Reprod. Nutr. Dev. 46 (2006) 219-226
DOI: 10.1051/rnd:2006002

Demecolcine-induced enucleation of sheep meiotically maturing oocytes

Jian Hou, Tinghua Lei, Lei Liu, Xiuhong Cui, Xiaorong An and Yongfu Chen

State Key Laboratory for Agrobiotechnology and Department of Animal Physiology, College of Biological Science, China Agricultural University, Beijing, 100094, China

(Received 02 March 2005; accepted 15 November 2005; published online 6 April 2006)

Abstract - The objective of this study was to investigate the possible effect of demecolcine, a microtubule-disrupting reagent, on induced enucleation (IE) of sheep meiotically maturing oocytes. Immunofluorescent staining with anti-tubulin antibodies was used to examine the spindle status of the oocytes. When the oocytes with intact germinal vesicles (GV) were cultured in the medium containing various concentrations of demecolcine (0.01 to 0.4 µg·mL^-1) for 20 to 22 h, the spindle microtubule organization and first polar body (PB1) extrusion were inhibited by demecolcine in a dose-dependent manner. The highest IE rate (58.1%) was from the treatment with 0.04 µg·mL^-1 demecolcine. Demecolcine treatment applied after germinal vesicle breakdown (GVBD) or at metaphase (M) yielded a PB1 extrusion rate and IE efficiency similar to the treatment applied at the onset of maturation. Analysis by immunofluorescence showed that both nonspindle microtubules and spindle microtubules were significantly disorganized by demecolcine. Combination treatment with demecolcine and cycloheximide (CHX) or 6-dimethylaminopurine (6-DMAP) led to single pronuclear formation rather than PB1 extrusion. When demecolcine-treated oocytes were transferred into demecolcine-free medium, the ability to extrude PB1 was quickly restored and a 72.1% IE rate was obtained following such treatment. These results demonstrate that demecolcine can be used as a potential reagent for induced enucleation of sheep meiotically maturing oocytes and may greatly facilitate research in nuclear transfer.

Key words: enucleation / oocyte / sheep / demecolcine / meiosis / nuclear transfer

Corresponding author: Xiaorong An xra@cau.edu.cn

© INRA, EDP Sciences 2006