Kosior, Michal Andrzej (2022) Seasonal factors affecting oocyte competence in Italian Mediterranean Buffalo. [Tesi di dottorato]

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The purpose of this thesis was to investigate the causes and the molecular mechanisms underlying the phenomenon of reproductive seasonality in the Italian Mediterranean Buffalo, with specific reference to oocyte developmental competence. The focus of this thesis was paid on the oocyte and the follicular environment, to unravel the causes of reduced oocyte competence during the non breading season, using novel approaches. Therefore, in Experiment 1 the miRNA and transcriptomic profiles of buffalo oocytes and corresponding follicular cells were compared between seasons. Then a further characterization of the follicular MiRNA content was carried out in Experiment 2. Specifically, the miRNA cargo of extracellular vescicles isolated from the follicular fluid was characterized both in relation to the stage of development (antral vs preovulatory follicles) and to the season. Finally, in Experiment 3 it was evaluated whether season influences the metabolome of the follicle. In particular, to further unveil molecular differences related to season, the metabolomic profile of various components of the ovarian follicle, such as follicular fluid, follicular cells, cumulus cells and immature oocytes was investigated. In addition, the metabolome of cumulus cells and oocytes after in vitro maturation, as well as that of the spent medium, was characterized. The results showed a seasonal variation in both oocytes and follicular cells in the expression of miRNA, known to be involved in follicular maturation and development regulation. Furthemore specific miRNAs contained in extracellular vescicles isolated from follicular fluid are involved in regulating follicular development and modulating seasonal effects on oocyte competence. In particular, a differential expression of miRNAs involved in lipid and steroid metabolism, response to glucocorticoid and estradiol stimulus was observed in relation to follicular growth. Interestingly, differences in miRNAs cargo were also shown between seasons in both antral and pre-ovulatury follicles. The results of Experiment 3 demonstrated substantial seasonal differences in metabolites content in all the follicular components, suggesting that the reduced oocyte competence during the non breading season is due to alterations of several metabolic pathways, such as glutathione metabolism, energy generating metabolism, phospholipid biosynthesis and amino acid metabolism. The lipid composition of the follicular environment also varied significantly in relation to season. The findings suggest that during non breading season metabolic changes take place, likely related to a condition of negative energetic balance, influencing the follicular environment and are in part reflected at the somatic cells and the oocyte in the attempt to counteract suboptimal conditions. The third experiment also permitted to identify potential positive markers of oocyte competence as glutathione, glutamate, lactate and cholines, whereas a negative association with competence was found for leucine, isoleucine and β-hydroxybutyrate. Taken together, these results pay the way to develop corrective strategies to modulate the follicular environment or, more easily the IVM medium to improve the competence of oocytes retrieved during the NBS. However, the amount of data produced confirm the complexity of the phenomenon of seasonality and undoubtedly suggest to carry out further studies to better interpret some of the changes observed.

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