You probably heard the phrase “opposites attract”—well, in sperm and egg meetings, this is literal! The sperm is small, has a tail, and swims, whereas the egg is big, immotile, and spherical. However, fertilization of the egg by the sperm is fundamental for all sexually reproducing animals. Despite its significance, scientists still do not fully understand the molecular mechanisms behind the sperm-egg cell binding.

Over the last 20 years, scientists identified proteins necessary for fertilization including four vertebrate sperm proteins in both fish (e.g. zebrafish) and mammals (e.g. mice and humans): Izumo1, Spaca6, Dcst1, and Dcst2. Yet, these proteins’ roles or even if, and how, they interact with each other was a mystery.

In a recent study, scientists attempted to answer these questions using zebrafish and mice. Mice and zebrafish are good model organisms because both of their genomes are fully sequenced. Mice have physiological similarities to humans, and zebrafish are small and inexpensive to work with.

Scientists used Google’s Nobel-prize-winning AlphaFold in this study. AlphaFold is an AI system developed by DeepMind that predicts a protein’s 3D structure and its interactions based on the proteins’ amino acid sequences. Previously, this process took years and hundreds of thousands of dollars. Nowadays, it can take a few minutes. However, AlphaFold took two to three weeks for this project. To identify the proteins’ functions, scientists inputted their amino acid sequences (FASTA format) into AlfaFold which predicted the proteins’ 3D structures and compared them against a library of about 1,400 proteins expressed in zebrafish testes to identify potential protein complexes being formed.

The study uncovered a molecular “key” on the sperm’s membrane consisting of a trimer, a 3 molecule compound, of two previously known proteins,  Izumo1, and Spaca6, and a protein not previously known to be involved in fertilization, Tmem81. All three proteins interact with each other in both fish and mammals including humans. Dcst1 and Dcst2, two other previously identified proteins, form a dimer, a 2-molecule compound, but are not part of this molecular “key”. Earlier this year, another group used AlfaFold and reached the same conclusion, validating these results.

To corroborate these results, scientists from the same study used CRISPR-Cas9, a gene editing tool, to deactivate the Tmem81 gene. The absence of this gene in both mice and zebrafish males caused sterility–their sperm were unable to interact with eggs. The females, however, remained fertile. These results were mirrored in males lacking the Spaca6 or Izumo1 genes, highlighting all these proteins’ importance in sperm-egg binding.

This sperm trimer has been described by Andrea Pauli, a molecular and developmental biologist at The Research Institute of Molecular Pathology in Vienna, as a “key” complex that fits into a lock molecule on the egg cell. In fish, this lock molecule is aptly named Bouncer because without it, the sperm can’t bind with the egg. In mammals, this lock molecule is an unrelated protein called Juno.

Juno and bouncer “lock molecules” bind to different sites on the newly discovered “key”. In zebrafish, Bouncer binds between the Izumo1 and Spaca6 proteins. In mice and humans, JUNO is bound to IZUMO1’s hinge.

The results of this study aid our understanding of infertility and fertilization. This research may lead to the discovery of drugs that act on the sperm’s key so males with infertile sperm could have a biological child using IVF.

Why the lock molecule differs between fish and mammals, yet the “key” on the sperm stayed the same remains a mystery that scientists have yet to fully understand.

One possible explanation is that sperm genes underwent duplication during evolution. Therefore as the sperm diverged, their structure remained unchanged. Another possible explanation is that in mammals, fertilization occurs inside their body. Whereas, in fish, it occurs outside their body. Therefore this can affect the outer egg cell membrane proteins. 

Despite this breakthrough, there are still many unanswered questions e.g., what are the other proteins on the sperm and eggs uninvolved in this “key” mechanism doing? Or do the Tmem81 proteins do more than ensure the sperm “key” is stable? These questions are met with few molecular answers.

However …
There is no denying the sperm and egg’s ancient, irrefutable bond!