Tesis Doctorado
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Browsing Tesis Doctorado by Author "Gutiérrez Reinoso, Miguel Ángel"
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Item 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, LleretnyThis 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.