58 EFFECTS OF CAFFEINE AND VANADATE ON NUCLEAR REMODELING AND MICROTUBULE DISTRIBUTION OF PORCINE NUCLEAR TRANSFER EMBRYOS

Abstract

The present study was conducted to control nuclear remodeling types by treatment with caffeine or vanadate, and to examine the microtubule distribution of nuclear transfer embryos (NTs) after nuclear remodeling control and the mitotic spindle assembly and its morphological changes during the first mitosis of NTs in the pig. Enucleated oocytes were treated with 5 mM caffeine or 0.5 mM sodium orthovanadate (vanadate) for 2.5 or 0.5 h to increase or decrease MPF activity before injection of fetal fibroblast cells. Reconstituted eggs were fused by an electric stimulation (ES, 1.5 kV cm^-1), activated by a combination of 2 pulses of ES (1.0 kV cm^-1), and cultured for 3 h with 2 mM 6-dimethylaminopurine (6-DMAP) at 1 h after fusion treatment. Some matured oocytes were also treated by the same chemicals before parthenogenetic activation under the same conditions as NTs, and cultured in vitro to evaluate the effects of these chemicals on embryo development. NTs and parthenogenetic embryos were cultured in PZM-3 for 20 h or 6 days at 39°C, 5% CO₂ in air, respectively. Nuclear remodeling types of NTs were examined at 1 h after fusion (before activation) by the whole-mount method. At least 3 replicates for each experiment were performed. Microtubules and DNA of NTs that were fixed at 1 h or 20 h after fusion were detected by indirect immunocytochemical technique. Images were captured using laser scanning confocal microscopy. Caffeine and vanadate did not affect the development to the blastocyst stage of porcine parthenogenetic embryos. When a nucleus was exposed to oocyte cytoplasm treated with caffeine, premature chromosome condensation (PCC) occurred at a higher rate (82/98, 83.7%) compared to control (42/73, 57.5%) and vanadate-treated (11/91, 12.1%) groups (P < 0.05). The proportion of embryos that did not undergo nuclear envelope breakdown (NEBD) was higher in the vanadate treatment group (87.9%) compared to the caffeine and control groups (16.3 and 42.5%, respectively; P < 0.05). The frequency of embryos showing a γ-tubulin only and both γ- and β-tubulins were 3.9–9.4% and 21.9–34.6%, respectively, in NTs (total 87 embryos) at 1 h after fusion regardless of caffeine and vanadate treatments. In the majority of NTs (61.5–68.6%), microtubules were not observed. At 20 h after fusion, the frequency of the embryos undergoing normal mitosis was similar in the control (17/45, 37.8%) and caffeine (19/43, 44.2%) groups, but it was significantly lower in the vanadate group (7/37, 18.9%; P < 0.05). The present study demonstrates that the nuclear remodeling type of NTs can be controlled by treatment with MPF regulators, caffeine and vanadate, and such treatment is not related to the microtubule distribution in porcine NTs. The finding, however, that the vanadate can delay the mitotic progression of porcine NTs at the first cell cycle may be due to the lack of NEBD and PCC.

This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD;KRF-2005-042-F00030).