Compromised Spindle Assembly Check-point Function in Oocytes From Aged Mares Impairs Correct Chromosome Alignment

The incidence of early pregnancy loss in mares increases with age. In older women, embryonic aneuploidy of meiotic origin is the leading cause of miscarriage. Although oocytes from older mares have been proposed to be more prone to meiotic chromosome segregation errors, the incidence and causes of mis-segregation are not known. This study evaluated the effect of advanced maternal age on spindle assembly check-point (SAC) component expression and function in mares. Cumulus oocyte complexes from slaughtered mares were grouped by mare age (young, < 15y; old, !15y), matured in vitro for 26h and denuded. Only Metaphase II oocytes were studied further. mRNA expression for important SAC components and regulators (MAD2, BUB1, BUB3, BUB1B, MPS1, NDC80, SPC25, AURKB, AURKC) was evaluated in pools (n4 per group) of 10 MII oocytes using qRT-PCR. Spindle morphology and chromosome alignment at MII (n20 per group) was evaluated by confocal microscopy after immuno!uorescent staining (IF) for DNA (Hoechst 33342) and a-tubulin (AlexaFluor 488-conjugated anti-a-tubulin). Spindle morphology was scored as normal (fusi- form, bipolar) or abnormal (tri- or tetra-polar, asymmetric), chromosome misalignment was scored as mild (1-5 misaligned) or severe (>5 misaligned chromosomes). To study the roles of MPS1 and AURKC in meiotic spindle function, IF was repeated after treating oocytes (n20 per group) with either an MPS1 inhibitor (cpd5: 0, 200 and 500nM) or an AURK inhibitor (ZM447439: 0,5 and 10mM) for 2h following Nocodazole-induced tubulin depoly- merization (20mM for 10min). Oocytes from old mares displayed reduced expression of MPS1, AURKC and SPC25, and a higher inci- dence of mild (37 vs 5%) and severe (11 vs 0%) chromosome mis- alignments and spindle abnormalities (5 vs 0%), than oocytes from young mares. The incidence of abnormalities increased further after treating oocytes from old mares with Nocodazole (mild misalignment 42 vs 10%; severe 21 vs 0%; spindle abnormalities 16 vs 0%). Oocytes from old mares were also more susceptible to Mps1 inhibition as shown by the increased incidence of spindle abnormalities and chromosome mis-alignment after 200nM (33 vs 0% spindle abnormalities, 43 vs 17% mild and 10 vs 6% severe mis- alignment) and 500nM cpd5 treatment (36 vs 23% spindle ab- normalities, 36 vs 23% mild and 45 vs 13% severe mis-alignment). By contrast, AURK inhibition primarily impaired spindle repoly- merization, and to a similar extent in both groups (25 vs 27% and 48 vs 38% spindle abnormalities after 5 and 10 mM, respectively). In conclusion, MII oocytes from aged mares show an increased inci- dence of spindle abnormalities and chromosome misalignment, and a reduced ability to recover from spindle depolymerization. Since advanced maternal age is associated with reduced MPS1 expression and increased sensitivity to MPS1 inhibition, we pro- pose that reduced MPS1 activity contributes to maternal age- related aneuploidy in horse embryos.