79 MicroRNAs of extracellular vesicles secreted by embryos as an early biomarker of competence

2020 ◽  
Vol 32 (2) ◽  
pp. 166
Author(s):  
B. Melo-Baez ◽  
Y. S. Wong ◽  
J. Cabezas ◽  
C. J. Aguilera ◽  
F. O. Castro ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Extracellular Vesicles ◽  
Culture Media ◽  
Blastocyst Stage ◽  
Fold Change ◽  
Target Cells ◽  
Bovine Embryos ◽  
Log2 Fold Change ◽  
Transmission Electron

Extracellular vesicles (EVs), including exosomes and microvesicles, are secreted by different cell types and participate in cellular communication by carrying molecules as microRNAs (miRNAs) that can interfere with gene expression of target cells. Extracellular vesicles have become relevant as a mechanism of embryo-maternal communication. The aim of this study was to evaluate miRNA content in EVs secreted after embryonic genome activation, by bovine embryos with different developmental potential. Bovine embryos were produced invitro and cultured in group until Day 3.5 in synthetic oviductal fluid (SOF) medium. Only 8-16-cell embryos were cultured individually in EVs-depleted SOF until Day 5. The SOF was EV depleted by ultrafiltration. Culture media (CM) were collected at Day 5 and embryos continued in culture until Day 7 with fresh SOF. Collected media were conserved individually and identified with the corresponding embryo. Then, CM were classified according to capacity of its embryo to reach blastocyst stage at Day 7: G1-CM (blocked embryos in 8-16 cell) and G2-CM (embryos that reach blastocyst stage). The EV isolation was carried out using the protocol described by Mellisho et al. (2017). Recovered EVs were evaluated by nanoparticle tracking analysis (NTA), Transmission electron microscopy and the presence of surface markers (CD9, CD63, CD81, and CD40L). After NTA, individual CM were pooled to organise 3 replicates of 10CM each, for G1 and G2. The whole miRNA isolation, library preparation, and sequencing was performed by Norgen Biotek facilities (Canada). The quality of libraries was analysed using the FastQC program platform followed by Trimmomatic to remove remnant adapters. For the miRNA library it accepted reads with value above 30 Phreads and 22 to 30bp length. The reads were mapped against the reference genome ARS-UCD1.2 using Bowtie2 software and miRDeep2 mapper, and the gene counts were calculated using HTSeq. Differential expression analysis was performed in EdgeR package. To expand this information, principal component analysis, Heatmap, and Volcano plot were plotted and pathway enrichment analysis was conducted. The NTA, transmission electron microscopy, and flow cytometry confirmed the presence of exosomes and microvesicles in isolated EVs. According to NTA, the mean size of EVs was 102.1-176.2nm and concentration of 8.4×107-8.6×108 particlesmL−1 in G1 and G2, respectively. We identified 96 miRNAs significantly expressed across the samples. Only eight miRNAs in EVs were differentially expressed between groups (G2 vs. G1). The bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were up-regulated (Log2 fold-change>1), whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were down-regulated (Log2 fold-change<1). The more significant (P-value<0.01) up-regulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The EVs secreted by embryos to culture environment carry miRNAs that can reflect the molecular state of their parental cell. This lets us suggest culture media derived-EVs and their miRNA cargo as early biomarkers to select more competent bovine embryos. This research was supported by FONDECYT, Chile (1170310).

scholarly journals MicroRNAs from Extracellular Vesicles Secreted by Bovine Embryos as Early Biomarkers of Developmental Competence

2020 ◽  
Vol 21 (23) ◽  
pp. 8888
Author(s):  
Bárbara Melo-Baez ◽  
Yat S. Wong ◽  
Constanza J. Aguilera ◽  
Joel Cabezas ◽  
Ana C. F. Mançanares ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Fatty Acid Biosynthesis ◽  
Culture Media ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Target Cells ◽  
Bovine Embryos ◽  
Developmental Potential ◽  
Maternal Communication

