Freeze-dried sperm
yields live rabbit

UH researchers apply pioneering
techniques used six years ago
to produce live mice

Ryuzo Yanagimachi, University of Hawaii's mouse-cloning pioneer, and colleagues have achieved another first: producing a live rabbit by using "dead" freeze-dried sperm.

The full-term pup was normal when it was born at the University of Connecticut, but its mother did not care for it and it soon died, said Yanagimachi, director of the Institute for Biogenesis Research in the John A. Burns School of Medicine.

This was the first live birth of a mammal other than a mouse from freeze-dried sperm, said Stefan Moisyadi, research coordinator at the institute.

Yanagimachi, a professor of anatomy and reproductive biology, developed freeze-dried sperm technology in 1998 to make live mice. The same year, his "Team Yana" became internationally known for the historic cloning of generations of mice, using what they called the "Honolulu Technique."

Yanagimachi has long been involved in basic research to improve the quality of sperm for infertile men, leading to a number of advances in biological development.

His institute has five research units investigating cloning, gene manipulation, cell differentiation, sperm and egg fertilization, and infertility.

In his freeze-dried process, sperm are technically dead, but the nucleus is viable and results in live births.

Yanagimachi and Xiangzhong Yang, of the Department of Animal Science/Center for Regenerative Biology at the University of Connecticut, headed the reproductive biologists who succeeded in fertilizing rabbit eggs with freeze-dried rabbit sperm.

Yanagimachi's lab supplied the rabbit sperm, which he said was more difficult than freeze-drying mouse sperm because rabbits are more like humans. All species except rodents have a centrosome, a specialized part of a cell essential for cell division, in the eggs and sperm, he said. Mice do not need it in sperm.

Some skeptics believed his success in creating mouse babies with freeze-dried sperm could not be duplicated in other species because the centrosome would be damaged.

Yanagimachi said the similarity between rabbit sperm and human sperm suggests freeze-drying could be used to preserve sperm from people and other animal species.

Freeze-drying breaks plasma membrane and causes fragmentation of sperm tails, but while the cell is dead, the DNA remains intact, he said.

His institute has no facility for rabbit experiments, and it is too expensive, so the sperm was freeze-dried at his lab and sent to the University of Connecticut, he said.

The rabbit sperm is frozen in liquid nitrogen to solidify, then liquid is removed slowly under vacuum and the dried sperm tissue remains behind.

Freeze-dried sperm keeps at room temperature, so it can be mailed without liquid nitrogen, which is troublesome and dangerous, Yanagimachi said.

Freeze-dried mouse sperm has remained active after being stored more than two years.

Ji-Long Liu, previously at the University of Connecticut and now with the Carnegie Institution of Washington's Department of Embryology in Baltimore, worked with the rabbits, Yanagimachi said.

One pup was born after 230 eggs were fertilized with freeze-dried sperm. The fertilized eggs developed into embryos, and some were transplanted into eight female rabbits.

The baby's death because of the mother's neglect often happens in single-birth pregnancies in species that usually give birth to multiple offspring, the scientists said. The mother saves her energy for the next litter.

Yanagimachi said the efficiency is low for production of live-birth rabbits, and the technique is "far from perfect." However, he said the concept has been proven, and he will continue working to improve the technology.

"If it works for rabbits, it should work for other species," he said.

Collaborating with him is David Whittingham, of England, a visiting scientist at the biogenesis institute.

The two scientists participated in an international project funded by the National Institutes of Health to preserve mice sperm more efficiently for studies of the role of genes in diseases.

Whittingham and associates in 1972 froze, thawed and implanted mouse embryos in surrogate mothers that delivered healthy mouse pups.


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