PARENT SESSION
GAMETE BIOLOGY AND GAMETOGENESIS - A

Monday, August 2, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(146) OPTIMIZATION OF CULTURE CONDITIONS FOR IMAGING LIVING MOUSE OOCYTES.

McGinnis, Lynda^{1, 2}, Lee, Gloria², Overstrom, Eric¹, Albertini, David², ¹ Dept. of Comparative Biomedical Sciences, North Grafton, MA² Dept. of Anatomy and Cellular Biology, Boston, MA

ABSTRACT- The increasing use of optical imaging and vital fluorescent molecules for evaluation of intracellular dynamics encourages development of a simplified, low cost, high throughput culture system that minimizes perturbations of oocyte viability. Towards this end, we have compared the effects of culture media in two different gas environments on in vitro maturation of mouse cumulus oocyte complexes (COC). Immature CF1 mice received 5 IU eCG. Intact COC were released from ovaries 40 h post-eCG and randomly allotted to one of 5 media: KSOM^aa_g, MEM, mL15+H, KSOM^aa_g+H or MEM+H. All media were supplemented with 10% FBS and all COC cultured at 37°C. The KSOM^aa_g and MEM cultures were gassed with 5% CO₂ in air while, COC cultured in HEPES buffered media were incubated in air without increased CO₂. COC were cultured 16 h, washed briefly in 0.3 mg/ml hyaluronidase and fixed with 2% formalin in microtubule stabilization buffer. Oocytes were labeled with antibodies against -tubulin, total tubulin and f-actin. DNA was stained with Hoechst. Oocytes were evaluated for stage of maturation, and measured for size of oocyte, MII spindle and extruded polar body (PB). Volumes were calculated for an oblate ellipsoid and the ratios of volume of oocyte to PB and oocyte to spindle were compared as indicators of oocyte quality. All media supported GVBD and progression to MII except mL15+H (Specialty Media, #F12B), which inhibited completion of meiosis (GVBD 60%, MII 13%). Data for the other 4 media show no obvious differences between the extent of maturation to MII (KSOM^aa_g 64%; MEM 61%; KSOM^aa_g+H 71%; MEM+H 63%) or in ration of oocyte to PB size(KSOM^aa_g 0.030 SEM 0.013; MEM 0.029 SEM 0.019; KSOM^aa_g+H 0.040 SEM 0.026;MEM+H 0.046 SEM 0.029)or ratio of oocyte to spindle size (KSOM^aa_g 0.013 SEM 0.007; MEM 0.011 SEM 0.007; KSOM^aa_g+H 0.013 SEM 0.005; MEM+H 0.014 SEM 0.005). These results suggest that mouse oocytes in HEPES buffered media can spontaneously mature to MII without the need for regulated CO₂ atmosphere. Studies are ongoing to determine the developmental competence of these oocytes.

KEY WORDS: polar body, medium, oocyte, spindle