Mouse study trumpets new way to preserve speciesWEST LAFAYETTE, Ind. -- In a popular Dr. Seuss book, Horton the elephant is tricked into hatching a bird's egg. Now, in a real-life story, scientists have "tricked" a mouse into producing an elephant egg. That development could boost breeding programs for endangered species and might address some fertility problems in humans.
The results, published in the October issue of the journal Animal Reproduction Science, indicate that transplanted ovarian tissue may be used to regenerate reproductive cells for a wide variety of female mammals, including less-studied, rare or endangered species.
The current research was performed as a follow-up to two studies published last year by Critser and others that showed cryopreserved ovarian tissue from different species could produce viable eggs in immune-compromised mice, a strain of mice that can readily accept foreign tissues because its immune system has been suppressed.
In those experiments, cryopreserved ovarian tissues from outbred mice or sheep were transplanted into immune-compromised mice. After hormone production was established, those mice with "mouse-to-mouse" transplants were mated and produced live offspring.
Critser says the elephant egg, in theory, could be fertilized in vitro and then transplanted into a female elephant. However, procedures to isolate and fertilize eggs from elephants and other exotic animals, and the techniques of transferring embryos into live animals, will require additional investigation and development, he says.
"We know a great deal about a few species, such as mice, humans and sheep. But as you begin to look at the simple, fundamental reproductive biology of a tiger or a cheetah or an African elephant, very little is known," Critser says.
"Using methods such as in vitro fertilization and embryo transfer for an animal requires detailed knowledge about the animal's physiology and how its reproductive tissues respond in various situations. Generally, this work must be done on a species-by-species basis."
The current findings also suggest that storing ovarian tissue at low temperatures may provide a fairly simple way of preserving genetic material from female mammals -- a feat that has proven to be extremely difficult.
"Preserving the genome from the female side is a difficult process, and has shown limited success, even in well-studied species and humans," says Critser, who is the scientific director of the Cryobiology Research Institute in Indianapolis. "Oocytes and embryos are very difficult and time-consuming to obtain from living animals, and they have proven to be equally difficult to preserve."
The ovarian tissue samples used in the latest study were collected from three healthy, mature African elephants in the Kruger National Park, South Africa. Critser collected the samples and stored them at ultra-low temperatures -- minus 320 degrees Fahrenheit -- using the same cryopreservation procedures used to store tissues for humans and domestic animals.
The tissue was later thawed and transplanted into immune-incompetent mice whose ovaries had been removed.
Though the elephant tissues initially contained only one or two immature follicles -- microscopic structures that contain the developing eggs -- the mice were able to develop mature follicles within 10 to 11 weeks.
"This indicates that hormone production from the elephant's ovarian tissue was established in all the mice," Critser says.
In addition, one mouse developed a mature egg two months after transplantation.
Critser's group at Purdue is now using rat ovarian tissue in an effort to preserve transgenic rat lines and to further develop methods of transplanting frozen tissue into mice. The group will then analyze the ovaries' ability to establish cycles and produce eggs.
The rat studies also may help the researchers develop optimal culture conditions for in vitro maturation and fertilization, and they may improve techniques for transferring embryos into live animals. These procedures then might be applied to rare or endangered species, Critser says.
He and his group also are looking at ways in which cryopreserved ovarian tissue from humans can be used to circumvent fertility problems caused by certain illnesses or medical conditions.
"Based on the earlier studies that showed ovarian tissue could be used to produce offspring in animals, some clinics have begun using cryopreservation methods to store human ovarian tissue, usually when a young woman is about to undergo radiation or chemotherapy for cancer," he says.
Critser and a group at Methodist Hospital have collected samples from a human patient and are applying for funding to expand their study.
Critser is a reproductive physiologist who serves as adjunct associate professor in Purdue's School of Veterinary Medicine. He is using cryobiology -- the study of low-temperature effects on biological systems -- to develop and improve ways of preserving reproductive cells and tissues, such as sperm, eggs, embryos and ovarian tissue.
Cryobiology routinely is used to preserve tissues such as blood and skin. Human sperm banks also use this method to distribute more than 100,000 units of preserved semen every year.
Since 1989, Critser has studied ways to apply cryobiology to preserve genetic materials of endangered species. He has worked with a number of zoos -- including Chicago's Brookfield Zoo, the Smithsonian National Zoo in Washington, D.C., and the Indianapolis Zoo -- to boost captive breeding programs using artificial reproduction techniques, and he has traveled to Africa to collect reproductive cells from nine different species.
