1 Preimplantation bovine embryos secrete extracellular vesicles that participate in embryo-maternal communication

2022 ◽  
Vol 34 (2) ◽  
pp. 234
Author(s):  
C. Aguilera ◽  
A. E. Velásquez ◽  
Y. Wong ◽  
M. A. Gutierrez-Reinoso ◽  
J. Cabezas ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Bovine Embryos ◽  
Maternal Communication

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.

88 MicroRNA profile of invitro bovine embryos cultured in the presence of amniotic extracellular vesicles shifts toward invivo-collected blastocysts

2020 ◽  
Vol 32 (2) ◽  
pp. 170
Author(s):  
A. Lange-Consiglio ◽  
B. Lazzari ◽  
F. Pizzi ◽  
A. Idda ◽  
F. Cremonesi ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Extracellular Vesicles ◽  
Embryo Implantation ◽  
Principal Component ◽  
Rna Isolation ◽  
Differentially Expressed ◽  
Bovine Embryos ◽  
Total Rna ◽  
Mirna Profiling ◽  
Genomic Study

The absence of maternal-embryo signals could be an important cause of the poor pregnancy rates of invitro-produced embryos, compared with those collected invivo. In the context of paracrine communication, co-culture of embryo with amniotic-derived extracellular vesicles (EVs) improved their quality compared with control (CTR) (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671), and after cryopreservation, provided higher invitro embryo hatching and recipient pregnancy rate (Lange-Consiglio et al. 2019 Reprod. Fertil. Dev. 31, 155). After these results, the aim of this study was to evaluate microRNA (miRNA) profiling of invitro-produced blastocysts with or without EV supplementation, using invivo-produced blastocysts as CTR. Invitro embryos were produced based on our protocol (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671) with or without 100×106 EVsmL−1 in synthetic oviductal fluid with amino acids (SOFaa) on Day 5 post-fertilisation (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671). Grade 1 blastocysts (B7) were immediately snap frozen in liquid nitrogen for genomic study. These embryos were obtained from three replicates. Invivo embryos were obtained from three cows superovulated by Folltropin and inseminated by the same cryopreserved semen. After flushing, only B7 were snap frozen for genomic study. Samples for RNA isolation were obtained from 3 pools of 10 embryos each for each condition (vivo, vitro-CTR, and vitro+EVs). Total RNA was isolated by a NucleoSpin1 miRNA kit. Concentration and quality of RNA were determined by an Agilent 2100 Bioanalyzer. Libraries were prepared using TruSeq Small RNA Library Preparation kits (Illumina). Differential expression analyses between samples were run with the Bioconductor edgeR package (false discovery rate<0.05). MicroRNA cluster analysis was performed with Genesis. The average quantity of total RNA extracted from each pool was 3.5ng. Our results show that the miRNAs identified were 1.74E5, 2.3E5, and 3.6E5 for vivo, vitro-CTR, and vitro+EVs, respectively. Principal component analysis calculated on differentially expressed miRNAs showed a separation of the three groups with a distinctive miRNA trait. The miRNAs differentially expressed among three comparisons (vivo vs. vitro-CTR, vivo vs. vitro+EVs, and vitro-CTR vs. vitro+EVs) were 20, 15, and 2, respectively. Principal component 1, which explains 62.4% of the variance, clearly separates invivo- and invitro-produced embryos even if EV addition seems to ameliorate the effect of invitro production, and this agrees with the embryo quality and pregnancy rate after EV supplementation (Perrini and Lange Consiglio 2018 Reprod. Fertil. Dev. 30, 658-671; Lange-Consiglio et al. 2019 Reprod. Fertil. Dev. 31, 155). Indeed, vitro-CTR and vitro+EVs embryos differ significantly for two miRNAs (miR-130a, miR-181b) that are found to be higher in our vitro-CTR embryos compared with vitro+EV ones. The miR-181b was also found to be higher in degenerate bovine embryos compared with good blastocysts (Kropp et al. 2014 Front. Genetics 24, 91). In conclusion, this is the first study reporting the complete miRNA profiling of invitro blastocysts compared with those obtained invivo. The addition of EVs during invitro production seems to influence the expression of specific miRNAs involved in the success of embryo implantation.

Signs of embryo-maternal communication: miRNAs in the maternal serum of pregnant pigs

Reproduction ◽  
2017 ◽  
Vol 154 (3) ◽  
pp. 217-228 ◽  
Author(s):  
Z P Reliszko ◽  
Z Gajewski ◽  
M M Kaczmarek
Keyword(s):  
Extracellular Vesicles ◽  
Dynamic Range ◽  
Target Prediction ◽  
Digital Pcr ◽  
Maternal Serum ◽  
Biological Processes ◽  
Circulating Mirnas ◽  
Maternal Communication ◽  
First Time ◽  
Reliable Methods

