In previous studies, we found the production of antibodies against cross-reactive bovine serum albumin (BSA) in D-galactose (D-gal) induced aging mouse models. We performed immunoblot analysis with mouse tissue lysates to investigate the changes in the overall autoantibody production in this animal model. And we were able to see the possibility of altering the activity of mouse natural antibodies in this process. In this study, we examined changes in existing natural antibodies in a D-gal-induced aging mouse model. Serum samples were collected from 3-week-old mice (3w), 13-week-old mice (13w), and 13-week-old mice that were treated with D-gal for 6 weeks (13wDG), beginning at the age of 8 weeks. Levels of immunoglobulins (IgM, IgG, and IgA) were quantitatively analyzed in serum samples. Tissue samples were obtained from skin, spleen, and ovary for Western blotting analyses. Natural antibody activity was examined by enzyme-linked immunosorbent assay analyses of anti- double-stranded DNA (dsDNA) antibody. Western blotting analyses using mouse tissue lysates showed that several protein bands detected by serum antibodies from 3w mice became increasingly thicker when detection was performed with serum samples from 13w and 13wDG mice, indicating quantitative increases in levels of natural antibodies. Relative amounts of total IgG, IgM, and IgA immunoglobulins sequentially increased in serum samples from 3w, 13w, and 13wDG mice. A similar tendency was observed regarding the levels of IgM and IgG antibodies against dsDNA. These results indicate increased levels of natural antibodies in the D-gal-induced aging mouse model. Therefore, this animal model could be useful for future natural antibody research.

Previous studies have shown that BMI-1026 is a potent inhibitor of the cyclin-dependent kinases (cdk). In cell culture, the compound also arrests G2/M strongly and G1/S and S weakly. Two key kinases, cdk1 (p34cdc2 kinase) and mitogen-activated protein (MAP) kinase (erk1 and 2), perform crucial roles during oocyte maturation and, later, metaphase II (MII) arrest. In mammalian oocytes, both kinases are activated gradually around the time of germinal vesicle breakdown (GVBD) and maintain high activity in eggs arrested at metaphase II. In this study, we examined the effects of BMI-1026 on GVBD and MII arrest in mouse oocytes. BMI-1026 inhibited GVBD of immature oocytes and activated MII-arrested oocytes in a concentration-dependent manner, with more than 90% of oocytes exhibiting GVBD inhibition and MII activation at 100 nM. This is approximately 500～1,000 times more potent than the activity reported for the cdk inhibitors roscovitine (~50 M) and butyrolactone (～100 M). Based on the results of previous in vitro kinase assays, we expected BMI-1026 to inhibit only cdk1 activation in oocytes and eggs, not MAP kinase. However, in our cell-based system, it inhibited the activity of both kinases. We also found that the effect of BMI-1026 is reversible. Our results suggest that BMI-1026 inhibits GVBD and activates MII-arrested oocytes efficiently and reversibly and that it also inhibits both cdk1/histone H1 kinase and MAP kinase in mouse oocytes.