97 POSITION CHANGES IN THE RELATIONSHIP BETWEEN FIRST POLAR BODY AND CHROMOSOME OF GOLDEN HAMSTER OOCYTES

Abstract

The golden hamster represents an attractive species for studying reproductive physiology, oncology, genetics, and virology. In an effort to establish experimental protocols necessary for cloning golden hamsters, positional changes in the relationship between the first polar body (FPB) and chromosomes of golden hamster oocytes were examined under different conditions. 1) Female hamsters (6 weeks of age) superovulated with eCG (30 IU, i.p.) followed by hCG, (30 IU, i.p.) at intervals of 72 h were sacrificed at different times (13.5, 18, and 23 h) following hCG injection. The cumulus–oocyte complexes (COC) were collected from oviducts, and cumulus cells were removed with 0.1% hyaluronidase; 2) The oocytes of 13.5 h after hCG injection, with or without cumulus cells, were collected and cultured in HECM-3 for 5 and 10 h; 3) The COC collected from small vesicular follicles on the ovarian surface 72 h after eCG administration were cultured in HECM-3 supplemented with 5 μg mL^–1 of FSH and 5 μg mL^–1 of LH for 18 and 23 h. After the above treatments, denuded oocytes were stained with 10 μg mL^–1 of Hoechst 33342 and observed on an inverted fluorescence microscope. During observation, the location of the FPB relative to the MII spindle was recorded as the angle (0–30°, 30–90°, 90–180°) between the line from the FPB to the center of the oocyte and the line from the spindle to the center of the oocyte. Oocytes were also stained with 10 μg mL^–1 of propidium iodide (PI) before observation. The FPB that stained with PI were considered to be degenerated. All data were statistically analyzed by one-way ANOVA. Our results showed that 82.10% of FPB in oocytes collected at 13.5 h (n = 73) post-hCG were within the zone of 0–30°, which was significantly greater (P < 0.05) than those of oocytes collected at 18 h (n = 50; FPB 46.32%) and 23 h (n = 82; FPB 33.33%) post-hCG. The degenerate percentage of FPB in oocytes (without cumulus) cultured in vitro for 5 h (n = 72) and 10 h (n = 63) was 45 and 63%, respectively; this was significantly greater (P < 0.05) than that of oocytes with cumulus (5 h, n = 46; 32%; 10 h, n = 46; 45%). The percentage of FPB in oocytes matured in vitro for 18 h (n = 36) was 77.94% within the zone of 0–30°, which was significantly greater (P < 0.05) than the 38.83% seen in oocytes cultured in vitro for 23 h (n = 36). In conclusion, the results of this study demonstrate that a change in position of FPB relative to the MII oocyte chromosome is age-dependent in in vivo-matured oocytes. Cumulus cells can protect the FPB of in vitro-cultured oocytes from degeneration but do not significantly affect its change in position. In vitro-matured oocytes age more slowly than those of in vivo maturation and in vitro culture. These results define conditions for changing the FPB position relative to the MII oocyte chromosome and should facilitate the development of cloned golden hamsters as an animal model for human diseases.