Definición del origen y potencial uso diagnóstico del ADN identificado en el medio de cultivo de embriones bovinos producidos in vitro
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Date
2025
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Universidad de Concepción
Abstract
El término vesícula extracelular (EVs) se refiere a cualquier tipo de nanopartícula rodeada por una bicapa lipídica. Estas vesículas son incapaces de replicarse debido a la ausencia de núcleo y son liberadas por todas las células. Las EVs son clasificadas según distintos criterios, como el tamaño, el órgano de origen o su biogénesis. Dependiendo de su proceso de formación, las células liberan tres tipos principales de EVs: exosomas, que derivan de cuerpos multivesiculares; microvesículas, que se generan por evaginación de la membrana plasmática; y cuerpos apoptóticos, que se producen durante la apoptosis. Las EVs desempeñan un papel crucial en la comunicación intercelular, determinado por su contenido molecular. Estas vesículas pueden transportar proteínas, lípidos y ácidos nucleicos. Este mensaje es protegido al interior de las EVs y es transferido a otra célula, modificando su función, participando en diversos procesos fisiopatológicos, incluidos trastornos neurodegenerativos, inmunológicos, vasculares, así como en el cáncer. Además, las EVs contribuyen a procesos biológicos normales, como la proliferación celular, la diferenciación celular y el desarrollo embrionario. Además, las EVs han estado implicadas en la diferenciación de ovocitos y espermatozoides, la maduración ovocitaria, la fertilización y la implantación embrionaria.
Los embriones preimplantatorios, al igual que otras células, pueden liberar EVs. Estas EVs embrionarias contienen cargo y participan en el diálogo materno-embrionario. Las características de las EVs, como su tamaño, concentración o contenido, varían en función de la competencia, calidad o estadio de desarrollo del embrión. Se ha identificado gDNA embrionario en medios de cultivo como herramienta para el diagnóstico genético, aunque su concordancia con el ADN embrionario ha mostrado una concordancia solo del 30-90%. Esta discordancia puede explicarse por la presencia de contaminantes de ADN derivados de células del cúmulo, espermatozoides y/o proteínas del medio de cultivo. Se ha reportado la presencia de gDNA y mtDNA en el interior de EVs liberadas por embriones humanos, murinos y porcinos, lo que sugiere un posible uso de estas vesículas como herramienta para la genotipificación embrionaria. Por esta razón, el objetivo general de este estudio fue determinar la presencia de gDNA dentro de EVs liberadas por embriones bovinos Pre-implantatorios y su potencial uso para el diagnóstico genético.
Para ello, se produjeron embriones bovinos mediante fertilización in vitro o partenogénesis (según el experimento específico) y cultivados en grupos hasta el día 5 (estadio de mórula). Posteriormente, las mórulas fueron cultivadas individualmente hasta el día 7 (estadio de blastocisto), momento en el cual fueron recolectadas junto con su medio de cultivo correspondiente. El medio condicionado fue utilizado para la obtención de EVs mediante tres protocolos diferentes de aislamiento con el fin de evaluar la eficiencia de cada método. Las EVs aisladas fueron caracterizadas mediante análisis de seguimiento de nanopartículas, microscopía electrónica de transmisión y Western blot para la detección de proteínas específicas. La presencia de ADN fue determinada mediante múltiples técnicas, y la genotipificación embrionaria se llevó a cabo mediante PCR en tiempo real. Además, algunas muestras fueron secuenciadas y analizadas para evaluar la representación génica. Finalmente, se determinó la tasa de apoptosis y la concentración total de ADN mediante espectrofotometría y análisis por NTA para evaluar la correlación entre ambas variables.
