No abstract

The process of sperm-egg fusion is critical for successful fertilization, yet the underpinning mechanisms that regulate these steps have remained unclear in vertebrates. Here, we show that both mouse and zebrafish DCST1 and DCST2 are necessary in sperm to fertilize the egg, similar to their orthologs SPE-42 and SPE-49 in C. elegans and Sneaky in D. melanogaster. Mouse Dcst1 and Dcst2 single knockout (KO) spermatozoa are able to undergo the acrosome reaction and show normal relocalization of IZUMO1, an essential factor for sperm-egg fusion, to the equatorial segment. While both single KO spermatozoa can bind to the oolemma, they rarely fuse with oocytes, resulting in male sterility. Similar to mice, zebrafish dcst1 KO males are subfertile and dcst2 and dcst1/2 double KO males are sterile. Zebrafish dcst1/2 KO spermatozoa are motile and can approach the egg, but rarely bind to the oolemma. These data demonstrate that DCST1/2 are essential for male fertility in two vertebrate species highlighting their crucial role as conserved factors in fertilization.

Fertilization is the fundamental process that initiates the development of a new individual in all sexually reproducing species. Despite its importance, our understanding of the molecular players that govern mammalian sperm–egg interaction is incomplete, partly because many of the essential factors found in nonmammalian species do not have obvious mammalian homologs. We have recently identified the lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) protein Bouncer as an essential fertilization factor in zebrafish [S. Herberg, K. R. Gert, A. Schleiffer, A. Pauli, Science 361, 1029–1033 (2018)]. Here, we show that Bouncer’s homolog in mammals, Sperm Acrosome Associated 4 (SPACA4), is also required for efficient fertilization in mice. In contrast to fish, in which Bouncer is expressed specifically in the egg, SPACA4 is expressed exclusively in the sperm. Male knockout mice are severely subfertile, and sperm lacking SPACA4 fail to fertilize wild-type eggs in vitro. Interestingly, removal of the zona pellucida rescues the fertilization defect of Spaca4-deficient sperm in vitro, indicating that SPACA4 is not required for the interaction of sperm and the oolemma but rather of sperm and the zona pellucida. Our work identifies SPACA4 as an important sperm protein necessary for zona pellucida penetration during mammalian fertilization.

The process of sperm-egg fusion is critical for successful fertilization, yet the underlying mechanisms that regulate these steps have remained unclear in vertebrates. Here, we show that both mouse and zebrafish DCST1 and DCST2 are necessary in sperm to fertilize the egg, similar to their orthologs SPE-42 and SPE-49 in C. elegans and Sneaky in D. melanogaster. Mouse Dcst1 and Dcst2 single knockout (KO) sperm are able to undergo the acrosome reaction and show normal relocalization of IZUMO1, an essential factor for sperm-egg fusion, to the equatorial segment. While both single KO sperm can bind to the oolemma, they show the fusion defect, resulting that Dcst1 KO males become almost sterile and Dcst2 KO males become sterile. Similar to mice, zebrafish dcst1 KO males are subfertile and dcst2 and dcst1/2 double KO males are sterile. Zebrafish dcst1/2 KO sperm are motile and can approach the egg, but are defective in binding to the oolemma. Furthermore, we find that DCST1 and DCST2 interact with each other and are interdependent. These data demonstrate that DCST1/2 are essential for male fertility in two vertebrate species, highlighting their crucial role as conserved factors in fertilization.

The successful production of recombinant protein for biochemical, biophysical, and structural biological studies critically depends on the correct expression organism. Currently, the most commonly used expression organisms for structural studies are Escherichia coli (70% of all PDB structures) and the baculovirus/ insect cell expression system (5% of all PDB structures). While insect cell expression is frequently successful for large eukaryotic proteins, it is relatively expensive and time-consuming compared to E. coli expression. Frequently the decision to carry out a baculovirus project means restarting cloning from scratch. Here we describe an integrated system that allows simultaneous cloning into E. coli and baculovirus expression vectors using the same PCR products. The system offers a flexible array of N-and C-terminal affinity, solubilization and utility tags, and the speed allows expression screening to be completed in E. coli, before carrying out time and cost-intensive experiments in baculovirus. Importantly, we describe a means of rapidly generating polycistronic bacterial constructs based on the hugely successful biGBac system, making InteBac of particular interest for researchers working on recombinant protein complexes.

