90 DESTABILIZATION OF COHESIN REC8 CAUSES ANEUPLOIDY AFTER THE SECOND MEIOSIS IN MURINE POST-OVULATORY AGED OOCYTES

Abstract

We have previously reported that the early embryos derived from post-ovulatory aged oocytes frequently exhibit aneuploidy, resulting from abnormalities in the cleavage apparatus of MII oocytes. Other studies have also described a potential mechanism that results in aneuploidy, which is attributed to the failure of a cell cycle checkpoint. The spindle assembly checkpoint (SAC), which acts during metaphase, is a monitoring system that equally distributes sister chromatids by correctly attaching spindle fibres to the appropriate centromere. Cohesin, a functional protein complex of the SAC, includes the REC8 subunit, and acts as an adhesion factor for sister chromatids. Segregation of sister chromatids occurs following the degradation of the cohesin complex by the separase enzyme. The segregation process can be mediated by meiosis-specific REC8, which contains a recognition site for separase. In this study, we examined the expression of meiosis-specific REC8 protein in murine post-ovulatory aged oocytes, and verified the association with aneuploidy induced during second meiosis. Superovulated oocytes from the ICR mouse strain were aged by culture for 3 to 24 h in vitro. To eliminate the male genome factor, chromosomal analysis was performed using oocytes activated by SrCl₂, without fertilization. The expression level of REC8 in oocytes, before and after activation, was analysed by Western blot, using a rabbit anti-REC8 antibody (primary) and horseradish peroxidase-conjugated anti-rabbit antibody (secondary). In the 6- and 12-h aged groups, 23.8% and 40.3% of oocytes, respectively, exhibited aneuploidy after the second meiosis. The rate of aneuploidy in the 12-h aged group was significantly higher than that in the fresh oocyte group (10.3%; P < 0.05). It could be speculated from our previous data that this fact contributed to the occurrence of aneuploidy in early embryos derived from the aged oocytes. In 3-, 6-, 12-, and 24-h aged groups, the results of semiquantitative analysis of REC8 levels in MII oocytes (non-activated) were 1.32 ± 0.38, 1.30 ± 0.58, 1.15 ± 0.21, and 0.98 ± 0.14, respectively. REC8 levels in the 24-h aged group were significantly lower than in the fresh group (1.86 ± 0.56, P < 0.05). The expression levels of REC8 in activated oocytes at 3 and 6 h were 0.53 ± 0.01 and 0.55 ± 0.04, respectively. REC8 levels in the 12-h (1.00 ± 0.03) and 24-h (0.95 ± 0.04) groups were significantly higher than in the fresh group (0.49 ± 0.09; P < 0.05). The significant reduction of REC8 levels at anaphase, after oocyte activation, was not observed in oocytes aged 12 h or more. In MII oocytes, REC8 levels tend to decrease gradually with post-ovulatory age. Destabilisation of the cohesin REC8 subunit may contribute to the nondisjunction of sister chromatids during second meiosis in post-ovulatory aged oocytes, ultimately resulting in aneuploidy.