Aneuploidy in horse oocytes and embryos; how late is too late? The 'fertility clock' doesn't only tick for career women

Abstract

Advanced mare age is known to be associated with reduced fertility and increased incidence of early pregnancy loss (EPL). Aberrations of embryonic chromosome number (aneuploidy) appear to be the primary contributor to this age-related increase in EPL. Age-related aneuploidy is the primary cause of developmental arrest, implantation failure, miscarriage and congenital birth defects in human pregnancy, and is mostly associated with loss of inter-chromosomal cohesion, abnormalities in chromosome-spindle interaction, and lack of stringency of the Spindle Assembly Checkpoint (SAC) mechanism. The aim of this thesis was to evaluate the effect of advanced mare age on the incidence of aneuploidy in in vitro matured oocytes, and to investigate the contributions to aneuploidy of the loss of chromosomal centromeric cohesion and of an abnormal SAC activity. In particular, we evaluated the effect of advanced mare age on gene expression for selected cohesin components, and on the incidence of aneuploidy and the strength of chromosome centromere cohesion by, respectively, counting the number of chromatid-kinetochore units and measuring the inter-kinetochore distance (iKD). Afterwards, we investigated the gene expression for the main SAC components in in vitro matured oocytes from young and old mares, and on the basis of these results, we investigated the function of MPS1 and AURKB/C during spindle assembly by analysing spindle morphology and chromosome alignment of in vitro matured MII oocytes from old and young mares, after nocodazole-induced microtubule depolymerization and spindle reassembly in the presence of specific MPS1 and AURKB/C inhibitors. Finally, we indirectly evaluated the incidence on aneuploidy in in vitro produced (IVP) and in vivo derived embryos, by evaluating presence of micronuclei and analysing nuclear morphology. At the same time, we investigated whether a correlation exists between bright field morphological characteristics of IVP embryos, the incidence of nuclear abnormalities and the likelihood of pregnancy after transfer. We showed that advanced mare age is associated with an increased incidence of chromosome misalignment and aneuploidy in in vitro matured oocytes. We found indeed premature separation and random segregation of the sister chromatids during the first meiotic division, which may originate from a weakened chromosomal centromeric cohesion, possibly explained by the increased iKD in oocytes from old mares. Moreover, MII oocytes from old mares showed a reduced mRNA expression for the SAC components MPS1, SPC25 and AURKC, and a higher incidence of spindle abnormalities after exposure to the MPS1 inhibitor CPD5. The defective function of the SAC protein MPS1 in oocytes from aged mares, may contribute to spindle instability which, in turn, could result in mis-segregation leading to aneuploidy. In addition, in vitro culture of horse embryos favours micronucleus formations, considered to be a proxy for mitotic aneuploidy. However, of all the bright field morphological characteristics, only the time of development and the expansion of the blastocyst resulted to be associated with the pregnancy success. All these finding may contribute and explain the higher incidence of EPL observed in aged mares and after transfer of IVP embryos.