Biology subjects

Blaha, A.

Publications and source records attributed to Blaha, A..

7 recordsLinked to original sources

bioRxiv2026

The SPARK complex forms the molecular basis of vertebrate fertilization

Fertilization requires gamete recognition and membrane fusion, yet the molecular basis of this process in vertebrates remains unknown. Here we identify SPARK (sperm protein assembly and receptor-binding key), a conserved multi-protein complex that integrates all known sperm fertilization factors, including TMEM81-IZUMO1-SPACA6 and DCST1/2, together with two newly identified components, TMDD1 and FAM187A. SPARK subunits are mutually dependent for stability in mature sperm, and disruption of any single component causes male sterility in zebrafish and mice. Incubating zebrafish sperm with soluble egg receptor Bouncer partially rescues fertilization of Bouncer-deficient eggs in a SPARK-dependent manner, consistent with egg receptor binding priming the complex for fusion. Thus, we propose SPARK as a con-served molecular machine that couples gamete recognition to membrane fusion.

developmental biology↗

bioRxiv2024

Bouncer's receptor on sperm: Investigating sperm-egg compatibility in fish

Fertilization requires the successful binding and fusion of sperm and egg. In zebrafish, sperm-egg binding is mediated by the Spaca6-Izumo1-Tmem81 complex on sperm interacting with Bouncer on the egg. We previously found that expressing medaka Bouncer on zebrafish eggs and vice versa allows hybridization between medaka and zebrafish, two species that can normally not hybridize. Here, we tested whether providing zebrafish Spaca6 and Izumo1 on medaka sperm and vice versa enables cross-species compatibility from the side of the sperm. To this end, we generated spaca6 and izumo1 knock-out (KO) lines in medaka, which are male sterile, and introduced zebrafish spaca6 and izumo1 transgenes. Transgenic medaka males did not fertilize zebrafish or medaka eggs with zebrafish Bouncer. Similarly, zebrafish males expressing medaka Spaca6 and Izumo1 failed to fertilize zebrafish eggs presenting medaka Bouncer. Unexpectedly, providing either full-length medaka Spaca6 or the Bouncer binding site of medaka Izumo1 in zebrafish sperm rescued the sterility of spaca6 and izumo1 KO, respectively. Therefore, medaka Spaca6 and Izumo1 can interact with zebrafish Bouncer when paired with their zebrafish sperm complex members underscoring the nuanced interplay between molecular restrictions and compatibilities during sperm-egg interaction across teleosts.

developmental biology↗

bioRxiv2023

A conserved fertilization complex of Izumo1, Spaca6, and Tmem81 mediates sperm-egg interaction in vertebrates

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.

developmental biology↗

bioRxiv2021

Rapid gene evolution in an ancient post-transcriptional and translationalregulatory system compensates for meiotic X chromosomal inactivation

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.

evolutionary biology↗

bioRxiv2021

The sperm protein SPACA4 is required for efficient fertilization in mice

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 non-mammalian species do not have obvious mammalian homologs. We have recently identified the Ly6/uPAR protein Bouncer as a new, essential fertilization factor in zebrafish (Herberg et al., 2018). Here, we show that Bouncers homolog in mammals, SPACA4, is also required for efficient fertilization in mice. In contrast to fish, where Bouncer is expressed specifically in the egg, SPACA4 is expressed exclusively in the sperm. Male knockout mice are severely sub-fertile, 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 zona pellucida. Our work identifies SPACA4 as an important sperm protein necessary for zona pellucida penetration during mammalian fertilization.

developmental biology↗

bioRxiv2021

Sperm membrane proteins DCST1 and DCST2 are required for the sperm-egg fusion process in mice and fish

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) 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.

developmental biology↗

bioRxiv2021

Fine-scale Population Structure of North American Arabidopsis thaliana Reveals Multiple Sources of Introduction from Across Eurasia

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.

evolutionary biology↗