Competence of oviductal and uterine Extracellular Vesicles in sequential culture of in vitro bovine embryos
[EN] In vitro embryo production (IVP) is a biotechnology applied to improve animal reproductive performance, efficiency and genetic gain. The primary focus of IVP is to maximize the number of offspring from genetically superior animals and disseminate germplasm worldwide. Despite the fact that IVP i...
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| Tipo de recurso: | tesis de maestría |
| Fecha de publicación: | 2020 |
| País: | España |
| Institución: | Universitat Politècnica de València (UPV) |
| Repositorio: | RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia |
| Idioma: | inglés |
| OAI Identifier: | oai:riunet.upv.es:10251/148960 |
| Acceso en línea: | https://riunet.upv.es/handle/10251/148960 |
| Access Level: | acceso abierto |
| Palabra clave: | Bovino Interacción materno-embrionaria Vesículas extracelulares In vitro Bovine Embryo-maternal interaction Extracellular Vesicles Boví Interacció matern-embrionària Vesícules extracelul·lars PRODUCCION ANIMAL Máster Universitario en Mejora Genética Animal y Biotecnología de la Reproducción-Màster Universitari en Millora Genètica Animal i Biotecnologia de la Reproducció |
| Sumario: | [EN] In vitro embryo production (IVP) is a biotechnology applied to improve animal reproductive performance, efficiency and genetic gain. The primary focus of IVP is to maximize the number of offspring from genetically superior animals and disseminate germplasm worldwide. Despite the fact that IVP is used commercially successfully, it still has some limiting factors for cost reduction and greater dissemination related to the lower quality of the embryos generated in vitro and the consequent greater sensitivity to cryopreservation. It is known that embryo-maternal communication that occurs before implantation plays a vital role in the establishment and maintenance of pregnancy in mammals, so it is necessary to imitate what happens during this communication in in vitro cultures to improve the quality of the obtained embryos. This maternal-embryonic communication is mediated by various growth factors and molecular signaling pathways between the oviduct, the uterus and the embryo, which affect its development in the short and long term. Small particles called extracellular vesicles (EVs), released naturally by cells and containing various products such as proteins and RNAs among other molecules, play an important role in this interaction. Currently, it is still unknown how they work and how they affect the embryo in vivo or in vitro, but it has already been observed that embryos are capable of capturing these EVs. Therefore, the objective of this study was to isolate and characterize the EVs of the oviductal and uterine fluids and to evaluate their effects in an in vitro sequential culture system on the development and quality of bovine embryos. For this, bovine zygotes were cultured in synthetic oviductal fluid (SOF) supplemented with 3 mg/ ml BSA or 5% serum depleted of EVs in the presence or absence of 3x105 EVs of the oviductal (Day 1-4) and uterine fluids (Day 4-9), mimicking the physiological conditions of early embryonic development in vivo. Pooled EVs from five oviducts (early-luteal phase) and five uterine horns (early-mid-luteal phase) from slaughtered heifers were isolated using the size exclusion chromatography kit (Hansa BioMed). The size and concentration of the EVs were evaluated by means of a nanotracking analysis system (NTA) and their morphology by transmission electron microscopy (TEM). Cleavage rate and blastocyst yield was recorded on day 2 and days 7 to 9 respectively. On day 7/8 day blastocyst quality was evaluated by staining with Hoechst 33342 for total number of cells, Bodipy 493/503 for lipid content, Mitotracker Deep Red for mitochondrial activity and survival rate after vitrification/warming. The The concentration of EVs was 2.97x1010 and 7.98x1010 particles/ml, and the mean size of 177.5 and 216.5 nm for the oviductal (OF) and uterine fluids (UF), respectively. Through TEM we confirmed the presence of exosomes among EVs, showing the typical cup-shaped morphology. Western blotting confirmed the expression of classic EVs markers described for exosomes such as CD9 and CD63 tetraspanins and HSP70. Our results showed that supplementation of culture media with EVs from OF and UF has no effect on embryo development. However, their use had a positive effect on the quality of the produced embryos giving rise to blastocysts with a greater number of cells, lower lipid content and greater mitochondrial activity. In contrary the survival rate after vitrification / warming was not affected by the use of EVs in culture. In conclusion, mimicking physiological conditions using EVs from OF and UF in a sequential in vitro culture does not affect embryo development, but improves embryo quality by increasing total cell number, decreasing lipid content, and increasing mitochondrial activity. These results evidenced the association of the reproductive tract environment with the development of the early embryo, which confirms the maternal-embryonic communication. |
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