Esposito, Riccardo (2025) Formulation of an innovative system of oocyte maturation in vitro in cattle. [Tesi di dottorato]

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Abstract

The aim of this thesis was to create an innovative in vitro maturation (IVM) system for bovine oocytes by mimicking the follicular environment in which the oocyte grows and develops. In the first part of the thesis, the follicular fluid (FF) enclosing oocytes with different developmental competence was characterized by analyzing the miRNA cargo of extravesicles (EVs) (Chapter 1, Experiment 1a) and the follicular fluid cytokine profile (Chapter 1, Experiment 1b). In the second part, based on the results, the identified competence-associated factors, i.e., the EVs isolated from the FF enclosing competent oocytes (Chapter 2, Experiment 2a) and the miR-143, the most abundant miRNA detected in the FF of competent oocytes, were validated during Simulated Physiological Oocyte Maturation (SPOM) (Chapter 2, Experiment 2b). The FF was collected from individual follicles of abattoir-derived ovaries and diluted in PBS 1:20 (Chapter 1, Experiment 1a) and 1:10 Chapter 1, Experiment 1b), centrifuged, and stored at -80°C, while the corresponding cumulus-oocyte-complexes (COCs) were introduced in the VivaVitro WOW dish for individual culture. At the end of the culture, the FF samples were retrospectively assigned to three different categories corresponding to: oocytes that reached the blastocyst stage (B), cleaved oocytes (C), and uncleaved oocytes (U). Three pools of 20 follicles and 16 individual follicles per category were analyzed, respectively, in Experiment 1.a and 1.b. In Experiment 1.a EVs were isolated through ultracentrifugation, characterized by Nanosight, and subjected to Illumina miRNA sequencing. In Experiment 1.b cytokine analysis was performed using the Quantibody® Bovine Cytokine Array 30 kit (Ray Biotech, Georgia, USA), to quantitatively measure the concentration of 30 bovine cytokines with an intra-assay coefficient of variation (CV) of <20%. The EVs size and concentration were higher in the B group than in the U group (P<0.05), with no differences in the C group (213.4 ± 3.1, 202.1 ± 2.3, and 208.9 ± 1.2 nm and 4.0 ± 0.2, 2.8 ± 0.1 and 3.9 ± 0.6 particle/mL, respectively). Concerning miRNA cargo, EVs from the B group had higher expression of bta-miR-143 and lower expression of bta-miR-21-5p in comparison to group U. In the B group EVs bta-miR-19a was overexpressed compared to the C group. Finally, the EVs from the C group showed a lower expression of bta-miR-19a and a higher expression of bta-let-7f than the U group. Regarding cytokine profile, the tumor necrosis factor-α (TNF-α) was higher (P<0.05) in the B group compared to the U group, with similar values recorded in the C group. The concentration of Acidic Fibroblast Growth Factor (aFGF) was reduced (P<0.01) in the B group compared to the 5 C and U groups. Lower (P<0.05) levels of Interleukin-15 (IL-15) were found in the B group compared to the C group, while no significant differences were observed compared to the U group. The Interferon Gamma-Induced Protein 10 (IP-10) levels were lower (P<0.05) both in the B and C groups compared to the U group. A decrease in Interleukin-17a (IL-17a) was observed only in the C group compared to the other groups. In Experiment 2.a, abattoir-derived bovine COCs were in vitro matured in the SPOM system with 0 (n =134), 1 (n =123), 10 (n =124), and 100 (n = 124) x 106 mL-1 EVs isolated from FF from follicles enclosing competent oocytes, over 4 replicates. In Experiment 2.b, abattoir-derived bovine COCs were in vitro matured in the SPOM with no supplement (control; n =194), miR-143 mimic (n= 194), miR-143 inhibitor (n =189), negative control (NC) of mimic (n =191), and NC of miR-143 inhibitor (n =194), over 5 replicates. In both experiments, after IVM oocytes were fertilized and cultured according to standard in vitro embryo production(IVEP) procedure. Cleavage and blastocyst rates were evaluated after 4 and 8 days of culture. Data were analyzed by ANOVA. The inclusion of 10 x 106 ml-1 of group B-derived EVs during IVM did not affect the cleavage rate (82.7 ± 2.7 vs 79.4 ± 2.8) but increased blastocyst rates based on the number of COCs (59.1 ± 7.1 vs 41.8 ± 7.0 %; P<0.05) and on cleaved embryos (72.0 ± 6.4 vs 52.7 ± 5.2; P<0.05). The inclusion of miR-143 in the SPOM did not significantly increase blastocyst rates compared to the control (48.4 ± 0.4 vs 40.8 ± 5.4), but the COCs matured in the presence of miR-143 mimic gave higher blastocyst rates than those exposed to the inhibitor, based on the number of COCs (48.4 ± 0.4 vs 35.3 ± 1.4; P<0.05) and cleaved embryos (61.9 ± 3.0 vs 42.9 ± 2.4; P<0.05). In conclusion, it was demonstrated that there are differences in the miRNA profile of the EVS derived from the FF and in the cytokine profile of the FF among bovine oocytes with different developmental competences. Adding EVs isolated from FF of competent oocytes into a SPOM system improved in vitro bovine embryo development.

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