Periodic Reporting for period 1 - GaMe (Mechanism of vertebrate sperm-egg recognition and fusion)
Periodo di rendicontazione: 2023-01-01 al 2025-06-30
The life of every sexually reproducing being starts with the fusion of sperm and egg, a process called fertilization. For fertilization to occur, compatible gametes must recognize each other, bind to each other, and eventually fuse for form a new cell, the zygote that gives rise to a new embryo. Despite of its central position at the beginning of each organism’s life, the mechanisms underlying fertilization in vertebrates have remained mysterious.
Although a handful of factors have been identified to be essential for gamete interactions in mice, their precise roles and mechanisms are unclear, and they are insufficient to mediate gamete fusion. From other fusion contexts, e.g. viral-cell fusion, it is known that specialized proteins called fusogens facilitate fusion by remodeling membranes and orchestrating membrane fusion. However, no fusogen-like protein mimicking known viral fusogens has been found in vertebrate gametes, and none of the known vertebrate fertility factors share similarities with known viral fusogens, leaving the nature and mechanism of sperm-egg fusion in vertebrates completely unclear.
In this project we aim to uncover the mechanistic principles of vertebrate fertilization. What are the molecules on the surface of sperm and egg that are required for sperm-egg interaction, and how do these factors work together to enable sperm-egg fusion?
To address these questions, we will focus on the two key phases of fertilization, sperm-egg recognition & binding, and sperm-egg fusion, using two complementary approaches: On the one hand, we will take advantages of zebrafish as a vertebrate model with external fertilization for in vivo analyses. On the other hand, we will employ in vitro approaches to investigate the protein structure and complex formation of fertilization factors.
Overall, our goal is to mechanistically understand how sperm and egg recognize and bind to each other, and how their membranes subsequently fuse. The insights gained will form the basis for our ultimate vision, to ‘re-build’ the vertebrate fertilization interface in vitro, and by these means understand one of the most fundamental processes of life.
Due to the scarcity of mechanistic knowledge, our research will provide major conceptual and technological advances to the field of fertilization by elucidating the nature of the elusive sperm-egg recognition and fusogenic machinery in vertebrates and by providing mechanistic insights into the physiological role of known and novel factors. We anticipate that our findings will also impact other fields involving cell recognition and fusion mechanisms, including immunology, neurobiology, host-pathogen interactions, membrane dynamics, and signaling.
Although a handful of factors have been identified to be essential for gamete interactions in mice, their precise roles and mechanisms are unclear, and they are insufficient to mediate gamete fusion. From other fusion contexts, e.g. viral-cell fusion, it is known that specialized proteins called fusogens facilitate fusion by remodeling membranes and orchestrating membrane fusion. However, no fusogen-like protein mimicking known viral fusogens has been found in vertebrate gametes, and none of the known vertebrate fertility factors share similarities with known viral fusogens, leaving the nature and mechanism of sperm-egg fusion in vertebrates completely unclear.
In this project we aim to uncover the mechanistic principles of vertebrate fertilization. What are the molecules on the surface of sperm and egg that are required for sperm-egg interaction, and how do these factors work together to enable sperm-egg fusion?
To address these questions, we will focus on the two key phases of fertilization, sperm-egg recognition & binding, and sperm-egg fusion, using two complementary approaches: On the one hand, we will take advantages of zebrafish as a vertebrate model with external fertilization for in vivo analyses. On the other hand, we will employ in vitro approaches to investigate the protein structure and complex formation of fertilization factors.
Overall, our goal is to mechanistically understand how sperm and egg recognize and bind to each other, and how their membranes subsequently fuse. The insights gained will form the basis for our ultimate vision, to ‘re-build’ the vertebrate fertilization interface in vitro, and by these means understand one of the most fundamental processes of life.
Due to the scarcity of mechanistic knowledge, our research will provide major conceptual and technological advances to the field of fertilization by elucidating the nature of the elusive sperm-egg recognition and fusogenic machinery in vertebrates and by providing mechanistic insights into the physiological role of known and novel factors. We anticipate that our findings will also impact other fields involving cell recognition and fusion mechanisms, including immunology, neurobiology, host-pathogen interactions, membrane dynamics, and signaling.
