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Oviductal Extracellular Vesicles Enhance Porcine In Vitro Embryo Development by Modulating the Embryonic Transcriptome.

ABSTRACT: Oviductal extracellular vesicles (oEVs) have been identified as important components of the oviductal fluid (OF) and have been pointed to as key modulators of gamete/embryo-maternal interactions. Here, we determined the functional impact of oEVs on embryo development and the embryonic transcriptome in porcine. Experiment 1 examined the effect of oEVs and OF on embryo development. In vitro-produced embryos were cultured with oEVs or OF for 2 or 7 days using an in vitro sequential system or without supplementation (control). Experiment 2 analyzed transcriptomic alterations of EV-treated embryos versus control and the oEVs RNA cargo by RNA-sequencing. Two days of EV treatment enhanced embryo development over time when compared to other treatments. Different RNA expression profiles between embryos treated with EVs for two or seven days and untreated controls were obtained, with 54 and 59 differentially expressed (DE) genes and six and seven DE miRNAs, respectively. In oEV RNA cargo, 12,998 RNAs and 163 miRNAs were identified. Integrative analyses pointed to specific oEV components that might act as modulators of the embryonic transcriptome, such as S100A11, ANXA2 or miR-21-5p. Overall, the findings suggested that oEVs could be a potential strategy to improve porcine IVP outcomes, particularly by using two days of EV treatment.

SUBMITTER: de Alcantara-Neto AS

PROVIDER: S-EPMC9496104 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Oviductal Extracellular Vesicles Enhance Porcine In Vitro Embryo Development by Modulating the Embryonic Transcriptome.

de Alcântara-Neto Agostinho Soares AS Cuello Cristina C Uzbekov Rustem R Bauersachs Stefan S Mermillod Pascal P Almiñana Carmen C

Biomolecules 20220915 9

Oviductal extracellular vesicles (oEVs) have been identified as important components of the oviductal fluid (OF) and have been pointed to as key modulators of gamete/embryo-maternal interactions. Here, we determined the functional impact of oEVs on embryo development and the embryonic transcriptome in porcine. Experiment 1 examined the effect of oEVs and OF on embryo development. In vitro-produced embryos were cultured with oEVs or OF for 2 or 7 days using an in vitro sequential system or withou ...[more]

PMID: 36139139

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Project description:BackgroundExtracellular vesicles (EVs) and their cargoes, including MicroRNAs (miRNAs) play a crucial role in cell-to-cell communication. We previously demonstrated the upregulation of bta-mir-148b in EVs from oviductal fluid of cyclic cows. This miRNA is linked to the TGF-β pathway in the cell proliferation. Our aim was to verify whether miR-148b is taken up by embryos through gymnosis, validate its target genes, and investigate the effect of miR-148b supplementation on early embryo development and quality.MethodsZygotes were cultured in SOF + 0.3% BSA (Control) or supplemented with: 1 µM miR-148b mimics during: D1-D7 (miR148b) or D1-D4 (miR148b-OV: representing miRNA effect in the oviduct) or D4-D7 (miR148b-UT: representing miRNA effect in the uterus) or 1 µM control mimics was used during: D1-D7 (CMimic). Embryos at ≥ 16-cells and D7 blastocysts (BD7) were collected to examine the mRNA abundance of transcripts linked to the TGF-β pathway (TGFBR2, SMAD1, SMAD2, SMAD3, SMAD5, BMPR2, RPS6KB1, POU5F1, NANOG), total cell number (TC), trophectoderm (TE), and inner cell mass (ICM) were also evaluated. One-way ANOVA was used for all analyses.ResultsWe demonstrated that miR-148b can be taken up in both 16-cell embryos and BD7 by gymnosis, and we observed a decrease in SMAD5 mRNA, suggesting it's a potential target of miR-148b. Cleavage and blastocysts rates were not affected in any groups; however, supplementation of miR-148b mimics had a positive effect on TC, TE and ICM, with values of 136.4 ± 1.6, 92.5 ± 0.9, 43.9 ± 1.3 for miR148b and 135.3 ± 1.5, 92.6 ± 1.2, 42.7 ± 0.8, for miR148b-OV group. Furthermore, mRNA transcripts of SMAD1 and SMAD5 were decreased (P ≤ 0.001) in 16-cell embryos and BD7 from miR148b and miR148b-OV groups, while POU5F1 and NANOG were upregulated (P ≤ 0.001) in BD7 and TGFBR2 was only downregulated in 16-cell embryos. pSMAD1/5 levels were higher in the miR148b and miR148b-OV groups.ConclusionsOur findings suggest that supplementation of bta-miR-148b mimics during the entire culture period (D1 - D7) or from D1 - D4 improves embryo quality and influences the TGF-β signaling pathway by altering the transcription of genes associated with cellular differentiation and proliferation. This highlights the importance of miR-148b on embryo quality and development.