During early development, embryos secrete extracellular vesicles (EVs) that participate in embryo–maternal communication. Among other molecules, EVs carry microRNAs (miRNAs) that interfere with gene expression in target cells; miRNAs participate in embryo–maternal communication. Embryo selection based on secreted miRNAs may have an impact on bovine breeding programs. This research aimed to evaluate the size, concentration, and miRNA content of EVs secreted by bovine embryos with different developmental potential, during the compaction period (days 3.5–5). Individual culture media from in vitro–produced embryos were collected at day 5, while embryos were further cultured and classified at day 7, as G1 (conditioned-culture media by embryos arrested in the 8–16-cells stage) and G2 (conditioned-culture media by embryos that reached blastocyst stages at day 7). Collected nanoparticles from embryo conditioned culture media were cataloged as EVs by their morphology and the presence of classical molecular markers. Size and concentration of EVs from G1 were higher than EVs secreted by G2. We identified 95 miRNAs; bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were upregulated in G1, whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were downregulated. The most significant upregulated pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The characteristics of EVs secreted by bovine embryos during the compaction period vary according to embryo competence. Embryos that reach the blastocyst stage secrete fewer and smaller vesicles. Furthermore, the loading of specific miRNAs into the EVs depends on embryo developmental competence.

Monitoring the dynamics of cell-derived extracellular vesicles at the nanoscale by liquid-cell transmission electron microscopy

Nanoscale ◽  
2018 ◽  
Vol 10 (3) ◽  
pp. 1234-1244 ◽  
Author(s):  
Max Piffoux ◽  
Nabeel Ahmad ◽  
Jaysen Nelayah ◽  
Claire Wilhelm ◽  
Amanda Silva ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Extracellular Vesicles ◽  
Transmission Electron ◽  
Cell Transmission ◽  
Liquid Cell

Visualizing the behavior of extracellular vesicles and synthetic liposomes in their native environment at the nanoscale.

scholarly journals Morphological Features andIn VitroCytopathic Effect ofAcanthamoeba griffiniTrophozoites Isolated from a Clinical Case

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Arturo González-Robles ◽  
Lizbeth Salazar-Villatoro ◽  
Maritza Omaña-Molina ◽  
Maria Reyes-Batlle ◽  
Carmen M. Martín-Navarro ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Clinical Case ◽  
Cell Injury ◽  
Cytopathic Effect ◽  
Mdck Cells ◽  
Target Cells ◽  
Small Areas ◽  
Transmission Electron

Light and transmission electron microscopy observations are reported on the structure andin vitrocytopathic effect ofAcanthamoeba griffinitrophozoites isolated from a clinical case. Live trophozoites were moderately active with a remarkable pleomorphism which changed from ovoid to quite elongated shapes. When moving, amoebae formed cytoplasmic projections such as wide lamellae and acanthopodia of diverse size and thickness which contain a significant amount of actin. Ultrastructurally, the cytoplasm showed the main organelles found in other free-living amoebae. Coincubation of trophozoites with MDCK cell monolayers resulted in a local damage to target cells after 24 h of interaction, suggesting that the cytopathic effect is contact-dependent. By transmission electron microscopy, amoebae appeared to engulf small portions of the MDCK cells; however, the cells that were not in contact with trophozoites had an unaltered morphology. When epithelial monolayers were incubated with conditioned medium for 24 h, small areas of cell injury were also observed. The phylogenetical analysis as well as the sequencing of the acquired amplified product for the DF3 region of the amoebae isolate confirmed that it belongs to genotype T3, which includes other pathogenic amoebae; besides the activity of two drugs currently used againstAcanthamoebawas tested onA. griffini.

52 Blocking of embryonic development by nanoparticles derived from endometrial and oviductal cells isolated with an Amicon filter system

2020 ◽  
Vol 32 (2) ◽  
pp. 151
Author(s):  
J. Cabezas ◽  
D. Rojas ◽  
B. Melo-Baez ◽  
M. Gutierrez ◽  
F. Castro ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Embryonic Development ◽  
Embryo Development ◽  
Extracellular Vesicles ◽  
Primary Cultures ◽  
Nanoparticle Tracking Analysis ◽  
Endometrial Cells ◽  
Filter System ◽  
Transmission Electron