Following my lab’s discovery of the essential fertilization factor Bouncer in zebrafish and its species-specific mode of action in zebrafish and medaka (Herberg et al., 2018; Gert et al., 2021 and 2023), we initially focused on establishing zebrafish as a model system for studying fertilization in vertebrates (Fujihara & Herberg et al., 2021; Binner & Kogan et al., 2022; Noda et al., 2022), to ultimately tackle the fundamental open question regarding the molecular mechanism of fertilization in vertebrates.
In the past two years, we have made major progress in our quest to gain molecular insights into the mechanism of fertilization in vertebrates. The key technological advance that enabled our breakthrough findings was the release of AlphaFold. While not part of my original research strategy/methodology in my submitted proposal, we immediately recognized the power and huge potential of AlphaFold for our purposes, namely to screen not genetically or biochemically but ‘in silico’ for protein-protein interactions between known fertilization factors and a library of 1400 secreted/transmembrane proteins expressed in zebrafish sperm and testis. This screen predicted that (1) the conserved and already known essential fertilization factors Izumo1 and Spaca6 form a trimeric complex together with a previously uncharacterized factor, Tmem81, in both mammals and fish; and (2) it predicted that this trimer generates the composite binding interface for the egg protein Bouncer in fish. By combining in vivo genetic experiments in fish and mice with cellular and in vitro assays, we demonstrated that Tmem81 is indeed essential for male fertility in zebrafish and mice. Consistent with trimer formation, we found that Izumo1, Spaca6, and Tmem81 interact in zebrafish sperm, and that the human sperm factor orthologs interact in vitro when expressed in heterologous cells in culture. Moreover, neither Izumo1 nor Spaca6 can bind Bouncer on their own, but complex formation is required to create the binding site for Bouncer. Hence, our work presents an intriguing model for fertilization across vertebrates, where a conserved sperm complex binds to divergent egg proteins, Bouncer in fish and JUNO in mammals, to mediate sperm-egg interaction. This landmark study was published at the end of last year in Cell (Deneke & Blaha et al., 2024).
In the past two years, we have made major progress in our quest to gain molecular insights into the mechanism of fertilization in vertebrates. The key technological advance that enabled our breakthrough findings was the release of AlphaFold. While not part of my original research strategy/methodology in my submitted proposal, we immediately recognized the power and huge potential of AlphaFold for our purposes, namely to screen not genetically or biochemically but ‘in silico’ for protein-protein interactions between known fertilization factors and a library of 1400 secreted/transmembrane proteins expressed in zebrafish sperm and testis. This screen predicted that (1) the conserved and already known essential fertilization factors Izumo1 and Spaca6 form a trimeric complex together with a previously uncharacterized factor, Tmem81, in both mammals and fish; and (2) it predicted that this trimer generates the composite binding interface for the egg protein Bouncer in fish. By combining in vivo genetic experiments in fish and mice with cellular and in vitro assays, we demonstrated that Tmem81 is indeed essential for male fertility in zebrafish and mice. Consistent with trimer formation, we found that Izumo1, Spaca6, and Tmem81 interact in zebrafish sperm, and that the human sperm factor orthologs interact in vitro when expressed in heterologous cells in culture. Moreover, neither Izumo1 nor Spaca6 can bind Bouncer on their own, but complex formation is required to create the binding site for Bouncer. Hence, our work presents an intriguing model for fertilization across vertebrates, where a conserved sperm complex binds to divergent egg proteins, Bouncer in fish and JUNO in mammals, to mediate sperm-egg interaction. This landmark study was published at the end of last year in Cell (Deneke & Blaha et al., 2024).
Our landmark publication in Cell (Deneke & Blaha et al., 2024) was extremely well received not only in the scientific community but also by the general public as evidenced by extensive news coverages in Science, Nature, New York Times, GEO, and major international newspapers and news outlets.
We have also presented the work at international meetings (Gordon Research Conference in summer 2023; CSHL Fusion meeting in November 2024), and received excellent feedback from the scientific community.
We have also presented the work at international meetings (Gordon Research Conference in summer 2023; CSHL Fusion meeting in November 2024), and received excellent feedback from the scientific community.