No se encontraron diferencias significativas entre los tres protocolos de aislamiento en cuanto a las características poblacionales de las EVs, aunque sí en el tiempo requerido. Se logró detectar la presencia de ADN en el interior de las EVs mediante distintas técnicas, confirmando su existencia. El análisis de secuenciación reveló la presencia de 5,664 genes, incluyendo algunos involucrados en el desarrollo embrionario. El análisis por PCR confirmó la presencia e identificación de múltiples genes, incluyendo genes ligados al sexo (SRY y USP9Y). Todos los embriones presentaron al menos una célula en apoptosis. Las mórulas bloqueadas mostraron una mayor tasa de apoptosis, y los embriones derivados de partenogénesis presentaron una mayor concentración de ADN. No se encontró correlación entre la tasa de apoptosis y la concentración de ADN en las EVs.
Los resultados confirman que los embriones bovinos preimplantacionales liberan EVs que contienen ADN en su interior, independientemente de su calidad. Además, se demostró que este ADN puede utilizarse para la genotipificación embrionaria mediante PCR en tiempo real, aunque con ciertas limitaciones en función de la cantidad de ADN disponible. Finalmente, se determinó que la concentración de ADN en las EVs es independiente de la tasa de apoptosis embrionaria y la cantidad detectada y genes identificado puede variar según la competencia o calidad del embrión.
The term extracellular vesicle refers to any type of nanoparticle surrounded by a lipid bilayer. They cannot replicate itself due to the lack of nucleus and are released by all cells. These vesicles are classified according to multiple criteria, such us, size, origin organ and biogenesis. Depending on their formation process, cells release three types of EVs: exosomes, derived from multivesicular bodies; microvesicles, formed by plasma membrane evagination; and apoptotic bodies, produced during apoptosis. EVs play a crucial role in intercellular communication, largely depending on their cargo. The EVs can carry proteins, lipids, and nucleic acids. The message protected by these vesicles is loaded to another cell and it is capable to change their function, participating in several pathological processes, including neurodegenerative, immune, and vascular diseases, as well as cancer. Additionally, they contribute to normal biological processes, including cell proliferation, cell differentiation and embryo development. Besides, EVs participate in oocyte/spermdifferenti ation, oocyte maturation, fertilization, and embryo implantation. Pre-implantation embryos, likewise other cells, can release EVs. These embryo-derived EVs also can contain cargo and are implicate in the embryo-maternal crosstalk. The EVs characteristics, such a size, concentration, or content, vary according to embryo competence, quality, or stage of development. Embryo gDNA has been used to genetic diagnosis, found in culture media, but their concordance with the embryo DNA is only of 30-90%. This discordance can be explained for the presence of DNA contaminants, derivative of cumulus cell, sperm and/or DNA from culture media proteins. It has been found gDNA and mtDNA inside of EVs released by human, murine, and porcine embryos, suggesting a potential tool to embryo genotyping. This is why, the general aim of this study was determinate the presence of gDNA inside of EVs released by pre implantation bovine embryos and their potential to genetic diagnosis. To do this, bovine embryos were produced by in vitro fertilization or parthenogenesis (according to the specific experiment) on groups until day 5 (morula stage). After that, morula were transferred to individual culture until day 7 (blastocyst stage), and they were collected with their respective media. The conditioned media was used to the EVs isolation by, initially, three different types of isolation to determinate the EVs efficiency. The isolated EVs were characterized using nanoparticle tracking analysis, transmission electron microscopy, and Western blotting for EVspecific proteins. The DNA presence was measure using multiple techniques and the embryo genetic testing was performed by real time PCR. Also, a few samples were sequencing and analyzed to evaluate all the genes representation. Finally, it was conducted the determination of the apoptotic rate and the total DNA concentration using spectrophotometers and NTA analysis to determinate the correlation between them. There is not a huge different between the three isolation protocols in terms of EVs characteristics population, only in the time of whole process of the samples. We were capable to measure the DNA inside of EVs with different techniques, ensuring their presence. The sequencing analysis showed the presence of 5.664 genes, including genes involved on embryo development. The PCR analysis validate the presence and gene identification in the EVs, in many different genes, even in the sex-linked genes (SRY and USP9Y). All the embryos had almost one cell on apoptosis. The arrested morulae had a higher apoptotic rate and there was produced by parthenogenesis the higher DNA concentration. The apoptotic rate showed no correlation with the DNA concentration in the EVs. Our results confirm that pre implantation bovine embryos can releases EVs with DNA inside them independent of their quality. Furthermore, we demonstrate this DNA can be used to embryo genotyping by a simple real time PCR, with certain limitation according to their quantity. Finally, we found the DNA concentration was independent of the apoptotic rate and can vary according to the embryo competence or quality.