The success of development of invitro embryo production needs to mimic culture conditions in the maternal environment. Recently, it has been seen that extracellular vesicles (EVs) secreted by oviducal or endometrial cells may improve development and quality of embryos produced invitro. Extracellular vesicles are a mechanism of cellular communication; they carry molecules that are delivered into the target cells changing gene expression and function. Due to the size range and characteristics of EVs, they require specific methods for purification and characterisation. However, the possible contamination with other nanoparticles and their effect on embryo development have not been considered. Based on that, the goal of this work was to evaluate the effect on invitro bovine embryo development, of the addition to culture medium EVs secreted by oviducal and endometrial cells and isolated by centrifugation and concentrates with Amicon filters. For this purpose, cells were isolated from bovine oviduct and endometrium collected in local abattoir and primary cultures of epithelial and stromal cells were derived. The primary cultures from both sources were exposed or not to progesterone (P4; 15ngmL−1) for 4 days and then cultured for 24h in EV depleted media. The supernatant was harvested and EVs were isolated by serial centrifugations and subsequently concentrated by a 100 kDa Amicon filter system. The isolated EVs were characterised by transmission electron microscopy, nanoparticle tracking analysis, and flow cytometry. Oocytes were obtained from ovaries collected in the abattoir. The cumulus-oocyte complexes were matured invitro for 22h and subsequently fertilised for 18h. Presumptive zygotes were invitro cultured in synthetic oviducal fluid with EVs (1000ngmL−1 of total proteins) or not according to experimental group (1: EVs− (control); 2: EVs−OP4+; 3: EVs−OP4−; 4: EVs−EP4+ and 4: EVs−EP4−). Embryos were cultured for 7 days in 5% CO2, 5% O2, and 90% N2 (25 embryos/well in 4-well plates). At Day 7, embryo development was evaluated considering the blastocyst yield. Transmission electron microscopy showed typical structures and morphology of EVs and they were positive for CD9, CD63, and CD81 markers, and negative for CD40. According to nanoparticle tracking analysis, the mean size of EVs was 160±62nm and concentration of 3.29×1011 particlesmL−1 for oviducal and endometrial cells, respectively. A significant reduction of blastocyst rate was observed when embryos were cultured with cell-derived EVs; control: 152/44 (28.9%) vs. treatments with EVs; OP4+: 74/3 (4.1%), OP4−:76/2 (2.6%), EP4+: 74/6 (8.1%), and EP4−: 73/2 (2.7%) (P ≤ 0.01). Our results indicate that the use of nanoparticles, including EVs, isolated from cells of oviduct or endometrium, has a blocking effect on embryonic development and compromises the performance of blastocysts on Day 7 when used at concentrations of 1000ngmL−1 total protein, independent of the use or not of P4 and the source. These data provide insights regarding the use and protocols of acquiring exosomes for embryo supplementation. This research was supported by FONDECYT, Chile-1170310.

76 Extracellular vesicles from oviduct and uterus in sequential culture improve the quality of bovine embryos produced invitro

2020 ◽  
Vol 32 (2) ◽  
pp. 164
Author(s):  
C. Leal ◽  
K. Cañon-Beltrán ◽  
Y. Cajas ◽  
P. Gallego ◽  
P. Beltrán-Breña ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Lipid Content ◽  
Luteal Phase ◽  
Cell Number ◽  
Total Cell ◽  
Bovine Embryos ◽  
Total Cell Number ◽  
Transmission Electron

