Chemical signals at the most critical moment for new life in mammals -- when sperm meets egg and attempts fertilization -- evolve rapidly in a process driven by positive Darwinian selection, according to a Cornell University study.

New statistical studies of sequence divergence among egg-surface and oviduct proteins, reported by Willie J. Swanson, Mariana F. Wolfner and Charles F. Aquadro of Cornell University and Ziheng Yang of University College London (UCL) in the Feb. 27, 2001, Proceedings of the National Academy of Sciences , demonstrate for the first time that natural selection drives rapid changes in these female proteins at the site of sperm-egg interaction.

Beyond providing long-sought molecular evidence in female proteins for evolutionary theories about sperm competition, sexual conflict and cryptic female choice, this study's findings could have at least one practical application concerning infertility and also could help explain how new species evolve, says Aquadro, a Cornell professor of population genetics.

"We think we're seeing a cat-and-mouse game happening, on the molecular level, with the co-evolution of female and male reproductive proteins," says Swanson, a postdoctoral associate in Cornell's Department of Molecular Biology and Genetics.

"However," Swanson emphasizes, "the basic process of conception is clearly conserved throughout evolution: A sperm with proteinaceous chemical signals on its surface finds an egg. Then other proteins on the surface of the egg check out the sperm proteins for species compatibility."

Says Wolfner, a professor of developmental biology in Cornell's Department of Molecular Biology and Genetics: "If a sperm of the right species finds an egg, proteins on the egg's surface let the sperm bind, penetrate and begin fertilization. What is new about our finding is that portions of the female proteins involved in these interactions are shown to have been driven to change rapidly between species."

Previous studies had only detected rapid evolutionary change in male reproductive proteins, suggesting that some form of natural selection drove male proteins to change, according to Wolfner. The mechanism driving the changes remains unknown. "Since male proteins interact with female proteins such as ZP2 and ZP3, several theories of evolution have predicted that female proteins must also change rapidly, but this had never been detected," says Swanson. "This motivated us to examine in detail the rates of evolutionary change in female proteins predicted to interact with sperm during reproduction."

In collaboration with UCL's Ziheng Yang, the Cornell scientists applied powerful statistical methods developed by Yang and by Rasmus Nielsen, now a professor of statistical genomics at Cornell, to analyze the differences in three female reproductive proteins. (See further background attached.)Yang's computer software was a crucial ingredient in the study, Aquadro says, because it permitted detection of rapidly changing regions of the female proteins without their being obscured by the slower rate of change of other regions of the proteins.

Pointing to a possible application of the findings, Aquadro says: "Anything we can learn about signaling proteins in eggs and sperm might help us to understand the molecular basis of some infertility problems. For example, the rapid evolution of the ZP3 protein opens the possibility that sperm/egg incompatibilities might occur that could be detected by sequence-based tests."

Moreover, the subtle but significant changes in signaling proteins of sperm and eggs could turn out to be an "engine of speciation," Aquadro says, referring to the origin of new species through evolution. Just a few changes in amino acid sequences in recognition of surface proteins may be enough to make egg and sperm proteins incompatible, he notes. One of the distinguishing marks of different species is that they cannot cross-reproduce. The study of the evolution of reproductive proteins might thus provide important insight into how new species arise.

The protein study could herald a fundamental shift from only regarding commonality between species as evidence for functional importance: "Here, we have focused on what is different, with the idea that evolutionary novelty can also indicate an important function," Aquadro says. "As genomic sequences are compared between closely related species, attention paid not just to conserved, but also to rapidly evolving, sequences may help to identify what is functionally important and represent the molecular basis of biological diversity."

The study was supported by grants from the National Science Foundation, National Institutes of Health, Biotechnology and Biological Sciences Research Council, and the Alfred P. Sloan Foundation.

Further background: Keeping score in evolutionary cat-and-mouse game

Two of the reproductive proteins analyzed, ZP2 and ZP3 (for zona pellucida proteins 2 and 3), are components of the mammalian egg's surface that are known to bind to sperm in a species-specific manner. The third protein, OGP, is an oviductal glycoprotein. The researchers used statistical methods to search for similarities and differences among reported ZP2, ZP3 and OGP sequences in a variety of mammalian species, including cats, dogs, rats, mice, marmosets, macaques and humans.

They compared the number of DNA changes seen between species that cause protein sequence differences (replacement changes) to those DNA changes that still encoded the same protein sequence (silent changes). Silent changes are used as a measure for the background of neutral changes; detection of a level of replacement changes above this background indicates that evolution is forcing the protein to change. For all three female proteins tested, the researchers found that some segments of the proteins changed significantly faster than neutral, indicating positive Darwinian selection at work on the most basic, molecular level.

In addition to detecting the rapid evolutionary change of the female proteins, the new methods also allowed the researchers to identify the particular parts of the proteins that had changed rapidly during evolution. "Remarkably, one of the regions of the egg-surface protein ZP3 that we detected as being driven to change during evolution was the same region that other groups' previous functional studies had identified as critical for species-specific binding of sperm," says Wolfner.

"This implies that the evolutionary force driving the changes is related to the interaction between the sperm and the egg," adds Swanson. "We seem to have a cat-and-mouse game going on here, with competing evolutionary forces at play in the interactions of reproductive proteins." To test the "cat-and-mouse," as well as other, evolutionary hypotheses, Swanson and colleagues are now examining the molecular evolution of sperm proteins that are candidates for binding to the egg-surface proteins.

Other related websites:

• PNAS article: http://www.pnas.org/cgi/content/full/98/5/2509
• Molecular Biology and Genetics at Cornell: http://www.mbg.cornell.edu/
• Ziheng Yang's Group at University College London: http://abacus.gene.ucl.ac.uk/