The Oviductal Extracellular Vesicles' RNA Cargo Regulates the Bovine Embryonic Transcriptome.
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ABSTRACT:
SUBMITTER: Bauersachs S
PROVIDER: S-EPMC7072903 | biostudies-literature | 2020 Feb
REPOSITORIES: biostudies-literature
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The Oviductal Extracellular Vesicles' RNA Cargo Regulates the Bovine Embryonic Transcriptome.
Bauersachs Stefan S Mermillod Pascal P Almiñana Carmen C
International journal of molecular sciences 20200214 4
Oviductal extracellular vesicles (oEVs) are emerging as key players in the gamete/embryo-oviduct interactions that contribute to successful pregnancy. Various positive effects of oEVs on gametes and early embryos have been found in vitro. To determine whether these effects are associated with changes of embryonic gene expression, the transcriptomes of embryos supplemented with bovine fresh (FeEVs) or frozen (FoEVs) oEVs during in vitro culture compared to controls without oEVs were analyzed by l ...[more]
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Project description:While follicular fluid (FF) is well known to provide an optimal environment for oogenesis, its functional roles following its release into the oviduct during ovulation are currently elusive. We hypothesized that FF and FF-derived extracellular vesicles (EVs) may be conveyors of signals capable of inducing functionally-relevant transcriptional responses in oviductal cells. The aim of this study was, therefore, to evaluate the effect of FF and FF-derived EVs on the transcriptome of primary bovine oviductal epithelial cells (BOECs). We examined the gene expression of BOECs in three conditions: BOECs cultured with FF, FF-derived EVs, and without supplementations. For each condition, cells were cultured for 6 and 24 h. RNA sequencing results revealed that FF had a stronger effect on BOECs gene expression compared to EVs. We detected 488 and 1998 differentially expressed genes (DEGs) with FF treatment in 6 and 24 h, respectively, whereas only 41 DEGs were detected at 6 h following EV treatment. Pathway analysis of the FF-induced DEGs showed that several pathways were highly enriched, notably oxidative phosphorylation, thermogenesis, arachidonic acid metabolism, and steroid hormone biosynthesis. Some of these pathways have a role in sperm survival, fertilization, and early embryo development. In conclusion, the findings of our study demonstrate for the first time that bovine FF and FF-derived EVs can induce changes in the gene expression of the bovine oviductal cells which, although observed in vitro, may be reflective of in vivo responses which may contribute to a favorable periconceptional microenvironment for sperm survival, fertilization, and early embryo development.
| S-EPMC7432463 | biostudies-literature
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.
| S-EPMC10960404 | biostudies-literature
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.
| S-EPMC9594899 | biostudies-literature
Project description:Oviductal extracellular vesicles (oEVs) are emerging as key players in gamete/embryo-oviduct interactions contributing to successful pregnancy. Various positive effects of oEVs on gametes and early embryos have been found in vitro. To find out if these effects are associated with changes of embryonic gene expression, the embryonic transcriptome was analyzed after supplementation of bovine embryos with bovine oEVs during in vitro culture (IVC) in comparison to corresponding controls without oEVs. Embryos were cultured during 7 days of IVC with fresh oEVs, oEVs frozen after isolation from oviductal fluid or without oEVs (control), since a previous study showed different effects of fresh and frozen oEVs. Five pools of 10-14 embryos were analyzed for each group by low-input Illumina RNA-sequencing. Analysis of differential gene expression revealed 221 differentially expressed genes (DEGs) for the comparison of frozen oEVs vs control, 67 DEGs for fresh vs frozen oEVs, and only minor differences for the comparison fresh oEVs vs control (28 DEGs). An integrative analysis with the previously studied mRNA and small ncRNA content of bovine oEVs suggested direct effects of oEVs content on the embryonic transcriptome for the supplementation with frozen oEVs, i.e., increase of transcripts found in higher concentrations in oEVs and decrease of transcripts which are potential targets of miRNAs found in oEVs.
2020-03-21 | GSE143596 | GEO
Project description:Extracellular vesicles (EVs) and their microRNA (miRNA) cargo have been proposed as possible mammary gland health biomarkers in cattle. However, throughout the day, the biologically active milk components, such as miRNAs, may change due to the dynamic nature of milk. The current study aimed to evaluate the circadian fluctuation of milk EVs miRNA cargo to assess the feasibility of milk EVs as future biomarkers for mammary gland health management. Milk from four healthy dairy cows was collected for four consecutive days in the two daily milking sessions in the morning and the evening. The isolated EVs were heterogeneous, intact, and carried the EV protein markers CD9, CD81, and TSG101, as shown by transmission electron microscopy and western blot. The miRNA sequencing results demonstrate that the abundance of miRNA cargo in milk EVs remained stable, unlike other milk components, such as somatic cells, that changed during milking sessions. These findings indicated that the miRNA cargo within milk EVs remains stable irrespective of the time of day, suggesting their potential utility as diagnostic markers for mammary gland health.
