Chemical modification and chemical cross-linking for protein/enzyme stabilization

The developmental competence of bovine follicular oocytes that had been meiotically arrested with the phosphokinase inhibitor 6-dimethylaminopurine (6-DMAP) was studied. After 24 h in vitro culture with 2 mM 6-DMAP, 85 +/- 12% of the oocytes were at the germinal vesicle stage compared to 97 +/- 3% at the start of culture (P > 0.05). After release of the 6-DMAP inhibition, followed by 24 h IVM, 82 +/- 18% were at MII stage, compared with 93 +/- 7% in the control group (P > 0.05). The 6-DMAP oocytes displayed a much higher frequency of abnormal MII configurations than the control oocytes (67% vs 23%; P < 0.0001). In addition spontaneous oocyte activation was more frequent than among control oocytes (5% vs 0.3%; P 0.0006). After IVF of 6-DMAP oocytes, normal fertilization was lower (76 +/- 8% vs 89 +/- 7%; P < 0.01), oocyte activation higher (11 +/- 5% vs 2 +/- 2%; P < 0.01), and polyspermy slightly but not significantly higher (8 +/- 7% vs 4 +/- 4%; P > 0.05), compared with the control group. Cleavage was lower (61 +/- 13% vs 81 +/- 6%; P < 0.001), as well as day 8 blastocyst formation (17 +/- 7% vs 36 +/- 8%; P < 0.001). The MII kinetics was different for 6-DMAP and control oocytes. Maximum MII levels were reached at 22 h IVM in both groups, but 50% MII was reached at 17 h in 6-DMAP oocytes, compared to 20 h in control oocytes. Ultrastructure of MII oocytes was similar in the two groups, but in 6-DMAP oocytes the ooplasmic vesicle pattern at GV was at a more advanced stage than in control oocytes. In conclusion, 6-DMAP exposure of GV oocytes prior to IVM induce asynchronous cytoplasmic maturation, leading to aberrant MII kinetics. Thus, at the time of insemination a smaller cohort of oocytes will be at the optimal stage for normal fertilization and subsequent blastocyst development.