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Environmental Factor, April 2012

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The importance of calcium entry in mammalian reproduction

By Robin Arnette

Illustration of a sperm and egg
Yi-Liang Miao, Ph.D.

Miao has been at NIEHS since August 2009. Williams gave him much of the credit for this research by saying, “There are very few people who could have technically done this work.” (Photo courtesy of Steve McCaw)

Carmen Williams, M.D., Ph.D.

Williams holds dual appointments in the Laboratory of Reproductive and Developmental Toxicology and the Clinical Research Program at NIEHS. (Photo courtesy of Steve McCaw)

The intricate steps that a fertilized egg takes in route to becoming an embryo are true marvels of nature. Reproductive biologists explain the phenomenon by saying the merging of an egg and sperm triggers signals deep inside the egg that allow it to begin cell division. These signaling events are known as egg activation, but new research suggests that some of the cues that induce egg development actually occur elsewhere.

Scientists from NIEHS, led by Carmen Williams, M.D., Ph.D., and the University of Pennsylvania are the first to show that calcium ions entering the egg from the outside are needed for key events that propel the egg to the two-cell stage. The Williams team published the results online Feb. 27 in the Proceedings of the National Academy of Sciences. Since calcium is a common ingredient in solutions used for in vitro fertilization, the work has significant implications for assisted reproductive procedures.

Back and forth

According to Williams, during fertilization of a mammalian egg, the sperm delivers a protein called phospholipase C (PLC), which causes the egg to release calcium from internal storage areas to the egg’s cytoplasm. Since too much calcium in the cytoplasm is toxic to the egg, excess calcium is either pumped back into storage or out of the egg. The back and forth movement of calcium from storage or outside the cell into the cytoplasm is known as oscillation, and PLC keeps it going for 4-6 hours.

"The signaling that happens as a result of calcium entry occurs directly under the plasma membrane, rather than the entire inside of the egg," Williams said. "Our work shows that the calcium being pumped back into the egg from the outside helps spur egg activation."

Williams said she became interested in signaling as a result of calcium entry, after hearing about the work of NIEHS researcher James Putney, Ph.D. Putney studies store-operated calcium entry (SOCE), one of the pathways that calcium uses to re-enter a cell. Williams' work determined that SOCE probably happens at fertilization, but it isn't necessary. There are other unidentified channels that permit calcium reuptake and generate signals. Putney was pleased with Williams' findings.

"It is exciting to see that the process of calcium entry, so extensively studied in simple cell lines, plays such a fundamental role in the complex signaling underlying egg fertilization," Putney said.

Williams said this study wasn’t possible without the technical expertise of visiting fellow and lead author on the paper Yi-Liang Miao, Ph.D. Miao only had five minutes to microinject mouse sperm into mouse eggs, load the eggs into a device that measures calcium levels, and record. He also had to figure out the proper buffer solution for the experiments. He needed something that had the correct pH, kept all of the ingredients in solution, and wasn’t toxic to the eggs. It took several months, but he developed a buffer that worked.

What’s in the media?

This research sets the stage for a new way of thinking when it comes to handling eggs for human in vitro fertilization. Williams explained that after eggs are removed from a woman, they are placed into a solution called culture medium that normally contains calcium. She also noted that many reproductive specialists freeze eggs and embryos and need to add chemicals, such as the cryoprotectant dimethyl sulfoxide (DMSO), to prevent the eggs from forming ice crystals. She urged these practitioners to pay attention to what's in the media.

“We’ve demonstrated that calcium and other chemicals in the media may impact the egg’s ability to receive signals from the outside, or may let calcium leak in too soon, which is also a problem,” Williams maintained.

Eggs depend on calcium to receive signals from the outside world and to communicate with other parts of the cell. Thanks to the Williams research team, we now know how important this abundant mineral can be.

Citation: Miao YL, Stein P, Jefferson WN, Padilla-Banks E, Williams CJ. 2012. Calcium influx-mediated signaling is required for complete mouse egg activation. Proc Natl Acad Sci U S A; doi:10.1073/pnas.1112333109 [Online 27 February 2012].

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