Project description:BackgroundIn vitro production of bovine embryos is a well-established technology, but the in vitro culture (IVC) system still warrants improvements, especially regarding embryo quality. This study aimed to evaluate the effect of extracellular vesicles (EVs) isolated from oviductal (OF) and uterine fluid (UF) in sequential IVC on the development and quality of bovine embryos. Zygotes were cultured in SOF supplemented with either BSA or EVs-depleted fetal calf serum (dFCS) in the presence (BSA-EV and dFCS-EV) or absence of EVs from OF (D1 to D4) and UF (D5 to D8), mimicking in vivo conditions. EVs from oviducts (early luteal phase) and uterine horns (mid-luteal phase) from slaughtered heifers were isolated by size exclusion chromatography. Blastocyst rate was recorded on days 7-8 and their quality was assessed based on lipid contents, mitochondrial activity and total cell numbers, as well as survival rate after vitrification. Relative mRNA abundance for lipid metabolism-related transcripts and levels of phosphorylated hormone-sensitive lipase (pHSL) proteins were also determined. Additionally, the expression levels of 383 miRNA in OF- and UF-EVs were assessed by qRT-PCR.ResultsBlastocyst yield was lower (P < 0.05) in BSA treatments compared with dFCS treatments. Survival rates after vitrification/warming were improved in dFCS-EVs (P < 0.05). EVs increased (P < 0.05) blastocysts total cell number in dFCS-EV and BSA-EV compared with respective controls (dFCS and BSA), while lipid content was decreased in dFCS-EV (P < 0.05) and mitochondrial activity did not change (P > 0.05). Lipid metabolism transcripts were affected by EVs and showed interaction with type of protein source in medium (PPARGC1B, LDLR, CD36, FASN and PNPLA2, P < 0.05). Levels of pHSL were lower in dFCS (P < 0.05). Twenty miRNA were differentially expressed between OF- and UF-EVs and only bta-miR-148b was increased in OF-EVs (P < 0.05).ConclusionsMimicking physiological conditions using EVs from OF and UF in sequential IVC does not affect embryo development but improves blastocyst quality regarding survival rate after vitrification/warming, total cell number, lipid content, and relative changes in expression of lipid metabolism transcripts and lipase activation. Finally, EVs miRNA contents may contribute to the observed effects.

Project description:To evaluate the effect of conditioned media (CM) and Extracellular Vesicles (EVs) derived from bovine oviduct epithelial cell (BOEC) lines on the developmental capacity of bovine zygotes and the quality of embryos produced in vitro, presumptive zygotes were cultured under specific conditions. In experiment 1, zygotes were cultured either on monolayers from BOEC extended culture (E), together with fresh BOEC suspension cells, or with BOEC-CM from fresh or E-monolayers. In experiment 2, EVs were isolated from BOEC-CM and characterized (150-200 nm) by Nanosight® and electron microscopy. Zygotes were cultured in the presence of 3x10(5) EVs/mL, 1.5x10(5) EVs/mL or 7.5x10(4) EVs/mL of fresh or frozen BOEC-EVs. In experiment 3, zygotes were cultured in absence of FCS but with EVs from BOEC-E that had been cultured in different culture media. In experiment 4, zygotes were cultured in SOF+5% normal-FCS, or EV-depleted-FCS. In all cases, cleavage rate (Day 2) and blastocyst development (Day 7-9) was assessed. Blastocysts on Days 7/8 were used for quality evaluation through differential cell count, cryotolerance and gene expression patterns. No differences were found among all FCS-containing groups in cleavage rate or blastocyst yield. However, embryos derived from BOEC-CM had more trophectoderm cells, while embryos derived from BOEC-EVs, both fresh and frozen, has more trophectoderm and total cells. More embryos survived vitrification in the BOEC-CM and BOEC-EV groups. In contrast, more embryos survived in the EV-depleted-FCS than in normal-FCS group. Gene expression patterns were modified for PAG1 for embryos cultured with EVs in the presence of FCS and for IFN-T, PLAC8, PAG1, CX43, and GAPDH in the absence of FCS. In conclusion, EVs from FCS have a deleterious effect on embryo quality. BOEC-CM and EVs during in vitro culture had a positive effect on the quality of in vitro produced bovine embryos, suggesting that EVs have functional communication between the oviduct and the embryo in the early stages of development.

Dataset Information

Oviductal Extracellular Vesicles Enhance Porcine In Vitro Embryo Development by Modulating the Embryonic Transcriptome.

Publications

Oviductal Extracellular Vesicles Enhance Porcine In Vitro Embryo Development by Modulating the Embryonic Transcriptome.

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