RIKEN Center for Developmental Biology

2003 Annual Report

The experimental cloning of animals has a history that extends back more than fifty years, to when Briggs and King successfully produced tadpoles by transplanting the nuclei from embryonic cells into frogs' eggs whose own nuclei had been removed. At the time, cloning was used not as a technique to be studied for its immediate application, but as a means of testing a fundamental question of reproductive biology: whether the processes of fertilization and later development, in which the body's cells become more specialized and functionally distinct, involves a loss of genetic information, or whether all cells retain the full set of genetic code, even after differentiation has proceeded. The success of these initial experiments in cloning by nuclear transfer demonstrated conclusively that cells do maintain intact genomes even after differentiation, as the genetic code in a specialized cell's nucleus is sufficient to instruct an egg into which it is transplanted to give rise to a normal individual.

Although questions of whether genetic information is lost or irreversibly altered during development were laid to rest, new questions arose to take their place. For Teruhiko Wakayama, the most intriguing issue raised by the ability of animals to be cloned using the nuclei of specialized somatic cells (which form the body and, unlike sperm and eggs, cannot normally produce a new individual), is that of reprogramming, the processes by which the genome receives new sets of coding instructions enabling it to order the development of all the cells in a new individual while remaining intact and fundamentally unchanged in each of those cells. The Wakayama lab studies mammalian cloning and fertilization with the same basic goal as drove Briggs and King in their nuclear transfer studies a half century ago: to answer central questions in the biology of animal reproduction.

Cloning efficiencies

In all species and in all experimental methods tested to date, cloning by nuclear transfer has consistently low efficiency rates – generally below 5% of enucleated eggs fertilized by nuclear transfer go on to develop into live-born offspring. Many hypotheses have been proposed to explain the inefficiency of this procedure, while accounting for the fact that cloning is not altogether impossible. It has been suggested that the process of removing the nucleus from an oocyte (an unfertilized egg) or the absence of chromosomal information during the several hours in which the egg is missing a nucleus may somehow damage or cause the loss of factors that would normally act to reprogram the genetic information in the fertilizing (or transplanted) cell's nucleus. To test this possibility, Wakayama re-ordered conventional cloning methodology by first transferring nuclei from cumulus cells into oocytes whose own nuclei were still present, and only then removing the mitotic spindle derived from the native nucleus. These experiments resulted in the generation of live offspring at a rate of efficiency similar to that of standard cloning by nuclear transfer, providing evidence that tends to counter the hypothesis that the temporary absence of a nucleus is responsible for the poor developmental prospects of NT oocytes.

Scientists in the Wakayama lab have adopted similar approaches to the study of fertilization – experiments that involve the substitution of components or the reordering of natural sequences of events to test for specific biological function. In nature, fertilization occurs after a mature spermatozoon fuses with and activates an oocyte, but some laboratory techniques achieve fertilization by artificially activating the egg and then injecting a spermatid, which is an incompletely maturated sperm cell. Such techniques are known to be less efficient than fertilization using mature spermatozoa, but the reason for this lower developmental competency remains unknown. The Laboratory for Genomic Reprogramming is now comparing in vitro fertilization using spermatozoa and spermatids under controlled conditions in an effort to identify the differences between these stages in the developing sperm cell. Future research will look at changes in epigenetic modifications to the sperm genome over time as a possible explanation for their disparate potentials.

New technologies

Sperm preserved for use in experiments and in vitro fertilization is traditionally frozen in liquid nitrogen at extremely low temperatures (around -190 degrees C). This cryopreservation allows the sperm to be maintained viably for very long periods, but requires expensive facilities and some degree of technical skill in handling. Oocytes and fertilized eggs are also extremely labile, and must likewise be stored cryogenically. But these requirements tend to limit the access of germ cells to researchers unequipped with liquid nitrogen facilities, preventing the spread of the technology and the development of research using these cells. Wakayama hopes to develop new, less expensive and less technically demanding methods for the storage and maintenance of germ cells for experimental use. Recent tests using a modified commercially available culture medium showed that under the right conditions spermatozoa can be preserved for long periods, up to 70 days, at 4 degrees Celsius, a temperature that can be maintained using ordinary and inexpensive refrigeration equipment. This new preservation method opens up opportunities in animal breeding and reproductive biology research to scientists working under limited budgets, which Wakayama hopes may help to make these fields of science more accessible to developing countries and smaller labs.