Extracellular vesicles (EVs) are released by cells and transport cargo that affect functions of other cells. Oviductal fluid (OF) and uterine fluid (UF) have been shown to improve quality of embryos during invitro culture (Hamdi et al. 2017 Reprod. Fertil. Dev. 30, 935-945) which may be due to their content of EV (Lopera-Vásquez et al. 2017 Reproduction 153, 461-470). Thus, the aim of this study was to evaluate the effect of EVs from OF and UF on a sequential invitro culture system on the development and quality of bovine embryos. Zygotes were cultured in synthetic oviduct fluid (SOF) supplemented with 3mgmL−1 bovine serum albumin (BSA; n=1228) or 5% EV-depleted fetal calf serum (dFCS, n=1261) in the presence (BSAEV, n=1265 and dFCSEV, n=1253) or absence of 3×105 EVmL−1 from OF (Day 1 to Day 4) and UF (Day 4 to Day 9), mimicking invivo conditions. The EVs pooled from 5 oviducts (early luteal phase) and 5 uterine horns (middle luteal phase) from slaughtered heifers were isolated by a Size Exclusion Chromatography kit (Hansa BioMed). The EV size and concentration were assessed by the nanotracking analysis system and morphology by transmission electron microscopy. Embryo development was recorded on Days 7/9. Day 7/8 blastocysts were assessed for quality by staining with (a) Hoechst 33342 (10 µgmL−1, 30min) for total cell number, (b) Bodipy 493/503 (20 µgmL−1, 1h) for lipid content (lipid droplet area in µm2), and (c) for survival rate after vitrification/warming. Data were analysed by one-way ANOVA and Tukey test. The EV concentration was 2.97 and 7.98×1010 particlesmL−1, and mode size 137.2 and 151.2nm for OF and UF, respectively. Transmission electron microscopy confirmed EV presence and size, showing typical cup-shaped morphology. Blastocyst yield was lower (P<0.05) on Day 7 in the BSA groups (BSA: 15.7±1.9 and BSAEV: 15.2.4%) compared with serum groups (dFCS: 28.1±2.6 and dFCSEV: 30.1±2.9%) irrespective of EV supplementation; however, these differences were compensated at Days 8 and 9 (range: 30.0±3.2-40.8±3.9%). The EVs increased (P<0.05) blastocyst total cell number in dFCSEV (152.6±2.9) and BSAEV (140.5±1.5) compared with dFCS (117.9±2.0) and BSA groups (122.4±1.1). However, lipid content was decreased (P<0.05) in the presence of EVs only in dFCSEV (0.231±0.05µm2) compared with BSA (0.393±0.03µm2) and BSAEV (0.379±0.03µm2) groups. The dFCS did not differ from any group (0.371±0.05µm2; P>0.05). Blastocyst survival after vitrification/warming was high in all groups up to 72h (range: 80.0±3.8-100%; P>0.05). In conclusion, mimicking physiological conditions using EV from OF and UF during invitro culture does not affect development but improves embryo quality by increasing blastocyst total cell numbers and decreasing lipid contents. These results provide evidence of the association of the reproductive tract environment and developing embryo, confirming embryo-maternal communication. Funding was provided by MINECO-Spain AGL2015-70140-R; Y. N. Cajas, SENESCYT-Ecuador; C. L. V. Leal, FAPESP-Brazil 2017/20339-3.

scholarly journals Deep-Learning-Based Segmentation of Small Extracellular Vesicles in Transmission Electron Microscopy Images

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Estibaliz Gómez-de-Mariscal ◽  
Martin Maška ◽  
Anna Kotrbová ◽  
Vendula Pospíchalová ◽  
Pavel Matula ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Deep Learning ◽  
Extracellular Vesicles ◽  
Physiological Condition ◽  
State Of The Art ◽  
Automatic Quantification ◽  
Transmission Electron ◽  
Complex Procedure ◽  
Microscopy Images

Abstract Small extracellular vesicles (sEVs) are cell-derived vesicles of nanoscale size (~30–200 nm) that function as conveyors of information between cells, reflecting the cell of their origin and its physiological condition in their content. Valuable information on the shape and even on the composition of individual sEVs can be recorded using transmission electron microscopy (TEM). Unfortunately, sample preparation for TEM image acquisition is a complex procedure, which often leads to noisy images and renders automatic quantification of sEVs an extremely difficult task. We present a completely deep-learning-based pipeline for the segmentation of sEVs in TEM images. Our method applies a residual convolutional neural network to obtain fine masks and use the Radon transform for splitting clustered sEVs. Using three manually annotated datasets that cover a natural variability typical for sEV studies, we show that the proposed method outperforms two different state-of-the-art approaches in terms of detection and segmentation performance. Furthermore, the diameter and roundness of the segmented vesicles are estimated with an error of less than 10%, which supports the high potential of our method in biological applications.

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