Fertilization and Early Embryogenesis
(M280) DEVELOPMENT AND DNA DAMAGE IN PARTHENOGENETICALLY ACTIVATED BOVINE EMBRYOS PRODUCED FROM OOCYTES DERIVED FROM SMALL AND LARGE FOLLICLES.
Bastos, Guilherme1, 2, Gonçalves, Paulo Bayard1, Bordignon, Vilceu2, 1 Federal University of Santa Maria, Santa Maria, RS, Brazil2 McGill University, Ste-Anne-De-Bellevue, QC, Canada
ABSTRACT- It has been demonstrated that oocytes of several species acquire the capacity to complete meiotic maturation and support early embryonic development during the final stages of follicular growth. The objective in this study was to verify the expression pattern of the phosphorylated histone H2A.X (H2A.X), which is an indicator of DNA double-strand breaks, in parthenogenetically activated embryos derived from oocytes collected from small (1-2 mm) and large (4-8 mm) follicles. Oocytes were matured in vitro for 24 h, selected for the presence of a polar body, activated with Ionomycin (5 M/5min) followed by incubation in 6-DMAP (2.8 mM/5h), and then cultured in synthetic oviduct fluid medium. The pattern of H2A.X was determined at 36 h post-activation using an anti-phospho-H2A.X (Ser 139) primary antibody and a Cy3-conjugated secondary antibody. The rates of meiotic maturation (84% vs. 69%) and development to blastocyst stage at day 7 (34% vs. 19%) were significantly higher in oocytes from large compared to small follicles. However, there was no difference in both early (before 24h) and late (from 24-36h) cleavage rates. A high proportion of cleaved embryos fixed at 36 h had detectable amounts of H2A.X, ranging from few foci to a complete diffuse staining of the nuclei. Signals indicating H2A.X were detected in 68.8% (small follicles) and 75.8% (large follicles) of nuclei. The average number of nuclei at 36 h was greater (4.5±1.5 vs. 3.8±1.3; p=0.02) in early-cleaved embryos derived from small compared to large follicles. However, the number of nuclei tended to be higher (7.75±3.6 vs. 5.92±2.75; P=0.09) in the embryos from large follicles at 50 h. The immunolocalization of the High-mobility group protein (HMG-17) at 50 and 60 h revealed that a greater proportion of nuclei were more intensively stained in embryos derived from small, compared to large follicles. In summary, these data suggest a difference in the timing of embryo cleavage after the 2-cell stage, in the timing of nuclear assembly of the HMG-17 protein, but not in the expression of the H2A.X in parthenogenetically activated embryos from small and large follicles. Supported by CNPq and NSERC.
KEY WORDS: bovine oocytes, follicle size, embryonic development