"Biodiversity on the planet is rapidly declining. We may be losing one animal species every hour or every two hours every day," Critser says. "There is a growing interest in finding ways to preserve the genetic material from rare and endangered species, with the intent to both study the genetic information and to reintroduce it into the population through planned breeding programs."
In most cases, scientists have been working with sperm because it's easier to collect and store. Although sperm from some species can be preserved using the methods developed for humans, these procedures can cause cellular damage in other species.
In a second study published in the same issue of Animal Reproduction Science , Critser analyzes the properties of semen from elephants, lions, warthogs and impalas, and he discusses ways to improve the preservation procedures for these species.
"Damage can occur if cells are cooled either too quickly or too slowly," Critser says." If cooled too quickly, ice crystals will form inside the cells, and just as water in an ice cube tray expands, the ice inside the cell expands, causing the cell membrane to rupture."
Cells also may be damaged if cooled too slowly, as they become susceptible to high concentrations of salts and other compounds that are left behind as the water turns into ice.
The fragile reproductive tissues in females -- namely eggs and embryos -- have proven to be even more susceptible to cellular damage caused by freezing and thawing, Critser says.
Ovarian tissue, however, appears to be less susceptible to the rigors of freezing and thawing. Critser says his group's findings suggest that a single method for cryopreserving ovarian tissue may be effective for a wide variety of species.
"The major advantage of cryopreserving ovarian tissue, as opposed to oocytes or embryos, is that fairly generic protocols appear to be successful in freezing such tissue," Critser says. "These findings may be a boon to conservation biologists working to collect and preserve genetic resources from those species that are most threatened by extinction."
Critser is president and director of the Biodiversity Research Institute, a not-for-profit research organization made up of universities and zoos worldwide to establish a network of researchers interested in collecting and preserving genetic resources from endangered species. Purdue's School of Veterinary Medicine is a member of the institute.
Working with Critser on the studies were: Karen Gunasena, Patty Villines and Beth Critser of the Advanced Fertility Institute; Jonathan Lakey of the Cryobiology Research Institute; and Mitchell Bush of the Smithsonian Institute's National Zoological Park. Other contributors were Cobus Raath of the Kruger National Park and Locksley McGann of the Department of Medicine and Pathology at the University of Alberta.
The studies were funded by the Cryobiology Research Institute and the Biodiversity Research Institute of Indianapolis.
Source: John K. Critser, (317) 278-3215; e-mail, firstname.lastname@example.org
Writer: Susan Gaidos, (765) 494-2081; e-mail, email@example.com
Purdue News Service: (765) 494-2096; e-mail, firstname.lastname@example.org
NOTE TO JOURNALISTS: Copies of the two journal articles from Animal Reproduction Science are available from Purdue News Service. A color photo of John Critser showing samples of cryopreserved ovarian tissue and spermatozoa also is available.
Antral follicles develop in xenografted cryopreserved African elephant (Loxodonta africana)
The preservation of germ plasm from endangered species could augment captive breeding programs aimed at maintaining genetic diversity. Mammalian female germ plasm (oocytes) is extremely difficult to collect and cryopreserve; however, a promising alternative is the cryopreservation of ovarian tissue. In the present study, athymic nude (nu/nu) Balb/C mice were used to evaluate in vivo viability of cryopreserved ovarian tissue from Institute of Cancer Research genotype (ICR) mice or elephants. Female mice were ovariectomized prior to transplant of cryopreserved-thawed ovarian tissue from ICR mice (n = 4) or elephants (n = 6). Control mice were sham operated (n = 4) or ovariectomized (n = 5). Transplants were in the ovarian bursa, enabling in vivo ovulation and pregnancies from allografts. Vaginal cytology was monitored daily, and the intervals between and duration of epithelial cells present in smears were evaluated. Appearance of epithelial cells in sham-operated and allografted mice were at intervals of 4.3 + 0.6 and 3.3 + 0.5 days, lasting for 1.4 .+ 0.1 and 1.6 + 0.2 days, respectively. Sporadic incidence of epithelial cells in ovariectomized animals occurred at longer intervals (8.6 + 3.8 days). Females with xenografted elephant ovarian tissue demonstrated epithelial cells in vaginal smears at intervals of 4.5 + 1.0 days, for 2.5 + 0.5 days duration, which was significantly longer than the other groups ( P < 0.05). Histological evaluation of tissues at the time of epithelial cells in smears demonstrated well-developed antral follicles, although oocytes were of poor morphological appearance or only cumulus-like complexes were seen. The nude mouse model is effective for assessing cryopreserved ovarian tissue xenograft function which can support the development of antral follicles.