Tesi di dottorato

Egg quality is affected by several factors, which are determined by the intrinsic properties of the egg itself and by the environment in which the egg grows and matures. Up to date, some of the factors that can influence egg's quality are known, but most of them are still to be highlighted. Usually the formation of a good quality egg is determined by the right achievement of several physical, genetic and chemical parameters, as well as the initial physiological processes occurring in the egg. The main objectives of this PhD thesis have been addressed with the purpose to increase knowledge in respect to egg quality, thereby;
Basic information in regards of those molecules associated with oocyte growth and maturation (cathepsin B, D and L), in terms of gene expression timing and the relative enzymatic activity, were obtained in two experimental models Sparus aurata and Danio rerio. The enzymatic activities were investigated trough the development of specific biochemical assays. Moreover, gene expression of these molecules during oogenesis has been determined in Sparus aurata. Furthermore, in order to better understand the relation between two concomitant physiological steps, such as the second proteolytic yolk protein processing and the resumption of the meiosis during GVBD, an in vitro Danio rerio oocyte maturation protocol has been setup. These results indicated that the GVBD process is independent of the occurrence of the second proteolytic process and that yolk proteolysis is related to the temporal and specific activation of cathepsins by acidification of yolk spheres. These results taken together with previous results strengthen the use of cathepsins as biomarkers for egg quality evaluation. Furthermore, the characterization of the functional levels of cathepsin activities provided evidence for their possible application as biomarkers in the biotechnology applied to reproduction. As a matter of fact, these biomarkers have been used to monitor oocytes viability after manipulation and/or cryopreservation.
Up to date there is little agreement for commonly shared egg quality biomarkers, thus there is the absence of standard protocols and/or biomarkers that can be generally applied to the large variety of teleosts; as a matter of fact egg quality evaluation may be different in different species. In the present thesis Sparus aurata has been used as experimental model in order to highlight possible differential presence of maternal messenger RNA in floating and non-floating eggs. The results obtained evidenced in sinking eggs (bad quality egg) a higher expression of apoptotic factors taken into account (P53, IL-1, IL-8, TNFα, cathepsin L and cathepsin D) and lower levels of survival ones (IGF-I and IGFBP2). Just the opposite was observed in floating eggs, in which high levels of survival factors were associated with low levels of apoptotic inducers. Therefore, the genes that have been here investigated can be considered suitable biomarkers for egg quality evaluation on gilthead sea bream.
In vertebrates, reproductive behavior, gonad development, spawning, fecundity and fertility are influenced by a multitude of both external (e.g. photoperiod, temperature) and internal stimuli (e.g. hormonal levels). The alteration of one or more of these stimuli leads to adverse effects on reproduction. In my PhD thesis, for the first time on Danio rerio females, the effect of the EDC di-2-ethylhexyl phthalate (DEHP), via water exposure to (0.2, 2, 20, and 40 µg/L) has been evaluated. Overall the results obtained were similar to those obtained in the positive control group exposed to the synthetic oestrogen EE2. The results obtained evidenced the ability of DEHP in negatively influences Danio rerio female reproduction. In particular the negative biological effects have been assessed on hatching rate, GSI, plasma VTG levels, reproductive performance. Moreover, the molecular mechanisms by which DEHP exerts his negative reproductive effect has been evaluated by real time PCR on the ovarian gene expression of peroxisome proliferator-activated receptor (PPAR-α and β), oestrogen receptor (ER-α and β1) and the aromatase CYP19a and by a DNA microarray analysis carried out on the liver. Effects have been found to be similar in both tissues, and since the similarity observed between EE2 and DEHP, in modulating the genes taken in account, the results here evidenced the ability of DEHP in mimicking oestrogens molecular and physiological effects.