27 CYTOPLASM OF A GERMINAL VESICLE OOCYTE CAN PROMOTE SOMATIC NUCLEAR REPROGRAMMING IN MICE
Authors: Bui H.T. , Van Thuan N. , Kishigami S. , Wakayama S. , Hikichi T. , Ohta H. , Mizutani E. , Wakayama T. 
Published: December 2006

A number of studies have shown that epigenetic reprogramming is severely deficient in cloned embryos, the majority of which exhibit histone hypermethylation. Xenopus geminal vesicle (GV) oocytes have been reported to have a DNA demethylating activity (Simonsson and Gurdon 2004 Nat. Cell Biol. 6, 984–990). In an attempt to develop a new method for erasing or reprogramming the epigenetic status of the donor cell prior to nuclear transfer, we examined whether the mammalian GV oocyte cytoplasm can demethylate H3-K9 of somatic cell nuclei and improve the quality of reconstructed embryos. In the first series of experiments, cumulus nuclei were injected into enucleated GV oocytes and cultured for various times, 0 h, 3 h, 7 h, 9 h, 11 h, and 16 h, before examining the chromosome morphology of somatic nuclei together with Me-H3-K9. In the second series of experiments, permeabilized cumulus cells were immersed with GV oocyte extract and injected into enucleated MII oocytes. These reconstructed oocytes were activated and cultured until the blastocyst stage. Preparation of oocyte extracts was as follows: 200 GV oocytes were collected and zonae pellucidae removed using Tyrode's solution. These zona-free oocytes were broken down in 5 �L of HEPES medium containing the ATP-generating system (1 mM ATP, 10 mM creatine phosphate, 25 �g mL-1 creatine kinase, 100 �M GTP). Permeabilized cumulus was incubated in oocyte extract for 45 min at 37�C. The demethylation of H3-K9 was analyzed in nuclear transfer embryos at the pronucleus, 2-cell, 8-cell, morula, and blastocyst stages. In 2 experiments, we examined a total of 234 enucleated GV oocytes and 358 enucleated MII oocytes. The results of first experiment showed that histone H3-K9 begins demethylation in the somatic chromosome 1 h after injection into GV oocytes (90%) and, importantly, this state was maintained until the MII-like stage. The donor somatic chromosomes in the enucleated GV oocyte can condense and undergo stages GVI (0–3 h), MI (7 h), and AI-TI (9–11 h), and the MII (16 h)-like stage. These results clearly show that enucleated GV oocytes can carry out spindle assembly and extrude the first polar body. However, some of these chromosomes are located not only on the metaphase plate of the spindle but also on spindle poles or dispersed on the spindle. In the second experiment, reconstructed embryos with GV extract-treated somatic cells showed a strong H3-K9 demethylation that was significantly different to nontreated somatic cells at the blastocyst stage. The demethylation was expressed at a rate comparable to that observed in ICSI embryos. Although there was no increase in the frequency of development at the blastocyst stage, we found an increase in cell number at the blastocyst stage. Our findings show that the GV oocyte cytoplasm has the ability to erase methylation of H3-K9 in somatic nuclei, which suggests that the incorporation of GV mammalian oocyte components may contribute to the reprogramming of somatic cell nuclei.