| S-EPMC10299634 | biostudies-literature
Project description:The study of early maternal-embryonic cross-talk remains one of the most challenging topics in reproductive biology. Understanding the physiological mechanisms involved in the interactions between the maternal reproductive tract and the developing embryo is essential for enhancing bovine reproductive efficiency. This complex communication starts within the oviduct, where the modulation of biological processes important for ensuring embryo quality is partially facilitated through extracellular vesicles (EVs). Utilizing a combination of in vivo and in vitro models this study had three main objectives: (i) to examine the protein cargo of EVs isolated from the oviductal fluid (OF) of cyclic and pregnant heifers to understand their role in maternal-embryonic communication in vivo; (ii) to characterize the protein profile of EVs in conditioned medium (CM) resulting from the culture of oviductal explants alone (Exp) or in the presence of 8- to 16-cell stage embryos (Exp+Emb); and (iii) to compare the protein cargo of EVs from Exp with EVs from cyclic heifers and EVs from Exp+Emb with EVs from pregnant heifers. Proteins were considered ‘present’ if detected in at least three out of five replicates and considered ‘exclusive’ if detected in at least three out of five replicates within one group but absent in all samples of other groups. We identified 670 and 1508 proteins in the OF-EVs of cyclic and pregnant heifers, respectively. Among these, 651 proteins were found in both groups, while 42 were exclusive to OF-EVs from the pregnant group. Within the 651 proteins detected in both groups, 32 were identified as differently abundant proteins (DAPs). In pregnant heifers, DAPs were mainly related to genome activation, DNA repair, embryonic cell differentiation, migration, and immune tolerance. In vitro, we identified 859 proteins in the CM-EVs from Exp alone, 627 from Exp+Emb, and 111 in the CM-EVs from Emb alone. In the qualitative analysis between the three groups, 81 proteins were commonly detected, 485 were common to Exp and Exp+Emb, 17 were common to Exp and Emb, 5 were common to Exp+Emb and Emb, 5 were unique to Exp, 6 were unique to Exp+Emb, and none were unique to Emb. Proteins identified when there is an interaction between the oviduct and the embryo in vitro, corresponding to Exp+Emb group, were associated with immune tolerance, structural activity, binding, and cytoskeletal regulation. In vivo and in vitro EVs exhibit distinct qualitative and quantitative protein contents, both when comparing EVs produced in the absence of an embryo (Cyclic and Exp) and those that have undergone embryo-oviduct interaction (Pregnant and Exp+Emb). The observed changes in the protein cargo of EVs due to maternal-embryonic communication in vitro and in vitro suggest that in vivo, the interaction between the embryo and the maternal milieu initiates within the oviduct and is potentially facilitated by EVs and their protein contents.
2024-07-04 | PXD053670 |
Project description:The study of early maternal-embryonic cross-talk remains one of the most challenging topics in reproductive biology. Understanding the physiological mechanisms involved in the interactions between the maternal reproductive tract and the developing embryo is essential for enhancing bovine reproductive efficiency. This complex communication starts within the oviduct, where the modulation of biological processes important for ensuring embryo quality is partially facilitated through extracellular vesicles (EVs). Utilizing a combination of in vivo and in vitro models this study had three main objectives: (i) to examine the protein cargo of EVs isolated from the oviductal fluid (OF) of cyclic and pregnant heifers to understand their role in maternal-embryonic communication in vivo; (ii) to characterize the protein profile of EVs in conditioned medium (CM) resulting from the culture of oviductal explants alone (Exp) or in the presence of 8- to 16-cell stage embryos (Exp+Emb); and (iii) to compare the protein cargo of EVs from Exp with EVs from cyclic heifers and EVs from Exp+Emb with EVs from pregnant heifers. Proteins were considered ‘present’ if detected in at least three out of five replicates and considered ‘exclusive’ if detected in at least three out of five replicates within one group but absent in all samples of other groups. We identified 670 and 1508 proteins in the OF-EVs of cyclic and pregnant heifers, respectively. Among these, 651 proteins were found in both groups, while 42 were exclusive to OF-EVs from the pregnant group. Within the 651 proteins detected in both groups, 32 were identified as differently abundant proteins (DAPs). In pregnant heifers, DAPs were mainly related to genome activation, DNA repair, embryonic cell differentiation, migration, and immune tolerance. In vitro, we identified 859 proteins in the CM-EVs from Exp alone, 627 from Exp+Emb, and 111 in the CM-EVs from Emb alone. In the qualitative analysis between the three groups, 81 proteins were commonly detected, 485 were common to Exp and Exp+Emb, 17 were common to Exp and Emb, 5 were common to Exp+Emb and Emb, 5 were unique to Exp, 6 were unique to Exp+Emb, and none were unique to Emb. Proteins identified when there is an interaction between the oviduct and the embryo in vitro, corresponding to Exp+Emb group, were associated with immune tolerance, structural activity, binding, and cytoskeletal regulation. In vivo and in vitro EVs exhibit distinct qualitative and quantitative protein contents, both when comparing EVs produced in the absence of an embryo (Cyclic and Exp) and those that have undergone embryo-oviduct interaction (Pregnant and Exp+Emb). The observed changes in the protein cargo of EVs due to maternal-embryonic communication in vitro and in vitro suggest that in vivo, the interaction between the embryo and the maternal milieu initiates within the oviduct and is potentially facilitated by EVs and their protein contents.
