Dr Peter Nagy, from Reproductive Biology Associates, Atlanta, collaborating with the University of Connecticut, USA, told the European Society of Human Reproduction and Embryology annual conference that former approaches to haploidisation using a fully mature oocyte and a resting (interphase) somatic cell had caused misaligned chromosomes during cell division. However, he was confident from his team's latest experiments that this difficulty could be overcome, even though their new approach also ran into some problems.
"We decided to initiate haploidisation at an earlier stage in the oocyte's cell cycle, when it was still immature, but this time using a somatic cell in its active (metaphase or G2/M) stage. Essentially, we took the control of the first nuclear division away from the oocyte and gave it to the somatic cell," he said.
The US-Brazilian research team , working with mouse cells, removed the nucleus of the immature oocyte, then transformed the somatic cell from its diploid (46 chromosome or 2n) stage to its next (4n) stage and transferred it to the immature enucleated oocyte (ooplast).
"What we expected by doing this was that the DNA in the somatic cell would condense into chromosomes inside the somatic cell - not in the ooplast - and that the somatic cell would direct the chromosome alignment and initial spindle formation, which would then be normal. The nucleus of the somatic cell, at its second stage of division and correctly assembled, would then undergo chromosome segregation in the ooplast, resulting in twice its diploid nuclear content during in-vitro maturation. As a result of an artificial activation, a second round of chromosome segregation provides the haploid (23 chromosome) normal oocyte content. This is a novel strategy that cannot be used with a mature ooplast because mature ooplasts can support only one round of chromosome segregation."
However, the researchers found that there were still some misaligned chromosomes and problems with the integrity of the spindle - the chromosomes' 'holding' mechanism. But, they are confident that these will be overcome.
"This initial set of experiments shows that it is possible to induce haploidisation with our approach," said Dr Nagy. "This is the first time that this has been tried so we are still learning. Now we have to check how frequently the chromosomal problems occur and whether there is an easy solution or whether it is a fundamental difficulty."
But, even in a worst case scenario, he said, it does not mean that they were back to the drawing board because his team was already developing new techniques to overcome the problem.
"I'm really confident - not simply optimistic - that haploidisation will work and if everything goes well we will be able to obtain artificial gametes in one or two years. Even if we encounter more problems it should still be possible within three to five years."
Haploidisation is not cloning because it is the production of a reconstituted egg (which can then be fertilised by the sperm) in a situation where a woman has no eggs of her own. One of the woman's own somatic cells would be the source of the chromosome-carrying nucleus, which would be transferred into a donated 'shelled-out' oocyte.
Abstract no: O-169 (Tuesday 16.00hrs CET Roma room)
 Haploid: a cell with only one set of chromosomes - in humans 23. Only the egg and sperm are haploid.
 University of Connecticut, Animal Science, Storrs, USA; Reproductive Biology Associates, Atlanta, USA; Clinical e Centro de Pesquisa em Reprodução Humana Roger Abdelmassih, São Paulo, Brazil.
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