47 CHROMATIN AND CYTOSKELETAL REORGANIZATION OF RABBIT OOCYTES AFTER CUMULUS CELL NUCLEAR TRANSFER

Abstract

It has been reported that a preincubation of the reconstructed oocytes prior to activation treatment might enhance the efficiency of nuclear reprogramming after somatic cell nuclear transfer in many species. The objectives of this study were to evaluate chromatin and cytoskeletal reorganization of nuclear-transferred rabbit oocytes before activation treatment. Sexually mature New Zealand White rabbits (6-month-old) were superovulated by six consecutive injections of FSH (0.4 mg) at 12-h intervals, followed by a single hCG (200-IU) injection. Mature oocytes were collected by flushing the oviducts 14–15 h post-hCG treatment. Cumulus cells were introduced into enucleated oocytes by piezo-driven microinjection. The reconstructed oocytes were randomly allocated to two treatment groups, with or without electric pulses (EP, 2 kV cm⁻¹), and the progressive changes of the chromatin and cytoskeleton were examined by immunocytochemical staining at 0, 2, and 4 h after EP. The oocytes (n = 59) without successful enucleation served as the control, in which 30 out of 59 oocytes were electrically pulsed. After immunocytochemical staining, the proportions of reconstructed oocytes possessing dense chromatin decreased from 0 to 4 h (40-0% vs. 82-9%, respectively) in both groups, but only the oocytes without EP showed a significant reduction (P < 0.05). In contrast, the proportions of reconstructed oocytes with spread chromatin increased significantly from 0 to 4 h in both groups (20–95% vs. 5–82%, respectively; P < 0.05). The oocytes at 0 h after EP had the greatest percentage (35%) of normal chromosome alignment on the spindle compared to the other EP groups. The EP treatment also caused significant increase of the cytasters in both the reconstructed (5 to 45%, P < 0.05) and the control oocytes (20 to 78%) at 0 h. These changes were recovered at 2 h after EP, but the proportions of cytasters in the reconstructed oocytes increased to 59% in the non-EP group and 81% in EP group at 4 h after EP. The percentages of reconstructed oocytes with multiple spindles increased from 0 to 27% and 43% in EP and non-EP groups, respectively. These results demonstrated that EP changed the proportions of chromatin configurations from the condensed form to normal alignment on the metaphase plate. The extended preincubation period for the reconstructed oocytes prior to activation treatment might increase the incidences of chromatin and spindle abnormalities of the reconstructed rabbit embryos.