2024-07-04 | PXD053667 |
Project description:Extracellular vesicles are a heterogeneous group of membrane-limited vesicles loaded with various proteins, lipids, and nucleic acids. Release of extracellular vesicles from its cell of origin occurs either through the outward budding of the plasma membrane or through the inward budding of the endosomal membrane, resulting in the formation of multivesicular bodies, which release vesicles upon fusion with the plasma membrane. The release of vesicles can facilitate intercellular communication by contact with or by internalization of contents, either by fusion with the plasma membrane or by endocytosis into "recipient" cells. Although the interest in extracellular vesicle research is increasing, there are still no real standards in place to separate or classify the different types of vesicles. This review provides an introduction into this expanding and complex field of research focusing on the biogenesis, nucleic acid cargo loading, content, release, and uptake of extracellular vesicles.
| S-EPMC5546313 | biostudies-other
Project description:The success of the early reproductive events depends on an appropriate communication between gametes/embryos and the oviduct. Extracellular vesicles (EVs) contained in oviductal secretions have been suggested as new players in mediating this crucial cross-talk by transferring their cargo (e.g. proteins, mRNA and small ncRNA) from cell to cell. However, little is known about the oviduct EVs composition and their implications in reproductive success. The objective of our study was to determine the changes of oviductal EVs mRNA content under the hormonal influence during the estrous cycle. Methods: EVs, exosomes and microvesicles, were isolated from bovine oviductal fluid at different stages of the estrous cycle (postovulatory-stage, early luteal phase, late luteal phase and pre-ovulatory stage). Total RNA was isolated and used for the preparation of RNA-seq libraries. RNA-sequencing was performed on an Illumina HiSeq 2500. The obtained sequence reads that passed quality filters were mapped to the bovine genome sequence using Hisat 2. Read counts were calculated with QuasR Qcount and statistical analysis with EdgeR to identify differential mRNA abundance across the different stages of the estrous cycle. Results: RNA-sequencing identified 903 differentially expressed transcripts (FDR<0.001) in bovine oviductal EVs across the estrous cycle. Major differences were found between post-ovulatory and the rest of the stages analyzed.Functional annotation of the differentially abundant mRNAs identified functions related to cilia expression, exosome/vesicles, and many transcripts encoding ribosomal proteins. Conclusions: Our findings represent the first extensive oviductal signature of bovine oviductal EVs mRNA content and contribute to a better understanding of the role of EVs as modulators of gamete/embryo-maternal interactions.
2018-07-31 | GSE110399 | GEO
Project description:The success of the early reproductive events depends on an appropriate communication between gametes/embryos and the oviduct. Extracellular vesicles (EVs) contained in oviductal secretions have been suggested as new players in mediating this crucial cross-talk by transferring their cargo (e.g. proteins, mRNA and small ncRNA) from cell to cell. However, little is known about the oviduct EVs composition and their implications in reproductive success. The objective of our study was to determine the changes of oviductal EVs small ncRNA content under the hormonal influence during the estrous cycle. Methods: EVs, exosomes and microvesicles, were isolated from bovine oviductal fluid at different stages of the estrous cycle (postovulatory-stage, early luteal phase, late luteal phase and pre-ovulatory stage). RNA enriched for short molecules was isolated and used for the preparation of small RNA-seq libraries. RNA-sequencing was performed on an Illumina HiSeq 2500. The obtained sequence reads that passed quality filters were annotated by BLAST alignment to various sets of non-coding and coding RNAs. Read counts were calculated based on the frequency of unique sequences after removal of PCR duplicates. Statistical analysis was done with EdgeR to identify differential small ncRNA abundance across the different stages of the estrous cycle. Results: Small RNA-Seq identified the presence of different types of ncRNAs (miRNAs, rRNA fragments, tRNA fragments, snRNA, snoRNA, and other ncRNAs), which were partially also under hormonal influence. Major differences were found between post-ovulatory and early luteal and the rest of the stages for small ncRNAs. Interesting miRNAs identified in oviductal EVs, miR-34b and miR-449a, showed differential abundance among stages. Dysregulation of these miRNAs has been associated with defective cilia in the oviduct and infertility. Conclusions: Our findings represent the first extensive oviductal signature of bovine oviductal EVs miRNA content and contribute to a better understanding of the role of EVs as modulators of gamete/embryo-maternal interactions.
2018-07-31 | GSE110443 | GEO