Differential gene expression, ultrastructural analysis and evaluation of the lipid profile of in vitro produced bovine blastocysts from oocytes matured conventionally or via SPOM system

Besides allowing for the large-scale in vitro production of embryos, the in vitro maturation (IVM) dismiss the use of superstimulation hormones, preventing excessive financial expenses and avoiding iatrogenic effects of ovarian overstimulation. However, the outcome of IVM is still below expectations due to the inability in simulating the physiological events occurring in vivo. An artifact of IVM consists on the spontaneous resumption of meiosis after oocyte removal from the follicular environment. In this regard, without the molecular apparatus for arresting meiosis, the nuclear maturation is triggered irrespective of the physiological hormonal stimulation. As a result, the cytoplasmic maturation, i.e., the adequate reorganization of organelles in the oocyte is bypassed, compromising the oocyte competence. The general idea in this thesis was to test the pre-maturation system (Pre-IVM), in which two drugs (forskolin and 3- isobutyl-methylxanthine) are used prior to IVM to sustain meiotic arrest. The Pre-IVM performance was compared to the conventional IVM in the context of either commercial routines or research laboratories. At first, we aimed to compare the conventional IVM and the Pre-IVM in a holistic way, i.e., challenging each other using commercial systems. In this approach, we evaluated the ultrastructural aspects of oocytes and blastocysts, and associated with the expression of 96 genes related to embryo quality. In the second approach, we adapted the Pre-IVM drugs in our own IVM. Those drugs are known to affect the lipid metabolism, which is extremely relevant for the cellular energy metabolism in oocytes and embryo. Hence, we focus on the lipid quantification in oocytes and blastocysts, and, in addition, we assessed the membrane lipid profile of the blastocysts, since it is directly related to embryo cryopreservation potential. We collected cumulus cells and blastocysts and assessed the expression of 96 genes of oocyte competence and 96 genes of embryo quality. Results indicated that the Pre-IVM affected oocyte ultrastructure, the gene expression of blastocysts was differentially affected, but there was variation according to the systems used. Also the Pre- IVM increased the lipid content in oocytes and reduced in blastocysts. The membrane lipid profile of blastocysts was positively modulated by the Pre-IVM. Finally, the Pre-IVM affected the gene expression of cumulus (positively) and blastocysts (differentially). (AU)