Gene expression and human oocyte maturation: transcriptional control and post-transcriptional mechanisms based on intercellular signalling via exosomal miRNAs in follicular microenvironment

This thesis aimed at characterizing mRNA and miRNA expression profiles in the female gamete and its microenvironment, respectively, under physiological and non-physiological conditions in order i) to assess the effect of vitrification on the biomolecular profile of the oocytes, ii) to understand the molecular basis of reproductive ageing and iii) to identify miRNAs in FF to be used as non-invasive biomarkers of oocyte quality. We demonstrated that vitrification technique might be very helpful for preserving women s fertility, since it keeps unaltered oocyte molecular profile and does not cause degradation of mRNAs essential for oocyte development (i.e. BMP15, FIGLA, GDF9, OCT4, TAF4B). We determined Apoptotic Machinery (AM) transcriptome in mature MII human oocyte pools from women aged more than 38 years (old) and compared it to those of women aged up to 35 years (young). Subsequently, some AM candidate genes with a key role in apoptotic regulation were selected and analysed in single oocytes. These studies led us to identify AM transcripts never reported in human oocytes so far (BAG3, CD40, CFLAR, TNFRSF21, TRAF2, TRAF3) and to find out other differentially expressed genes in oocytes from older women. In fact, we found a significant upregulation of proapoptotic CD40, TNFRSF10A, TNFRSF21 and downregulation of antiapoptotic BCL2 and CFLAR. Our results demonstrate that during maturation old oocytes selectively accumulate mRNAs potentially able to trigger the extrinsic apoptotic pathway and express at low levels some survival factors: this condition could make old oocytes more inclined to apoptosis. Moreover, we found TP73 among differentially expressed genes in human MII oocyte pools during reproductive ageing, a process closely related to the production of oocytes with a reduced developmental competence whose main hallmark is aneuploidy. In order to verify the potential involvement of TP73 isoforms in reproductive ageing, we determined their expression in single mature MII oocytes from women younger than 35 and older than 38 years. We found out that TAp73 isoforms are significantly downregulated in oocytes from women of advanced reproductive age. There is evidence that TAp73 interacts with some kinetochore proteins in order to stop the anaphase if chromosomes are not properly attached to the meiotic spindle. The absence of TAp73 removes this cell cycle brake, so causing genomic instability. Consequently, TAp73 downregulation in old oocytes could lead to aneuploidy in the developing embryos, explaining both the reduction of fertility and the increase of newborns with chromosomal abnormalities. Finally, we profiled the expression of 384 miRNAs in human follicular fluid and its purified exosomal fraction with respect to plasma from the same women, providing the first molecular evidence of these bioactive vesicles inside ovarian follicle. Among the 37 miRNAs that we found upregulated in follicular microenvironment, the majority of them are carried by exosomes (exosome Follicular Fluid miRNAs eFF-miRNAs) and are involved in signaling pathways critically important for follicle growth, oocyte maturation, and early embryo development. Moreover, eFF-miRNAs are able to negatively regulate genes encoding inhibitors of follicle maturation and meiosis resumption as PTEN, MTOR, P21 and RB1. These data could reveal new actors in the molecular communication among cells of ovarian follicle and eFF-miRNAs may represent valuable biomarkers of oocyte quality and reproductive disorders