Tesis Doctorado
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Browsing Tesis Doctorado by Author "Aguilera González, Constanza Javiera"
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Item 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, LleretnyEmbryo-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.