The term extracellular vesicle refers to any type of nanoparticle surrounded by a lipid bilayer. They cannot replicate itself due to the lack of nucleus and are released by all cells. These vesicles are classified according to multiple criteria, such us, size, origin organ and biogenesis. Depending on their formation process, cells release three types of EVs: exosomes, derived from multivesicular bodies; microvesicles, formed by plasma membrane evagination; and apoptotic bodies, produced during apoptosis. EVs play a crucial role in intercellular communication, largely depending on their cargo. The EVs can carry proteins, lipids, and nucleic acids. The message protected by these vesicles is loaded to another cell and it is capable to change their function, participating in several pathological processes, including neurodegenerative, immune, and vascular diseases, as well as cancer. Additionally, they contribute to normal biological processes, including cell proliferation, cell differentiation and embryo development. Besides, EVs participate in oocyte/spermdifferenti ation, oocyte maturation, fertilization, and embryo implantation. Pre-implantation embryos, likewise other cells, can release EVs. These embryo-derived EVs also can contain cargo and are implicate in the embryo-maternal crosstalk. The EVs characteristics, such a size, concentration, or content, vary according to embryo competence, quality, or stage of development. Embryo gDNA has been used to genetic diagnosis, found in culture media, but their concordance with the embryo DNA is only of 30-90%. This discordance can be explained for the presence of DNA contaminants, derivative of cumulus cell, sperm and/or DNA from culture media proteins. It has been found gDNA and mtDNA inside of EVs released by human, murine, and porcine embryos, suggesting a potential tool to embryo genotyping. This is why, the general aim of this study was determinate the presence of gDNA inside of EVs released by pre implantation bovine embryos and their potential to genetic diagnosis. To do this, bovine embryos were produced by in vitro fertilization or parthenogenesis (according to the specific experiment) on groups until day 5 (morula stage). After that, morula were transferred to individual culture until day 7 (blastocyst stage), and they were collected with their respective media. The conditioned media was used to the EVs isolation by, initially, three different types of isolation to determinate the EVs efficiency. The isolated EVs were characterized using nanoparticle tracking analysis, transmission electron microscopy, and Western blotting for EVspecific proteins. The DNA presence was measure using multiple techniques and the embryo genetic testing was performed by real time PCR. Also, a few samples were sequencing and analyzed to evaluate all the genes representation. Finally, it was conducted the determination of the apoptotic rate and the total DNA concentration using spectrophotometers and NTA analysis to determinate the correlation between them. There is not a huge different between the three isolation protocols in terms of EVs characteristics population, only in the time of whole process of the samples. We were capable to measure the DNA inside of EVs with different techniques, ensuring their presence. The sequencing analysis showed the presence of 5.664 genes, including genes involved on embryo development. The PCR analysis validate the presence and gene identification in the EVs, in many different genes, even in the sex-linked genes (SRY and USP9Y). All the embryos had almost one cell on apoptosis. The arrested morulae had a higher apoptotic rate and there was produced by parthenogenesis the higher DNA concentration. The apoptotic rate showed no correlation with the DNA concentration in the EVs. Our results confirm that pre implantation bovine embryos can releases EVs with DNA inside them independent of their quality. Furthermore, we demonstrate this DNA can be used to embryo genotyping by a simple real time PCR, with certain limitation according to their quantity. Finally, we found the DNA concentration was independent of the apoptotic rate and can vary according to the embryo competence or quality.
Description
Tesis presentada para optar al grado de Doctor en Ciencias Veterinarias
Keywords
Biogénesis, Vesículas extracelulares