BioEd Online

In a startling testimonial to the resilience of the male reproductive drive, a team of scientists has discovered that sperm recovered from dead, frozen mice are still capable of producing healthy pups.

The finding could help scientists - from fertility researchers using animal models to those attempting to create libraries of endangered species by giving them a simpler, more effective method to put DNA in a deep freeze. It could even offer some hope to those attempting to reawaken long-dead species such as the woolly mammoth.

Researchers currently harvest sperm by dissecting dead animals and isolating sperm from the epididymis, the site of sperm storage in the testes. The sperm are then purified and frozen in solution along with various chemicals that help to protect the cells' structure from the trials of freezing. When the time comes, samples are thawed out and injected into eggs for fertilization. The fertilization procedure works reasonably well even though the sperm are not alive and swimming.

Much the same storage technique can be used for human sperm: some samples have been frozen for more than 20 years and still produced healthy babies (see ' Baby breaks sperm-storage record').

The procedure is not ideal, however. It can get somewhat arduous when dealing with masses of frozen animal sperm, and freezing can sometimes damage the valuable DNA cargo: success rates vary from less than 1% to more than 30%.

Frozen whole

Atsuo Ogura, a professor at the Institute of Physical and Chemical Research (RIKEN) Bioresource Center in Ibaraki, Japan, and his colleague Ryuzo Yanagimachi of the University of Hawaii School of Medicine, were looking to simplify this process. So they and their co-workers tried freezing intact testes and, in some cases, the entire mouse.

In their most surprising experiment, the scientists harvested sperm from mice that had been frozen at -20°C for 15 years. The sperm themselves were long dead, their tiny tails motionless. And the freezing process wasn't perfect: their cell membranes were leaky and had sometimes ruptured. Nevertheless the sperm proved usable.

Over 80% of mouse eggs injected with this frozen sperm developed into two-celled embryos within a day. Furthermore, when the team transferred the embryos into female mice, 21% of the resulting offspring proved healthy, fertile pups, indicating that the genetic material recovered from the sperm was reasonably intact.

Bank on it

Robert Taft, associate director of reproductive sciences at the Jackson Laboratory in Bar Harbor, Maine, calls Ogura's findings "quite a feat".

Taft says that the Jackson Laboratory will stick to its current methods for preserving sperm, however. The lab maintains a collection of nearly 3,000 mouse strains and keeps its sperm collection in liquid nitrogen, safe from electrical failures that could thaw a freezer.

While he realizes that individual labs may find it convenient to simply toss their mice into a freezer, he urges every lab to deposit important strains into curated collections like the Jackson Laboratory's.

Bring it back

Ogura says that he especially hopes the method could be used to facilitate sperm collection from endangered species. As for the more far-flung idea of bringing back the woolly mammoth: "It's certainly an intriguing idea," says Taft. "But if you've only got the sperm, you've only got half the genome."

The Mammoth Creation Project, a privately funded group of Japanese scientists not including Ogura's team members - who aim to resurrect the animal, have said they intend to get around this problem by using mammoth sperm to fertilize elephant eggs.

Taft points out that getting usable sperm from a mouse kept at -20°C for 15 years is quite different from getting high-quality DNA from sperm frozen in permafrost for over 10,000 years. Large mammoths would have frozen quite slowly, for one thing, allowing time for bacteria to eat away at the tissue and damage its contents.

Ogura readily acknowledges these challenges, but says he is still keen to evaluate the quality of the sperm in the mammoth carcasses recovered from permafrost to date.

"I may find that recreating mammoths is impossible," he admits. "But I would be happy if animals that had been exterminated more recently could be restored."

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