BIOLOGY OF MALE AND FEMALE GAMETES
7:30 AM-10:00 AM
(193) EFFECT OF TRANSIENT SUPPRESSION OF MEIOSIS WITH ROSCOVITINE ON SUBSEQUENT DEVELOPMENTAL COMPETENCE OF EQUINE OOCYTES.
Franz, Lilian2, Choi, Young Ho1, Squires, Edward2, Seidel, George2, Hinrichs, Katrin1, 2 Animal Reproduction and Biotechnology Laboratory - Colorado State University, Fort Collins, CO1 Department of Veterinary Physiology and Pharmacology, College Station, TX
ABSTRACT- The effect of roscovitine on developmental competence of equine oocytes has not been evaluated. To study this, equine oocytes were classified by cumulus morphology alone (disregarding granulosa cell morphology); 54% were classified as compact (Com) and 43% as expanded (Exp). Oocytes were placed within 90 min of slaughter in either Equine Maturation I, or in the same medium but with 66 mM roscovitine and BSA instead of hormones, growth factors and FBS. Three culture groups were evaluated: Maturation (Mat), 30 h in EMMI medium; roscovitine (Ros), 24 h in roscovitine-containing medium then 30 h in EMMI; and 54 h control (54M), 54 h in EMMI. After culture, oocytes with a polar body were subjected to intracytoplasmic sperm injection using a Piezo drill, then cultured in G1.2 medium for 96 h, assessed for morphological cleavage, and stained with Hoechst 33258 to visualize nuclei. A subset of oocytes in the Ros treatment was fixed and stained at the end of 24 h roscovitine culture; of these, 26/31 (84%) of the Com and 16/28 (57%) of the Exp oocytes had a germinal vesicle. There were no differences in morphological cleavage rates among treatments within cumulus type (proportions of Com oocytes cleaving in the Mat, Ros, and 54M treatments were 82, 80, and 83%, respectively; for Exp oocytes 76, 71, and 62%, respectively). The proportion of embryos having > 2 normal nuclei was significantly higher for Com oocytes in the Ros than in the 54M treatment (63 vs. 36%; P < 0.05); and significantly higher for Exp oocytes in the Mat than in the Ros treatment (63 vs. 42%; P = 0.05). Significantly more Com oocytes yielded embryos having > 8 nuclei in the Ros and Mat treatments (63 and 50%) than in the 54M treatment (8%; P < 0.01). For Exp oocytes, the proportion of embryos with > 8 nuclei was significantly higher in the Mat treatment (44%) than in the 54h treatment (8%; P < 0.05) and tended to be higher in the Ros treatment (35%) than in 54M (P = 0.08). The average number of nuclei was significantly higher in Ros Com oocytes (13.5 + 2.1) than in any other group (3.6 + 0.9 to 9.3 + 1.4, P < 0.05). These results demonstrate that roscovitine can be used to maintain Com, but not Exp, equine oocytes in the germinal vesicle stage for up to 24 h without decreasing their developmental potential. This may aid in timing of assisted reproductive techniques in the horse.
KEY WORDS: roscovitine, equine, oocyte, ICSI