Sperm and egg fusion is older than sexual reproduction itself - study

The discovery that ancient creatures like archaea can also contain fusexin proteins raises the possibility that the Fsx1 protein is the ancestral version from which fusogens derived.

 An illustration by Prof. Luca Jovine of the Karolinska Institute shows the Fsx1 protein structure (on the right) deciphered by the researchers, belonging to an archaeon from a hyper-saline environment (represented by salt on the left). (photo credit: PROF. LUCA JOVINE/KAROLINSKA INSTITUTE)
An illustration by Prof. Luca Jovine of the Karolinska Institute shows the Fsx1 protein structure (on the right) deciphered by the researchers, belonging to an archaeon from a hyper-saline environment (represented by salt on the left).
(photo credit: PROF. LUCA JOVINE/KAROLINSKA INSTITUTE)

When did sexual reproduction appear in living things?

The cellular mechanisms that allow the fusion of sperm cells and egg cells originated three billion years ago in unicellular archaea – single-celled organisms that lack a nucleus – according to a hypothesis of researchers at the Technion-Israel Institute of Technology in Haifa and colleagues around the world. 

When did sperm and egg cells fuse?

Archaea were initially classified as bacteria but this term has fallen out of use. Archaea and bacteria are generally similar in size and shape, although a few archaea have very different shapes. 

The process of sexual reproduction in plants and animals, as is familiar to humans, requires the fusion between egg cells and sperm cells. The emergence of sexual reproduction is usually dated about one to two billion years ago, but researchers are now speculating that the mechanisms that allow this fusion event appeared as early as three billion years ago.

 Prof. Benjamin Podbilewicz and doctoral student Xiaohui Li. (credit: TECHNION-ISRAEL INSTITUTE OF TECHNOLOGY) Prof. Benjamin Podbilewicz and doctoral student Xiaohui Li. (credit: TECHNION-ISRAEL INSTITUTE OF TECHNOLOGY)

The Study

The study, published in Nature Communications under the title “Discovery of archaeal fusexins homologous to eukaryotic HAP2/GCS1 gamete fusion proteins,” was carried out by Prof. Beni Podbilewicz and doctoral student Xiaohui Li from the Technion’s Faculty of Biology, along with researchers from Uruguay, Switzerland, Sweden, France, Great Britain and Argentina. 

The fusion of sperm and egg marks the climax of fertilization and the onset of embryonic development. Since both cells contain exactly half of the genetic information needed for the offspring, deviant fusions of multiple sperm to one egg will have abnormal development.

 The research group from the Technion, from right to left: Prof. Benjamin Podbilewicz, Katerina Flyak, Clari Valansi, Xiaohui Li, and Dr. Nicolas Brukman. (credit: TECHNION-ISRAEL INSTITUTE OF TECHNOLOGY) The research group from the Technion, from right to left: Prof. Benjamin Podbilewicz, Katerina Flyak, Clari Valansi, Xiaohui Li, and Dr. Nicolas Brukman. (credit: TECHNION-ISRAEL INSTITUTE OF TECHNOLOGY)

The process is therefore tightly regulated. Specialized proteins called “fusogens” must be present at the precise time and place to allow the egg and the sperm to merge into one.

The Podbilewicz lab studies fusogens in several organisms. It first identified and characterized two such proteins in the nematode C. elegans (EFF-1 and AFF-1) worms, which are about a millimeter long with 959 somatic cells. Its transparent body consists of three layers: an epidermal layer, an intestinal layer and a muscle layer.

These proteins are involved in organ development but not in fertilization. Surprisingly, the structural analysis revealed that these proteins have a very similar three-dimensional structure to another fusogen involved in fertilization in plants (GCS1/HAP2).

This family of similarly structured fusion proteins has been named fusexins. It includes representatives in plants, animals, viruses, unicellular algae and parasites.

Connecting the familial dots

To expand upon and characterize the origin and evolution of the Fusexin family, research collaborator David Moi and a team of researchers from Argentina and Switzerland conducted a bioinformatic search on genetic sequences sampled from different environments. After screening samples from soil, saline lakes, freshwater, and marine sediments, they discovered 96 sequences belonging to archaea, which showed some similarities with known fusion proteins. 

The sequences were named fusexin1 (Fsx1), and an expert team led by Pablo Aguilar, Hector Romero and Martin Graña in Uruguay and Argentina confirmed that they belong to archaea species from lineages estimated to originate 3 billion years ago; however, it remained unclear whether the protein that Fsx1 encodes looks similar to members of the fusexin family and whether it is truly capable of mediating cell-to-cell fusion.

What's the next step?

To determine the structure of an Fsx1 protein, Shunsuke Nishio of the Karolinska Institute in Sweden used crystallographic methods to decipher the three-dimensional conformation of the Fsx1 protein.

Nishio showed that the Fsx1 protein contains three structural domains very similar to known Fusexin members and is arranged in a three-piece complex – known as a trimer – as do other known fusogens. Surprisingly, Fsx1 has an additional fourth domain not found in any known Fusexin member. Prof. Luca Jovine, leading the crystallographic structure analysis, also used novel machine-learning software (AlphaFold2) to determine the structure of the Fsx1 protein.

To prove that the protein Fsx1 carries a fusogen role, doctoral student Xiaohui Li from the Podbilewicz Laboratory conducted an experiment in which she expressed the Fsx1 protein in a cell culture derived from mammals, which typically do not fuse. In collaboration with lab manager Clari Valansi and lab members Dr. Nicolas Brukman and Kateryna Flyak, Li showed that Fsx1 from archaea does induce the fusion of these mammalian cells that diverged one to two billion years ago.

Known Fusexin proteins in viruses serve to mediate viral entry into the host cell (as is the case for coronavirus), while in eukaryotes (cells with nuclei) – plants, nematodes, and protists – they play roles in organ sculpting, neuronal repair and sex. 

But who came first? Was a fusogen used for sexual reproduction snatched by a virus, or was a viral protein used for infection adopted by plants?

The study by Moi, Nishio, Li and others raises a possible third scenario: all fusexins originate in archaea, from which the lineage split into a variety of functions, from viral infection to sperm and egg fusion – a billion years before sexual reproduction.

Where does this leave us?

An important next step will be to study what Fsx1 proteins are doing in nature. Do they fuse archaeal cells like their plant and animal fusexins counterparts fuse gametes (eggs and sperm) to promote a sex-like DNA exchange?

Parallel studies will also be needed to understand the evolutionary history connecting the Fsx1 protein and GCS1/HAP2 in order to establish what their origin is. Archaeal fusexins and other still undiscovered fusogens might help us to understand how cells evolved from apparently simple forms sharing discrete pieces of DNA between them to today’s complex life forms undergoing sexual reproduction. 

Thus, the discovery that ancient creatures like archaea can also contain fusexin proteins now raises the intriguing possibility whereby the Fsx1 protein is the ancestral version from which viral, plant, and animal fusogens derived.