Tesis Doctorado

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Browsing Tesis Doctorado by Subject "Bovinos"

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    Generación de embriones bovinos por transferencia nuclear somática: evaluación de la capacidad de desarrollo in vivo e in vitro y de la expresión génica en las etapas pre y peri-implantatorias
    (Universidad de Concepción, 2009) Rodríguez Álvarez, Lleretny; Castro Reboredo, Fidel Ovidio
    La clonación somática es una poderosa herramienta para la producción de animales con un genotipo específico, ya sea para fines productivos a través de la multiplicación de ejemplares de alto valor genético, la creación de animales transgénicos o para la conservación de especies en peligro de extinción. En la última década esta tecnología se ha ido perfeccionando y hoy en día se ha logrado la obtención de clones en dieciséis especies de mamíferos. La aplicación de la transferencia nuclear es limitada debido a la ineficiencia del proceso en término de animales nacidos y al desconocimiento de la expresión génica que gobierna el proceso de clonación, por lo que es sujeto de estudio a nivel mundial. En este trabajo se empleó una metodología novedosa de transferencia nuclear somática, conocida como Hand Made Cloning (HMC) para generar embriones bovinos clonados, usando dos líneas celulares distintas como donantes de núcleo, una de fibroblastos de piel de un animal adulto y la otra de un feto. Se evaluó el potencial de desarrollo de los embriones generados con ambas líneas, en términos de desarrollo in vitro hasta blastocistos, así como morfología y calidad. Se demostró que con la línea celular adulta es posible generar con mayor eficiencia y calidad embriones clonados en bovinos. Para caracterizar la expresión génica en blastocistos (etapa peri-implantatoria) generados a partir de ambas líneas se empleó PCR tiempo final y real y se comparó ésta con los patrones de expresión de embriones producidos mediante fecundación in vitro (FIV). Se estudiaron los patrones de expresión de genes cruciales para el desarrollo embrionario, concluyendo que los mismos son diferentes entre las líneas celulares y entre éstas y los embriones de FIV. Los patrones de expresión génica de los embriones producidos con la línea celular adulta fueron más parecidos a los de los controles, por ello y por el mayor potencial de desarrollo hasta blastocistos de calidad, se seleccionó esta línea para la producción de embriones elongados (día 17) y de clones a términos. Se transfirieron a hembras receptoras embriones clonados producidos a partir de la línea celular adulta, así como controles producidos por FIV y se recolectaron al día 17. A partir de estos embriones, se estudiaron los patrones de expresión génica por técnicas de PCR tanto a tiempo real como final de los embriones elongados (etapa peri-implantatoria) clonados y de FIV, siendo diferencial la expresión entre ambos y ésta a su vez diferente con respecto a los blastocistos de día 7, tanto para FIV como para los clones. Estos hallazgos, así como la cuantificación de los niveles de expresión de algunos genes cruciales para el desarrollo embrionario temprano, constituyen los primeros reportes de su tipo en la literatura del tema. Adicionalmente se estudió a nivel global, la expresión génica en embriones clonados a partir de células adultas y producidos por FIV, mediante microarreglos, se encontraron más de mil genes expresados en ambos grupos y de éstos alrededor del 4% se encontró diferencialmente expresado (hiper o hipo expresión) en los embriones clonados. Los datos del microarreglo fueron validados por PCR cuantitativo (PCR tiempo real), lo cual a su vez ofreció resultados novedosos en el tema, al reportarse por primera vez los niveles de expresión de ciertos genes en embriones bovinos elongados. Como elemento final, se transfirieron embriones clonados a hembras receptoras, lográndose gestación en la mitad de las hembras transferidas, lo que redundó en el nacimiento de dos terneras clonadas. La eficiencia del proceso fue similar a la descrita para la técnica de HMC y para la especie bovina. A pesar de las pérdidas gestacionales observadas, que también coinciden con lo reportado para la especie, se logró por primera vez en Chile el nacimiento de terneros clonados viables.