Fertilization, the fusion of sperm and egg, is essential for sexual reproduction. While several proteins have been demonstrated to be essential for the binding and fusion of gametes in vertebrates, the molecular mechanisms driving this key process are poorly understood. Here, we performed a protein interaction screen using AlphaFold-Multimer to uncover protein-protein interactions in fertilization. This screen resulted in the prediction of a trimeric complex composed of the essential fertilization factors Izumo1 and Spaca6, and Tmem81, a protein previously not implicated in fertilization. We show that Tmem81 is a conserved, testis-expressed transmembrane protein that is evolutionarily related to Izumo1 and Spaca6 and is essential for male fertility in fish and mice. Consistent with trimer formation in vivo, zebrafish izumo1-/-, spaca6-/-, and tmem81-/- mutants exhibit the same sperm-egg binding defect and show co-depletion of all three proteins in sperm. Moreover, we provide experimental evidence that Izumo1, Spaca6, and Tmem81 interact in zebrafish sperm. Strikingly, the Izumo1-Spaca6 interaction is predicted to form a cleft that serves as a binding site for Bouncer, the only identified egg protein essential for fertilization in zebrafish. Together, these results provide compelling evidence for a conserved sperm factor complex in vertebrates that forms a specific interface for the sperm-egg interaction required for successful fertilization.

It is conventionally assumed that conserved pathways evolve slowly with little participation of gene evolution. Nevertheless, it has been recently observed that young genes can take over fundamental functions in essential biological processes, for example, development and reproduction. It is unclear how newly duplicated genes are integrated into ancestral networks and reshape the conserved pathways of important functions. Here, we investigated origination and function of two autosomal genes that evolved recently in Drosophila: Poseidon and Zeus, which were created by RNA-based duplications from the X-linked CAF40, a subunit of the conserved CCR4-NOT deadenylase complex involved in post-transcriptional and translational regulation. Knockdown and knockout assays show that the two genes quickly evolved critically important functions in viability and male fertility. Moreover, our transcriptome analysis demonstrates that the three genes have a broad and distinct effect in the expression of hundreds of genes, with almost half of the differentially expressed genes being perturbed exclusively by one paralog, but not the others. Co-immunoprecipitation and tethering assays show that the CAF40 paralog Poseidon maintains the ability to interact with the CCR4-NOT deadenylase complex and might act in post-transcriptional mRNA regulation. The rapid gene evolution in the ancient post-transcriptional and translational regulatory system may be driven by evolution of sex chromosomes to compensate for the meiotic X chromosomal inactivation (MXCI) in Drosophila.

Large-scale movement of organisms across their habitable range, or migration, is an important evolutionary process that can contribute to observed patterns of genetic diversity and our understanding of the adaptive spread of alleles. While human migrations have been studied in great detail with modern and ancient genomes, recent anthropogenic influence on reducing the biogeographical constraints on the migration of non-native species has presented opportunities in several study systems to ask the questions about how repeated introductions shape genetic diversity in the introduced range. We present here the most comprehensive view of population structure of North American Arabidopsis thaliana by studying a set of 500 (whole-genome sequenced) and over 2800 (RAD-seq genotyped) individuals in the context of global diversity represented by Afro-Eurasian genomes. We use haplotype-sharing, phylogenetic modeling and rare-allele sharing based methods to identify putative sources of introductions of extant N. American A. thaliana from the native range of Afro-Eurasia. We find evidence of admixture among the introduced lineages that has resulted in the increased haplotype diversity and reduced mutational load. Further, we also present signals of selection in the immune-system related genes that impart qualitative disease resistance to pathogens of bacterial and oomycete origins. Thus, multiple introductions to a non-native range can quickly increase adaptive potential of a colonizing species by increasing haplotypic diversity through admixture. The results presented here lay the foundation for further investigations into the functional significance of admixture.