Circulating miRNAs were proposed to be indicators of normal or complicated pregnancies. Based on this knowledge and our recent transcriptomic approach showing expression of miRNAs in the porcine endometrium, conceptuses and uterine extracellular vesicles during pregnancy, we have hypothesized that signs of ongoing local embryo-maternal crosstalk involving miRNAs can be detected in the circulation of pregnant gilts as early as a few days after maternal recognition of pregnancy. By applying several molecular biology techniques that differ in dynamic range and precision in maternal serum of Day 16 pregnant pigs, we were able to show for the first time increased levels of several miRNAs, previously reported to be expressed in either conceptuses and extracellular vesicles (miR-26a and miR-125b) or pregnant endometrium (miR-23b). Our results clearly showed that real-time RT-PCR and digital PCR are the most reliable methods, being able to detect small-fold changes of low-abundant circulating miRNAs. Further validation in a separate group of gilts confirmed an increase in miR-23b and miR-125b levels.In silicoanalyses identified pregnancy-related biological processes and pathways affected by these miRNAs. Target prediction analysis revealed hundreds of porcine transcripts with conserved sites for these miRNAs, which were classified into signaling pathways relevant to pregnancy. We conclude that a unique set of miRNAs can already be observed in the circulation of pigs during the first weeks of pregnancy, as a result of the initiation of embryo-maternal communication.

scholarly journals Extracellular vesicles secreted during blastulation show viability of bovine embryos

Reproduction ◽  
2019 ◽  
Vol 158 (6) ◽  
pp. 477-492 ◽  
Author(s):  
Edwin A Mellisho ◽  
Mario A Briones ◽  
Alejandra E Velásquez ◽  
Joel Cabezas ◽  
Fidel O Castro ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Culture Media ◽  
Differential Expression Analysis ◽  
Binary Logistic Regression ◽  
Small Rna Sequencing ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Group V ◽  
Mean Size

Extracellular vesicles (EVs) secreted by blastocysts may be clinically relevant, as indicator of embryo viability on in vitro fertilization. We tested if the characteristics of EVs secreted during blastulation are related to embryo viability. Morulae were individually cultured in SOF media depleted of EVs until day 7.5 post IVF. Viable embryos were determined by a system of extended in vitro culture of bovine embryos until day 11 (post-hatching development). Afterward, a retrospective classification of blastocyst and culture media was performed based on blastulation time (early blastulation (EB) or late blastulation (LB)) and post-hatching development at day 11 (viable (V) or non-viable embryo (NV)). A total of 254 blastocysts and their culture media were classified in four groups (V-EB, NV-EB, V-LB, NV-LB). Group V-EB had a larger blastocyst diameter (170.8 μm), higher proportion of good-quality blastocysts (77%) and larger mean size of population of EVs (122.9 nm), although the highest concentration of EVs (5.75 × 109 particles/mL) were in group NV-EB. Furthermore, small RNA sequencing detected two biotypes, miRNA (86–91%) and snoRNA (9–14%), with a total of 182 and 32 respectively. In differential expression analysis of miRNAs between V versus NV blastocysts, there were 12 miRNAs upregulated and 15 miRNAs downregulated. Binary logistic regression was used to construct a non-invasive novel model to select viable embryos, based on a combination of variables of blastocyst morphokinetics and EVs characteristics, the ROC-AUC was 0.853. We concluded that characteristics of EVs secreted during blastulation vary depending on embryo quality.

139 BOVINE PREIMPLANTATION EMBRYOS SECRETE EXTRACELLULAR VESICLES, WHICH MAY INDICATE EMBRYO COMPETENCE

2017 ◽  
Vol 29 (1) ◽  
pp. 178
Author(s):  
E. Mellisho ◽  
A. Velasquez ◽  
M. J. Nuñez ◽  
L. Rodriguez-Alvarez
Keyword(s):  
Extracellular Vesicles ◽  
Culture Media ◽  
Total Cell ◽  
Developmental Competence ◽  
Preimplantation Embryos ◽  
Lower Amount ◽  
Bovine Embryos ◽  
Significant Difference ◽  
Blastulation Rate