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    Modulación de la comunicación Embrio-Materna en Bovinos, mediante mecanismos alternativos y complementarios a Interferon Tau
    (Universidad de Concepción, 2023) Aguilera González, Constanza Javiera; Rodríguez Álvarez, Lleretny
    Embryo-maternal communication is established from the first stages of embryonic development and is essential for implantation. In bovines, the key molecule for pregnancy recognition is interferon tau (IFNT), which is secreted by the conceptus on day 16 of gestation. However, it has been determined that the blastocyst stage embryois capable of producing IFNT. On the other hand, the embryo is capable of secreting extracellular vesicles (EVs) which, recently discovered, play an important role in the communication that the embryo establishes with the endometrium. This is because EVs are nanoparticles that have molecules with biological activity as charge. Recently, it has been determined that the embryo in early stages of development is capable of inducing a different response in endometrial epithelial cells (bEECs) through its EVs depending on the type of embryonic production (in vivo, in vitro). Likewise, it was shown that maternal EVs also present differences in their charge when obtained by cell culture (in vitro production) compared to their obtaining under in vivo conditions. The objective of this work was to determine if the in vitro production conditions could modify the embryomaternal communication established through the EVs. To do this, we first sought to determine if the EVs secreted by bovine embryos produced in vivo and in vitro during the pre-implantation period were able to induce transcriptomic modifications, activating IFNT signaling in bEECs. For this, it was evaluated whether during early embryonic development (blastulation, days 5-7 of development), the EVs of the embryos produced in vitro (EVs-IVP) and in vivo (EVs-IVV), induced a different response at the level of the embryo. transcriptomic in the bEECs. Along with this, we sought to determine the effect of EVs from embryos in a more advanced stage of development (day 7-9) produced both in vitro and in vivo, on the expression of genes linked to endometrial function and IFNT signaling in bEECs. Part of the objective of this work was also to determine the effect of the in vitro production system on EVs from endometrial cells. For this, the effect of EVs from uterine fluid (EV-UF) and cell culture (EVC) on pre-implantation development (Day 5-9 of development) of bovine embryos produced in vitro was evaluated. To obtain the embryonic EVs from the blastulation period (5-7 years of development), bovine morulae produced in vitro and in vivo were cultured individually for 48 hours to later isolate the embryonic EVs from the culture medium. Once obtained, the EVs were stained with PKH67 and added to the bEECs culture medium to assess their internalization. Subsequently, the transcriptomic profile of the bEECs was evaluated by messenger RNA sequencing. Both EVs-IVV and EVs-IVP induced the expression of interferon-induced classical and non-classical genes (ISGs) and other signaling pathways linked to endometrial function in bEECs. A greater number of differentially expressed genes were induced by EVs-IVP (3552) compared to EVs-IVV (1838). In gene ontology analysis, it was shown that EVs-IVP/IVV induce an upregulation of the exosome extracellular signaling pathway, cell response to stimuli, and protein modification processes. Regarding the effect on the bEECs of the EVsIVP/IVV from embryos on day 7-9 of development, expanded blastocysts were cultured individually for 48 hours for the isolation of their EVs. Subsequently, he evaluated the internalization of the embryonic EVs by the bEECs to later evaluate gene expression by RT-qPCR. It was determined that non-classical genes induced by IFNT were able to be activated in bEECs only by EVs-IVP. Finally, in relation to the effect of the EVs from EVC and EV-UF on embryonic development, the EVCs managed to induce an activation of genes linked to pluripotency, while the EV-UF induced a greater expression of IFNT by the bovine embryos, in addition to a sustained increase in diameter during development and a tendency to a greater number of expanded and protruded blastocysts.
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    Las vesículas extracelulares secretadas por embriones bovinos durante la etapa de blastulación y eclosión reflejan su competencia de desarrollo in vitro.
