracy of our method lies between 70% and 80% with few exceptions. We show that other known methods have lower classification performance than ours. We have investigated the images misclassified by our method and found several cases where the original annotation was not correct. This shows our method is robust against this kind of noise.Availability: The annotation result and the experimental dataset in the article can be freely accessed at http://www2.docm.mmu.ac.uk/STAFF/L.Han/geneannotation/.Contact: l.han@mmu.ac.ukSupplementary Information: Supplementary data are available at Bioinformatics online.

Bovine pretransfer endometrium and embryo transcriptome fingerprints as predictors of pregnancy success after embryo transfer

Aberrant gene expression in the uterine endometrium and embryo has been the major causes of pregnancy failure in cattle. However, selecting cows having adequate endometrial receptivity and embryos of better developmental competence based on the gene expression pattern has been a greater challenge. To investigate whether pretransfer endometrial and embryo gene expression pattern has a direct relation with upcoming pregnancy success, we performed a global endometrial and embryo transcriptome analysis using endometrial and embryo biopsy technology and the pregnancy outcome information. For this, endometrial samples were collected from Simmental heifers at day 7 and 14 of the estrous cycle, one cycle prior to embryo transfer. In the next cycle, blastocyst stage embryos were transferred to recipients at day 7 of the estrous cycle after taking 30-40% of the blastocyst as a biopsy for transcriptome analysis. The results revealed that at day 7 of the estrous cycle, the endometrial gene expression pattern of heifers whose pregnancy resulting in calf delivery was significantly different compared with those resulting in no pregnancy. These differences were accompanied by qualitative and quantitative alteration of major biological process and molecular pathways. However, the transcriptome difference was minimal between the two groups of animals at day 14 of the estrous cycle. Similarly, the transcriptome analysis between embryos biopsies that resulted in calf delivery and those resulted in no pregnancy revealed a total of 70 differentially expressed genes. Among these, the transcript levels of 32 genes including SPAG17, PF6, UBE2D3P, DFNB31, AMD1, DTNBP1, and ARL8B were higher in embryo biopsies resulting in calf delivery. Therefore, the present study highlights the potential of pretransfer endometrial and embryo gene expression patterns as predictors of pregnancy success in cattle.

The mammalian transcriptome and the cellular complexity of the brain

The mammalian transcriptome and the cellular complexity of the brain

Articles selected by Faculty of 1000: Xenopus embryo gene expression patterns; detecting SNPs; Arabidopsis circadian clock mutations; Deinococcus radiation resistant genes; Mycobacterium drug resistance

Articles selected by Faculty of 1000: Xenopus embryo gene expression patterns; detecting SNPs; Arabidopsis circadian clock mutations; Deinococcus radiation resistant genes; Mycobacterium drug resistance