UH team aids study of long-frozen sperm

Hope grows for reviving extinct species

Two University of Hawaii-Manoa researchers were part of a team that produced healthy offspring from sperm of a mouse dead and frozen for more than 15 years.

The finding raises the possibility of restoring extinct mammals such as the woolly mammoth if sperm could be retrieved from an animal frozen for thousands of years in permafrost, the researchers said.

Pioneering work in biological development over the years by Dr. Ryuzo Yanagimachi, University of Hawaii professor of anatomy and reproductive biology, led to the latest advance, reported in recent Proceedings of the National Academy of Sciences.

Yanagimachi and colleague Kazuto Morozumi of the Institute for Biogenesis Research in the UH John A. Burns School of Medicine participated with Japanese scientists in the research.

Leading the project was Atsuo Ogura of the Institute of Physical and Chemical Research Bioresource Center in Nibaraki, Japan. Ogura studied with Yanagimachi at UH about 10 years ago and the two have collaborated to simplify the sperm preservation technique.

Researchers now harvest sperm from dead animals and freeze it in a solution with chemicals to protect the cells.

The sperm usually is successful for fertilization when thawed and injected into an egg, but it's difficult to keep masses of sperm frozen without some damage to the genetic integrity, Yanagimachi said in an interview. He said temperature control is "troublesome."

It's simpler to put a mouse body in a freezer at minus 20 degrees centigrade and defrost the sperm when needed, he said.

The frozen mouse body used for the research in Japan was flown in dry ice from the Medical Research Council Mammalian Genetics Unit in England.

Yanagimachi said he and Morozumi were asked to review the first draft of a report on the work in Japan and he was surprised that the sperm was preserved in a minus 20 degree centigrade temperature.

"I didn't believe it," he said, explaining he thought the sperm had to be in liquid nitrogen at minus 196 degrees centigrade to preserve the DNA.

He said he asked Ogura to do more experiments, which took about half a year. He also did experiments in his Manoa lab, producing some pups with frozen mice sperm, to confirm Ogura's results.

Yanagimachi said he found there is no difference in fertilization rates whether the sperm is frozen for 15 years or one week.

"Simple freezing of animal bodies, using ordinary freezers, is perhaps the simplest procedure we can think of, and it can be done in any laboratory," the scientists reported in their paper.

Yanagimachi was recognized internationally in 1998 for historic cloning of generations of mice and also developing freeze-dried sperm technology. He produced a live rabbit from "dead" freeze-dried sperm, proving that while the sperm appears dead, the nucleus remains active.

The new study showed sperm can retain fertilizing ability in frozen reproductive organs or whole bodies longer than they anticipated, the researchers said.

"We found that the spermatozoa retrieved from the testes of mice frozen at minus 20 degrees centigrade for 15 years were able to produce normal offspring by microinsemination," they wrote.

"If spermatozoa of extinct mammalian species (e.g., woolly mammoth) can be retrieved from animal bodies that were kept frozen for millions of years in permanent frost, live animals might be restored by injecting them into oocytes from females of closely related species."

Yanagimachi said medically important animals and endangered species could be produced and preserved through this method.

It could also be used for in-vitro fertilization in humans, he said. For example, If a man dies without children, his testes or sperm could be frozen for future use, he said, noting he's had many requests from people to freeze sperm.

Yanagimachi recalled when the Discovery Channel sent a film crew to interview him after reports in 1997 that a woolly mammoth was found buried in permafrost in Siberia. A group of scientists planned to excavate and clone the animal, which died about 20,000 years ago, according to radiocarbon dating.

Yanagimachi said he had written to Northern Arizona University paleontologist Larry Agenbroad, one of the scientists involved in the project, asking for some tissue from the body. If the chromosomes were intact, they could be used to fertilize an elephant egg, he proposed.

Eventually, the elephant genes could be breeded out to get a mammoth, he said.

But he never heard from Agenbroad and the Discovery Channel in 2001 said the frozen ice block believed to contain the "Jarkov mammoth" did not have a body, just scattered remains.

Yanagimachi retired Dec. 31, giving him time to attend a recent international meeting in Brazil and to travel. Otherwise, he's still working in his Manoa office at the Institute for Biogenesis Research, created after his mouse-cloning feat.

He has a new goal. "I'm dreaming of keeping sperm at room temperature somehow forever," he said. "There's got to be a molecule or something to keep the DNA and protein intact."