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For ferrets, GPI means ’get pregnancy initiated’

26.06.2003


Embryo-implant protein exploited by tumors may help endangered species



Knowing what makes a ferret pregnancy take hold could help biologists save endangered species or understand how tumors spread.

Specifically, biologists examining early pregnancy in domestic ferrets report they have identified a protein necessary for embryos to implant successfully in the wall of the uterus, which is pregnancy’s first step in mammals.


Newly discovered as a molecular signal in ferret pregnancies, the protein-glucose-6-phosphate isomerase (GPI)-has long been known for its wide-ranging role in metabolism, where it breaks down sugars in organisms as diverse as bacteria and humans. Secreted by tumor cells, GPI also plays a role in metastasis, the invasion of cancer cells into healthy tissues.

Rsearchers Laura Clamon Schulz and Janice M. Bahr at the University of Illinois at Urbana-Champaign report their results in the Proceedings of the National Academies of Sciences. The article will appear online in the PNAS Early Edition on or before June 27 at (http://www.pnas.org) and in the journal’s July 8 print edition. (Schulz, a graduate student during the research project, is now doing post-doctoral studies at the Boston University.)

Their work was funded by the Division of Integrative Biology and Neurosciences at the National Science Foundation (NSF), an independent federal agency that supports fundamental research and education across all fields of science and engineering.

According to Bahr, "The identification of GPI as a key signal secreted from mother to embryo in the ferret is an important step forward in the understanding of pregnancy in this and related species. The fact that the protein has been so highly conserved throughout evolution means that it may well be useful in a wide variety of animals, as well as humans, for both reproductive and health issues."

In domestic ferrets, GPI might as well also mean "gets pregnancy initiated." That could likely go for other members of the mammalian order Carnivora, such as mink, skunks, badgers, the endangered black-footed ferret, and the more distantly related seals, sea lions, walruses, bears and pandas.

"The GPI work is basic science," said Schulz, the lead author. "But in order to do any sort of assisted reproduction, or to understand any reproductive problems in captive species, we need to understand normal reproductive functions in these animals. There are major gaps in our understanding of reproduction in carnivores. There isn’t even a pregnancy test available for carnivores."

When an embryo implants, its outer layer, called a trophoblast, invades the uterus to establish a supply link for nutrients from the mother. This link eventually becomes the placenta. Failure to implant is a major cause of doomed pregnancies in humans and other mammals.

In ferrets, mating with the male induces ovulation about 30 hours later. Then, triggered by the release of the heretofore-unidentified protein, the fertilized embryo implants in the uterus. Total gestation takes about 42 days.

The prospect of encouraging pregnancies with GPI could be crucial to boosting, through assisted reproduction, the populations of black-footed ferrets, giant pandas, sea otters or other threatened species.

In their report, Schulz and Bahr, citing others’ work, also note, "Metastatic (tumor-growing) processes are highly similar to the invasive process of implantation, suggesting a potential mechanism for GPI during implantation." Further study, they suggest, is needed to focus on whether GPI stimulation has similar results in uterine tissues and in tumors.

Before they could identify GPI, the biologists first had to isolate it from the corpus lutea, the ovarian glands responsible for secreting the implantation protein. They began with tissue taken from ferrets in a state of "pseudopregnancy." (In this condition, which can be induced artificially with hormones or arise naturally when ferrets produce an egg that is not fertilized, the animal produces hormone levels almost identical to those of a true pregnancy.)

Then Schulz and Bahr applied a sophisticated, high-tech series of techniques that included freezing, boiling, slicing, straining, gelling, washing, rinsing, drying and electrifying the samples.

First they froze the tissue in liquid nitrogen, and, through a process called sonication, blasted it with three 10-second bursts of high-frequency sound waves to break it into fragments. The fats and salts were removed. The proteins were extracted through "fast protein liquid chromatography" and boiled in a special solution.

The cellular proteins in this soup were separated, drawn onto a slab of special gel where, thanks to their varying electrical charges, they sorted by size. The gel was stained (purposefully), bathed in formaldehyde, rinsed with acetic acid (of vinegar fame), preserved in another solution and then dried.

Some proteins were broken up with trypsin, an enzyme in digestive juices that breaks down large food proteins into their amino-acid components. Appropriately called digests, samples of the protein fragments were then analyzed by laser-laden spectrometers.

From others’ investigations, Bahr and Schulz knew the size of the suspect protein. When they spotted that profile, they cut the section from the gel, determined the amino acid sequence of the sample, and then compared that sequence to those in existing computerized data banks.

The suspect’s sequence greatly resembled that of GPI already isolated and identified from pigs, mice and fruit flies. With other results, the collective evidence pointed to GPI as the ferret’s implantation protein.

With the protein identified, the biologists studied its effects in ferrets. They found, among other phenomena, that immunization of the mother ferret against GPI could severely reduce the number of embryos able to implant in her uterus.

Judith Verbeke, director of NSF’s Division of Integrative Biology and Neurosciences, said, "Through studies like this, we are becoming increasingly aware of the complexity of and variation in the implantation process. Several NSF-funded biologists are examining other factors that influence this critical aspect of mammalian pregnancy."

At Columbia University, for example, one is trying to determine how signaling molecules in stem cells of developing mice embryos might affect implantation. At Pittsburg State University in Kansas, researchers are shedding light on how the hormones estrogen and progesterone help prepare the rat uterus for implantation. At Tulane University, researchers are examining two genes-called Hoxa-10 and Hoxa-11-that may be responsible for many hormoneinduced physiological changes central to successful implantation in mice.

And, in a recent University of Virginia project, biologists looked at how the cells of the trophoblast, once implanted on the uterine wall, begin to differentiate into various roles-such as anchoring the embryo, developing a connecting network of blood vessels, secreting hormones and forming the placenta.

"It’s not just about ferrets, mice and rats," said Verbeke. "Collectively, efforts like these help us understand mammalian reproductive processes in general, and, of course, they offer fertile ground for further exploration, including outstanding opportunities for students to obtain quality training in whole-animal reproductive physiology."


###
NSF PR03-70

NSF Program Officer: Reynaldo Patino, program director, Integrative Animal Biology,
703-292-8421, rpatino@nsf.gov.

Principal Investigators:
Laura Clamon Schulz, Boston University, 617-353-2432, lcschulz@bu.edu,
Janice Bahr, University of Ilinois at Urbana-Champaign, 217-333-2900, jbahr@uiuc.edu.

The National Science Foundation is an independent federal agency that supports fundamental research and education across all fields of science and engineering, with an annual budget of nearly $5 billion. National Science Foundation funds reach all 50 states through grants to nearly 2,000 universities and institutions. Each year, NSF receives about 30,000 competitive requests for funding, and makes about 10,000 new funding awards. The National Science Foundation also awards over $200 million in professional and service contracts yearly.

Sean Kearns | National Science Foundation
Further information:
http://www.nsf.gov/
http://www.nsf.gov/od/lpa
http://www.nsf.gov/od/lpa/news/media/start.htm Science

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