Pre-implantation embryos secrete extracellular vesicles (EV) most likely to communicate with the surroundings. The objective of this study was to determine the distribution (size and concentration) of EV secreted by bovine pre-implantation embryos with different developmental competence. The IVF bovine embryos were produced from oocytes recovered from slaughterhouse ovaries. Presumptive zygotes were in vitro cultured (IVC) in groups in 4-well plates (30 zygotes per 500-µL well) using SOFaa medium at 39°C under 5% CO2, 5% O2, and 90% N2 until the morula stage (Day 5 post IVF). Morulae were cultured individually in 96 well at 39°C under until blastulation time (Day 6.5–7.5) in EV-free SOF medium. Culture medium was collected only from embryos that developed to the blastocyst stage that were classified in a group of early (Day 6.5) or late (Day 7.5) blastulation. Blastocysts were kept in culture until Day 11 to assess embryo developmental competence, considering embryo size (>350 µm) and total cell count (>500 blastomeres). For EV analysis, 4 groups were organised a posteriori: G1: Day 6.5-competent; G2: Day 6.5-not competent; G3: Day 7.5-competent; G4: Day 7.5-not competent. The EV in culture media were analysed using a nanoparticle tracking analysis (Nanosight NS300). Statistical analysis was performed using the InfoStat program (Buenos Aires, Argentina). Differences were considered significant at P < 0.05. Early blastulation rate (Day 6.5) was 40.3% (112/278), whereas late blastulation rate (Day 7.5) was 20.5% (57/278), showing a significant difference (P < 0.0001). Embryos derived from Day 6.5 blastocysts have a higher probability (39.3%: 44/112) of posthatching development [until Day 11; Day 7.5, 10.5% (6/57); P = 0.0001]. At Day 11, competent embryos (G1) derived from Day 6.5 blastocysts have a higher diameter and total cell number (447 µm; 688 cells) than those derived from Day 7.5 blastocysts (G3; 405 µm, 598 cells; P < 0.05 for both parameters). It was possible to detect EV from collected medium of individual embryos independent of their competence. Neither the EV size nor the EV concentration was statistically different between Day 6.5 and Day 7.5 blastocysts (without considering their further competence; 2.9 × 108, 147 nm; and 3.0 × 108, 149 nm, respectively). However, independent of the day of blastulation, competent embryos had a significantly lower concentration of EV (2.7 × 108 v. 3.3 × 108; P = 0.03). Moreover, competent embryos from early and late blastocysts (G1 and G3) tend to produce a lower amount of EV (G1: 2.8 × 108; G2: 3 × 108; G3: 2.6 × 108; G4: 3.5 × 108; P = 0.05). Furthermore, EV concentration was statistically different between G3 and G4 (P = 0.002). No differences in EV size were observed among groups (G1: 145 nm; G2: 148 nm; G3: 146 nm; G4: 151 nm). Our results provide an initial approach to study the EV secreted by individual pre-implantation embryos to assess their competence. From these results, we can conclude that blastulation time affects the future development of bovine embryos and a model based on blastulation time and EV secretion could be a simple noninvasive tool to improve embryo selection.

Placental miRNAs in feto-maternal communication mediated by extracellular vesicles

Placenta ◽  
2020 ◽  
Vol 102 ◽  
pp. 27-33 ◽  
Author(s):  
Diana M. Morales-Prieto ◽  
Rodolfo R. Favaro ◽  
Udo R. Markert
Keyword(s):  
Extracellular Vesicles ◽  
Maternal Communication

67 Optimizing a protocol for isolating extracellular vesicles from medium conditioned by bovine embryos in vitro

2019 ◽  
Vol 31 (1) ◽  
pp. 159
Author(s):  
K. C. Pavani ◽  
A. Hendrix ◽  
B. Leemans ◽  
A. Van Soom
Keyword(s):  
Conditioned Medium ◽  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Downstream Processing ◽  
Size Exclusion ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Isolation Method ◽  
Exclusion Chromatography

In the absence of the maternal tract, pre-implantation bovine embryos cultured in group are able to promote their own development in vitro by releasing autocrine embryotropins. Recently we have identified extracellular vesicles (EV) among these embryotropins as one of the communication mechanisms among embryos. Extracellular vesicles are nano-sized (25-250nm), with a lipid bilayer, and are functionally active, since they contain proteins, lipids, and nucleic acids, including RNA and miRNA. However, one of the major challenges in isolating EV is an inadequate volume of medium conditioned by bovine embryo. As it requires larger volumes of conditioned medium to isolate EV, our study mainly focused on isolating high yields of functional EV from a minimal volume. There are 3 known isolation methods for EV: differential ultracentrifugation (DU), OptiPrep™ density gradient ultracentrifugation (ODGU), and size-exclusion chromatography (SEC). We have used these 3 protocols to determine the method that yielded the highest number of EV. We used routine in vitro maturation and fertilization methods, but for in vitro culture presumed zygotes were cultured until 8 days post-insemination (dpi) in medium (synthetic oviducal fluid supplemented with insulin, transferrin, selenium, and bovine serum albumin) that was ultracentrifuged to remove any possible contaminating EV. In vitro embryo culture took place in groups of 25 presumed zygotes in 50-mL drops, covered with mineral oil and incubated at 38°C in 5% CO2, 5% O2, and 90% N2. On 8 dpi, medium conditioned by bovine embryo was collected and pooled until 3mL. For each isolation method, 1mL of conditioned medium was used, and next, EV isolated from each isolation method were analysed with nanoparticle tracking, electron microscopy, and Western blot (CD9, Flotillin 1, and AGO 2). We observed higher concentrations (1.03×109 particles mL−1) of EV were isolated from the SEC compared with the other 2 methods (301.5×108 particles mL−1 and 64.5×108 particles mL−1 for DU and ODGU, respectively; P&lt;0.05), whereas smaller size EV (20-50nm) were lost during the ultracentrifugation methods. Besides, it takes only 2h of time to perform size-exclusion chromatography for isolating EV, whereas it takes more than 1 day to perform ultracentrifugation methods. Therefore, we propose to use SEC for further downstream processing and sequencing of miRNA in isolated EV. We are currently focusing on optimizing an EV isolation protocol to extract EV from very low volumes of conditioned medium (less than 500 µL).

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&gt;1), whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were down-regulated (Log2 fold-change&lt;1). The more significant (P-value&lt;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).

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