Copper Is Crucial For Embryonic Development, Say University Of Michigan Scientists

In the June 5 issue of the Proceedings of the National Academy of Sciences, U-M scientists report that copper and a protein called Ctr1, which helps copper get inside cells, is essential for normal embryonic development in mice. Although scientists knew that Ctr1 was involved in copper transport in yeast microorganisms, no one knew exactly how the gene worked in mammals until now.

"Since the genetic structure and function of Ctr1 is nearly identical in mice and humans, it is very likely that Ctr1 is essential for human embryonic development, as well," says Dennis J. Thiele, Ph.D., a professor of biological chemistry in the U-M Medical School, who directed the PNAS study.

In recent studies with fruit flies, mice and human cells, Thiele found Ctr1 copper transport proteins and gene sequences in every species. "Ctr1 appears to have been conserved throughout evolutionary development, because it is so effective at bringing copper across membranes and into cells," he says.

"Ctr1 escorts copper through the cell's surface membrane and then hands it off to at least three other proteins, which deliver it to specific compartments inside the cell," Thiele says. "It's like a copper relay, and Ctr1 is the main gate."

A related paper by scientists from Washington University at St. Louis, published in the same issue of PNAS, describes the role of ATOX1, one of three other proteins in this intracellular copper relay.

"Copper is an essential micronutrient, which is required for vital biochemical reactions within cells," Thiele says. "Without copper, cells can't produce energy, metabolize iron or detoxify free radicals. Without copper, we can't grow blood vessels, synthesize neuropeptides that control muscle contractions, or make the collagen that gives our skin its elasticity."

Thiele studied yeast microorganisms for 17 years to learn how cells process copper. "Our work with yeast, and research by other scientists, was the key to finding the transporter proteins," he says.

In the PNAS study, Thiele's research team found that expression of the Ctr1 gene in human kidney cells stimulated a 30-fold increase in copper uptake by the cells. U-M scientists then used genetic engineering technology to create knock-out mice that were missing one of two alleles - or copies of the Ctr1 gene -- found in normal mice.

Although these heterozygous mice appeared and acted normal, U-M researchers found that their brains and spleens contained about half as much copper as was found in normal littermates. Since the ability to metabolize iron - another critical nutrient -- depends on copper, it was not a surprise to find that iron levels were lower in organs from heterozygous mice than from normal littermates.

The big surprise came when U-M researchers bred male and female heterozygous mice to see what would happen to mice without either copy of the Ctr1 gene. Of 378 mouse pups born, however, not one was found to be missing both copies of the gene. When Thiele examined mouse embryos from these crosses, he discovered that embryos without the Ctr1 gene all died 10 to 12 days after fertilization. In addition, all these embryos were much smaller than normal and had major abnormalities in organ and cell development.

"I anticipated the importance of copper in development, but I didn't expect it to be so critical that all the mouse embryos without Ctr1 would die before birth," Thiele said. "Based on these results, it wouldn't surprise me to find that human embryos lacking both copies of Ctr1 are aborted spontaneously during pregnancy."

To test whether copper supplements would help, U-M researchers added it to the drinking water of female experimental mice three weeks before and during their pregnancies. Although they received 50 to 100 times more copper than control mice, the effect on their embryos was unchanged.

"These data suggest there is no alternate system for copper uptake into cells that can compensate for loss of the plasma membrane Ctr1 transporter, and that the presence of at least one functional copy of the Ctr1 copper transporter gene is essential for normal embryonic development," Thiele says. "In addition, reduced copper accumulation in both brain and spleen from Ctr1 heterozygous mice indicates that two functional copies of Ctr1 are critical for maintaining normal copper balance."

Copper deprivation is particularly dangerous for children and infants, according to Thiele. Children born with a genetic condition called Menkes disease suffer irreversible damage, because copper remains trapped in their intestinal cells, where it is unavailable to the many copper-requiring enzymes in the body. Patients with Wilson disease develop cirrhosis and nerve degeneration due to their inability to distribute copper properly.

The U-M is offering non-exclusive licenses for use of Ctr1 knock-out mice as a commercial research tool and will provide mice without charge to academic researchers.

This research was supported by grants from the National Institutes of Health, the International Copper Association and the American Heart Association. Jaekwon Lee, Ph.D., U-M post-doctoral research fellow, and Joseph R. Prohaska, Ph.D., professor of biochemistry and molecular biology at the University of Minnesota-Duluth, were co-authors of the study.