Proteins that play a key role in sperm motility identified using electron microscopy
Study identifies promising candidates for future genetic research on infertility
By zooming in to near-atomic level using electron microscopy, Tzviya Zeev-Ben-Mordehai and her team were able to identify the proteins that form the molecular machinery that drives sperm motility. More than twenty of these proteins were not yet recognised as part of the sperm motor apparatus, all of which are promising candidates for future research on infertility. The results not only provide important insights about how sperm cells move and might dysfunction, but also about how they evolved. The study is published today in the scientific journal Cell. Zeev-Ben-Mordehai: “We open a new era for infertility diagnostics and male contraceptive development.”
A sperm cell is well adapted for its very specific task: to deliver the male genetic material to the female egg cell. Yet the journey of a sperm cell is highly challenging: in mammals, sperm must swim distances over more than thousandfold their own length in a viscous environment. When the mobility of sperm cells is in some way impaired, this will result in male infertility, a problem which is on the rise in humans globally.
The motor apparatus of sperm is in their tail: it is a ‘molecular machine’ called the axoneme. The axoneme is made of hundreds of different proteins anchored on cellular tubes called microtubules; each axoneme is built of nine double microtubules (so called doublet microtubules) that are arranged around one central pair of single microtubules.
Cryogenic electron microscopy
Previous work by the group of Zeev-Ben-Mordehai already revealed that the tubes of doublet microtubules of mammalian sperm cells are partly filled by proteins. The identity of these proteins, however, remained mostly unknown.
The group now found a way to identify these proteins without destroying the tubes and the structures within. They used a method called cryogenic electron microscopy (cryo-EM), where samples such as sperm cells are instantaneously frozen, providing the best preservation as samples retain their native condition most accurately.
To be able to identify the proteins within the tubes, the researchers had to zoom into the structures with unprecedented detail, close to atomic-level. This allowed them to discern individual amino acids, the building blocks of proteins. Once the amino acids in a protein are identified in the correct order, it is possible to determine which protein you are looking at.
To achieve this high level of detail, the researcher had to develop a new sample preparation strategy that they applied prior to freezing the samples: they removed the outer layer of the sperm cells, the cell membranes, and then added ATP, a molecule that provides energy to cells. The ATP triggered sliding of the microtubules relative to each other, thus causing them to come loose. The two tubes of each doublet, however, stayed connected, as well as all the proteins that are associated with them. This allowed the researchers to zoom in on individual doublets.
More than 60 proteins
Zeev-Ben-Mordehai and her team identified more than sixty proteins within the doublets of bovine sperm. At least twenty of these proteins are specific to sperm, and more than ten of the proteins are known to be associated with male infertility. These associations were discovered in earlier studies by analysing the DNA of infertile males, either in humans or other animals, but until now the exact position of these proteins in sperm cells was not yet known. Zeev-Ben-Mordehai: ”It is incredible that cryo-EM now allows for the identification of proteins in complex structures. And we not only identified the vast majority of the proteins in sperm doublet microtubules, we also accurately placed them in this majestic structure thus providing the framework to understand their function.”
Remarkably, 28 of the proteins found were not experimentally studied before and therefore did not have a meaningful name yet. Zeev-Ben-Mordehai points out that these previously unrecognised proteins would be good candidates for future genetic studies on male infertility and for the development of male contraceptives.
To gain insight into the evolution of the axoneme, the researchers also compared the doublet microtubules of bovine sperm cells with those of sea urchin sperm studied by collaborators in the US. They found that, while one of the tubes of bovine doublets is almost completely filled with proteins, in sea urchins both tubes are relatively empty. Zeev-Ben-Mordehai: “Sea urchins are external fertilizers, which means that the egg cells are fertilized in water. It seems that mammalian sperm need a substantial structural reinforcement to allow them to swim in the much more complex and viscous environment of the female reproductive tract.”
Zeev-Ben-Mordehai will continue her research on sperm cells in an ERC funded project GettinginShape, where she will use cryo-EM to provide a molecular understanding of how sperm cells transform from round precursor cells called spermatids into highly specialised motile sperm.
Cell, 15 June 2023
Miguel Ricardo Leung*, Jianwei Zeng, Xiangli Wang, Marc C. Roelofs*, Wei Huang, Riccardo Zenezini Chiozzi*, Johannes F. Hevler*, Albert J.R. Heck*, Susan K. Dutcher, Alan Brown, Rui Zhang, Tzviya Zeev-Ben-Mordehai*
*Researcher at Utrecht University