Male infertility is a major issue worldwide and its causes remain unclear. Now, an international team of researchers led by Hiroki Shibuya at the RIKEN Center for Biosystems Dynamics Research (BDR) in Japan has discovered a key structure in the germ cells of male mice that, when disturbed, leads to deformations in sperm flagellum—the tail that allows sperm to swim. Made possible by the first observation of the mouse flagellar base structure using ultrastructure expansion microscopy, this finding could explain some forms of infertility in human men. The study was published in the scientific journal Science Advances.
When conception fails, it is often due to abnormalities in egg or sperm cells that occur during their development. In males, this process is called spermatogenesis and continues throughout life after puberty. Although we know a little bit about this process, scientists have yet to map everything that happens at the subcellular level.
“While the causes of female infertility have been studied extensively,” says Shibuya, “the mechanisms underlying male infertility—which are known to account for about half of all infertility cases—remain poorly understood.”
Shibuya and his team tackled this problem using a relatively new technology called ultrastructure expansion microscopy. Detailed images of the insides of cells can already be taken with an electron microscope. But you cannot identify specific proteins or track how a structure changes over time. On the other hand, fluorescent microscopy can’t normally visualize ultrastructures inside a cell. With ultrastructure expansion microscopy, the cells of interest are placed on a gel. The gel is then expanded many times its original size. Now standard immunofluorescence labeling combined with a fluorescent microscope can be used to look at the giant specimen and specific ultrastructures can be imaged with high resolution.
The researchers adapted this relatively new technology for male mouse germ cells by gently fixing and drying the cells onto coverslips before putting them in the gel. This prevented the cells from moving around, which is a problem for male germ cells. They also applied a treatment to remove excess cytoplasm, which improved the resolution.
Having solved these issues, Shibuya and his team focused their efforts on the centriole, a tiny cylindrical structure—only 450 nanometers in length and 200 nanometers in diameter—that undergoes major changes during spermatogenesis that allow the flagellum to form. A correctly formed flagellum is critical because without it, sperm cannot move properly and will never even reach an egg cell, let alone fertilize it. Using their modified protocol, the researchers were able to visualize both the proximal and distal centriole throughout the entire transformation from germ cell to sperm.
They found that the inner scaffold within the distal centriole becomes stronger after the completion of meiosis, when germ cells divide and the new cells contain only one copy of each gene. Fluorescent labeling of key proteins that make up the distal centriole inner scaffold showed an increase in centrin-POC5 protein complexes. How important are these proteins for fertility? A complete knockout of POC5 using CRISPR gene editing produced normal male mice with zero viable sperm. Detailed analysis revealed that while centriole function in regular cells was unaffected, the lack of POC5 caused malformed flagella that disintegrated, explaining why the mice were completely infertile.
“Our modified expansion microscopy protocol can be extended to other analyses, including human sperm, opening new possibilities for investigating fine structural abnormalities that account for male infertility,” says Shibuya. “In the long-term, this could lead to novel diagnostic and therapeutic approaches in reproductive medicine.”