    (Universidad de Concepción, 2023) Gutiérrez Reinoso, Miguel Ángel; Rodríguez Álvarez, Lleretny
    This work was based on the fact that embryos produced In vitro are less competent than embryos generated In vivo. The objective of our research was to determine the population characteristics and miRNA profile of EVs secreted by bovine embryos produced In vitro and In vivo during the blastulation and hatching stages, and their relationship with embryo quality. To address this objective, a biological question was posed: Are the characteristics (morphological and miRNA content) of EVs different when morphologically competent embryos are produced in vitro - in vivo or in pre-implantation stages (blastulation vs hatching)? To resolve this question, two experiments were proposed that focused on the population characterization of EVs and miRNA content contained in EVs secreted by embryos in vitro and in vivo during blastulation (Exp 1) and hatching (Exp 2) by embryos qualified as competent regarding the competition criteria (Grades 1.2 IETS, for day 7 and diameter or hatching, for day 11). Pre-implantation bovine embryos were produced by PIV and IVV, and distributed into two blastulation and hatching groups (PIV5-7, IVV5-7; PIV7-9, IVV7-9). Morulas (Exp1: day 5) and blastocysts (Exp 2: day 7) collected in vivo on day 5 and day 7, respectively, were selected morphologically according to the IETS criteria (2010); In vitro embryos from day 5 and 7 of the group culture were cultured individually in SOFaa-depleted medium until day 7 and 9, respectively. In general, IVV and PIV embryos were cultured until day 11 in extended medium from their stages of 5-7 days and 7- 9 days. 88.3% and 43.05% of the PIV and IVV embryos reached the blastocyst stage on day 7. Development on day 11 was significantly higher in embryos produced IVV (d7-9) (P˂0.05) compared to to the PIV vs. IVV embryos in general had a higher hatching rate, being >50% compared to PIV. The kinetics of embryonic development presented significant differences between the groups of embryos associated both by the origin (In vitro-In vivo) and by the study window (blastulation-hatching). On day 11, the embryos from window 7-9 IVV had the largest diameter (PIV 5-7: 381.93 ±97.48; IVV 5-7: 345.68 ± 107.26; PIV 7-9: 471 0.72 ± 77.24, and IVV 7-9: 508.69 ± 90.56 μm). After this analysis, 133 culture media collected from embryos qualified as competent were selected and the populations of Extracellular Vesicles (EVs) were characterized, defined according to the three basic criteria recommended by the International Society of Extracellular Vesicles (ISEV) to define EVs: 1) presence of specific surface markers (cytometry), 2) morphology (Transmission Electron Microscopy-TEM), and 3) size and concentration (Nano Tracking Analysis, NTA). Embryo culture media EVs and positive control (fetal calf serum EVs) were positive for all four EV surface-specific protein markers tested (CD9, CD63, CD81, and CD40). Using TEM, structures with classic EVs morphology were identified, with a diameter between 100 and 500 nm, with a rounded or cup shape and the presence of a bilayer; Furthermore, no orphological differences were observed between the EVs of the embryos of the different experimental groups. The population of EVs was characterized according to their size (small (<120 nm) and large (>120 nm) and concentration. (NTA): the size and concentration of EVs. The variables of blastocyst morphology and morphological characteristics of the EVs they were submitted to principal component analysis (PCA) to discriminate the groups of embryos. The statistical analysis was performed with the SAS program, version eight for Windows and the IBM SPSS Statistics version 19 program. Populations of EVs were detected in the range of 25-620 nm, but with a higher concentration in the range of small vesicles (less than 200nm). Two populations of EVs were observed: large vesicles (>120 nm) and small vesicles (≤120 nm). For the analysis of the microRNA content, the samples were randomly obtained from each experimental replicate containing EVs of 10 embryos: 6 replicates from the PIV 5-7 group, 3 replicates IVV5-7, 3 replicates PIV 7-9 and 3 replicates IVV 7-9. Extraction and quality analysis of total RNA per replicate was performed. The RIN (“RNA Integrity Number”) was greater than 7, considered acceptable for sequencing analysis. The Fastp program detected reads of 75 ± 1 base pairs (Paired End) and with an insert of 149 base pairs, which were mapped against the genome available in Mirbase version 21. Principal component analysis (PCA) was performed to determine the variability of the counts between the replicates of each experimental group: greater inter-group separation was observed between the IVV and PIV groups (blastulation) and less inter-group separation between the IVV and PIV groups (hatching). 122 miRNAs with a CPM (counts per million) over 5 were identified; and 74 common miRNAs that are present in all experimental groups were detected. Analysis of differential expression of miRNAs in EVs based on embryonic origin shows 32 and 65 miRNAs mostly represented in EVs secreted during blastulation of IVV and PIV embryos, respectively, and 5 miRNAs differentially represented in EVs of IVV embryos. Regarding the content of miRNAs in the EVs, there was a decrease in the content of miRNAs mostly represented as embryonic development progressed, both for in vitro and in vivo tests. At the hatching stage, one exclusive miRNA was identified in the EVs of IVV embryos and 18 exclusives in the EVs of PIV embryos, while 2 miRNAs are equally represented in the EVs of embryos of both origins. Finally, an ontology analysis was performed to characterize the molecular function. Overexpressed miRNAs from EVs secreted during hatching by PIV embryos affect a greater number of biological functions than those overexpressed in EVs from IVV embryos, including 9 exclusively affected functions. In conclusion, in this work it was shown that the xix embryonic origin (In vivo vs In vitro) and its stage of development (blastulation and hatching) have an effect on the population characteristics (average size, concentration and content of miRNAs) of secreted extracellular vesicles. by bovine embryos. Therefore, a greater secretion of EVS and a greater number of differentially expressed microRNAs could be related to embryonic competence.
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