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Findings may yield methods for improving fertility as well as developing male contraceptives

For 40 percent of the estimated six million American couples battling infertility, the problem lies with the man. But help may be on the way.

New research in mice by scientists at Rockefeller University and the Population Council sheds light on the causes behind male infertility. The findings, reported in the March issue of Developmental Cell, also include potential targets for developing a reversible male contraceptive.

“The sperm from these mice have problems similar to many defects seen in human sperm that contribute to infertility,” says Hermann Steller, Ph.D., head of the Strang Laboratory of Apoptosis and Cancer Biology at Rockefeller. “We have found an event in the maturation of sperm that is extremely sensitive, and thus would be a good target for both improving fertility and quality of sperm for men who are infertile, and for the inhibition of fertility using a male contraceptive pill.”

Led by first author Holger Kissel, Ph.D., a postdoctoral researcher in Steller’s laboratory, the scientists created a mouse missing a gene called Septin 4; male mice that lack this gene are sterile. While these mice produce the same volume and number of sperm as normal mice, the sperm from the mutant mice is unable to fertilize eggs. The sperm has several defects, including severely bent tails and large droplets of cytoplasm at the area of the cell that surrounds the nucleus. These defects would not normally appear in fertile sperm. The appearance of the mutant mouse sperm is very similar to a human condition known as “droplet sperm.”

“The majority of sperm that even a fertile man makes appears abnormal,” says Kissel. “The sperm defects we are seeing in the mutant mice are probably an enhanced phenomenon that occurs in normal, healthy mice as well.”

This research follows previous work by Steller’s lab showing that caspases, known as death enzymes, are needed by fruit flies to make healthy sperm. Instead of killing the cell, as would normally occur, the caspases remove excess cytoplasm from sperm, helping to give them their nice streamlined shape. The new data indicates that the same events occur in mammalian sperm.

“Caspases are needed for the elimination of the majority of the cytoplasm and organelles to make mature sperm,” says Steller. “Rather than having death of the whole cell, there is death of only part of the cell. This is clear evidence that this process is conserved from fruit flies to mammals.”

The Septin 4 gene, which is missing in the mice with defective sperm, makes both the Septin 4 protein and a protein called ARTS. This second protein is important for activating the death enzymes. The Septin 4 protein, part of a family of proteins first discovered in yeast, is important for making large cytoplasmic scaffolds, similar to construction scaffolding, in cells where proteins can assemble. The scaffolds provide a framework in the cell that helps to compartmentalize different proteins in the cell.

“The Septin 4 protein is normally found in a structure called the annulus in the sperm,” says Steller. “The annulus was predicted to be a way to make different compartments in the sperm, and the mutant mice completely lack an annulus. If you normally have to keep all of the proteins in the right compartment, messing that up leads to a number of problems with the sperm, including bent tails and an inability to swim.”

Co-author Gary Hunnicutt, Ph.D., the senior collaborative scientist at the Population Council, studies surface compartmentalization and function of sperm.

“Sperm, unlike other cells in the body, must respond and react to a host of different environments in both the male and female reproductive tracts,” says Hunnicutt. “Yet they do this without making any new proteins. Instead, they appear to change their functions by rearranging the molecules on their surfaces. The annulus has been thought to act as a gatekeeper that separates two areas of the sperm tail. Studying sperm from mice lacking Septin 4 allows us to finally ask if this is really the case, and the findings will advance our understanding of how sperm become cells capable of fertilization.”

Research on the roles of both the ARTS and Septin 4 proteins in sperm maturation may further our understanding of male infertility and potentially improve the fertility and quality of sperm in infertile men. Also, these proteins are attractive targets for male contraceptive drugs, because while the sperm are still produced in the mutant mice, they are unable to fertilize an egg.

“There are not many mutations that cause complete sterility without affecting the anatomy of the testis, as with our mouse,” says Kissel. “The Septin 4 and ARTS proteins are great targets because interfering with them could accomplish inhibition of fertility without any negative side effects.”

“This is a really great starting point,” says Steller. “We were originally interested in the cell death aspects of these proteins, but the mouse has opened up many other nice opportunities—including insights into human sterility—that we are currently pursuing.”

Contributing authors include Maria Magdalena Georgescu, formerly at Rockefeller and now at the University of Texas M.D. Anderson Cancer Center; Sarit Larisch, a scientist visiting Rockefeller from the Rambam Medical Center in Israel; and Katia Manova at Memorial Sloan-Kettering Cancer Center.

This research was partially funded by a Fogarty International Research collaboration award grant from the National Institutes of Health. A Focused Giving Award from Johnson & Johnson to Steller and a grant from the National Institutes of Health to Hunnicutt also supported this research.