Gamete Biology

Reduced Expression of MAD2, Bcl-2, and MAP Kinase Activity in Pig Oocytes after In Vitro Aging Are Associated with Defects in Sister Chromatid Segregation During Meiosis II and Embryo Fragmentation after Activation

Abstract
This study was conducted to examine expression of centromere protein B (CENP-B), spindle checkpoint protein MAD2 (mitotic arrest deficient protein) and anti-apoptotic protein Bcl-2, activities of MAPK (mitogen-activated protein kinase) and mitochondria distribution in pig oocytes during ageing, and their relationship with sister chromatid separation during meiosis II and embryo fragmentation and apoptosis after activation. After immature oocytes were cultured for 40-72 h, CENP-B, MAD2, tubulin, Bcl-2 and MAPK in the oocytes were examined by immunoblotting. Spindles, chromosomes, kinetochores and mitochondria were examined by immunofluorescence staining and apoptosis was examined by TUNEL assay. It was found that tubulin and CENP-B was not changed during 40-72 h of culture. However, the expression of MAD2 and Bcl-2 and the activity of MAPK were gradually reduced during oocyte ageing. The percentages of oocytes with normal spindle, chromosomes and kinetochores were also reduced as oocyte aged from 9.5% at 40 h to 17.3, 34.6 and 42.9% at 48, 60 and 72 h, respectively. Aggregated mitochondria were found in the aged oocytes as compared with the uniform distribution in young oocytes. After activation, the proportions of oocytes with abnormal anaphase II were significantly increased in aged oocytes. More (P<0.001) oocytes cultured for 60-72 h fragmented and showed apoptosis after activation as compared with the oocytes cultured for 40-48h. This study indicates that ageing reduces expression in spindle checkpoint protein and anti-apoptosis protein, and MAPK activity in pig oocytes. These events in turn cause abnormal sister chromatid segregation during meiosis II, embryo fragmentation and apoptosis.

Key words: Gamete Biology • Gametogenesis • Meiosis • Oocyte development

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