166 MATURATION OF BOVINE OOCYTES IN POLY(DIMETHYLSILOXANE) MICROWELLS AND THEIR SUBSEQUENT DEVELOPMENT FOLLOWING IN VITRO FERTILIZATION

During bovine oocyte maturation, a lower density of cumulus cells surrounding oocytes reduces the developmental competence of the oocytes after IVF. Adding more cumulus cells (Hashimoto et al. 1998) rescues the developmental competence of the corona-enclosed oocytes. In this study, we examined the effects of poly(dimethylsiloxane) (PDMS) microwells (MW) for bovine oocyte maturation on the developmental competence of the oocytes following IVF. In experiment 1, MW were produced by making holes on 0.5-mm-thick PDMS plates using a 0.5-mm-diameter biopsy punch. The punched plates were placed on the bottoms of culture dishes. Bovine cumulus oocytes complexes (COC) were collected from slaughterhouse ovaries. Cumulus layers were removed from COC to prepare corona-enclosed oocytes (CEO) and denuded oocytes (DO). Then, COC, CEO, or DO were individually matured in single MW for 24 h at 39°C under 5% CO2 in air with high humidity. Ten oocytes of each group were matured in 50-μL droplets of maturation medium (group culture, GC) as controls. Maturation medium was TCM-199 supplemented with 10% FCS, 0.02 AU mL–1 FSH, and 1 μg mL–1 E2. The matured oocytes were fertilized with frozen–thawed spermatozoa. The embryos were cultured in CR1aa medium for 168 h under 5% CO2, 5% O2 and 90% N2 with high humidity. In experiment 2, effects of depth of MW for maturation on subsequent development following IVF were examined. Microwells were produced by making 0.5-mm-diameter holes on 0.5- or 1.5-mm-thick PDMS plates. Then, COC or CEO were individually matured in the MW for 24 h. Matured oocytes were fertilized in vitro and cultured for 168 h. Oocytes that were matured by GC were used as controls. In experiment 1(N = 4), rates of maturation (76–100%, n = 26 to 38), normal fertilization (53–70%, n = 44 to 49), and cleavage (61–77%, n = 114 to 117) were not different among all groups (P > 0.05; Fisher's PLSD test following ANOVA). Blastocyst rates were the same (P > 0.05) for COC matured in MW (50%) and by GC (43%). The rate for CEO that matured in MW (46%) tended to be higher (P = 0.061) than the rate for CEO that matured by GC (31%), and was comparable to the rate for COC matured by GC (43%). The blastocyst rates for DO that matured in MW and by GC were low (6%). In experiment 2 (N = 3), rates of maturation (86–100%, n = 13 to 28), normal fertilization (60–78%, n = 22 to 40), and cleavage (67–73%, n = 85 to 90) were not different among all groups (P > 0.05). However, the blastocyst rate for COC that matured in 1.5-mm-deep MW (53%) was significantly higher than the rates for COC that matured in 0.5-mm-deep MW (38%) and by GC (31%; P < 0.05). The results indicate that the developmental competence of oocytes that matured individually in PDMS MW was greater than that of oocytes that matured by GC. The deeper (1.5 mm) MW were found to be more effective for oocyte maturation than shallow (0.5 mm) MW and GC. The MW might increase density of cumulus cells surrounding oocytes, and the high cell-density enhanced the developmental competence of the oocytes.