The EGF-motif-containing protein SPE-36 is a secreted sperm protein required for fertilization in C. elegans.

Identified through forward genetics, spe-9 was the first gene to be identified in C. elegans as necessary for fertilization.1 Since then, genetic screens in C. elegans have led to the identification of nine additional sperm genes necessary for fertilization (including spe-51 reported by Mei et al.2 and the spe-36 gene reported here).3,4,5,6,7,8,9 This includes spe-45, which encodes an immunoglobulin-containing protein similar to the mammalian protein IZUMO1, and spe-42 and spe-49, which are homologous to vertebrate DCST2 and DCST1, respectively.4,7,8,10,11,12,13 Mutations in any one of these genes result in healthy adult animals that are sterile. Sperm from these mutants have normal morphology, migrate to and maintain their position at the site of fertilization in the reproductive tract, and make contact with eggs but fail to fertilize the eggs. This same phenotype is observed in mammals lacking Izumo1, Spaca6, Tmem95, Sof1, FIMP, or Dcst1 and Dcst2.10,14,15,16,17,18,19 Here we report the discovery of SPE-36 as a sperm-derived secreted protein that is necessary for fertilization. Mutations in the Caenorhabditis elegans spe-36 gene result in a sperm-specific fertilization defect. Sperm from spe-36 mutants look phenotypically normal, are motile, and can migrate to the site of fertilization. However, sperm that do not produce SPE-36 protein cannot fertilize. Surprisingly, spe-36 encodes a secreted EGF-motif-containing protein that functions cell autonomously. The genetic requirement for secreted sperm-derived proteins for fertilization sheds new light on the complex nature of fertilization and represents a paradigm-shifting discovery in the molecular understanding of fertilization.

SPE-51, a sperm-secreted protein with an immunoglobulin-like domain, is required for fertilization in C. elegans.

Fertilization is a fundamental process in sexual reproduction during which gametes fuse to combine their genetic material and start the next generation in their life cycle. Fertilization involves species-specific recognition, adhesion, and fusion between the gametes.1,2 In mammals and other model species, some proteins are known to be required for gamete interactions and have been validated with loss-of-function fertility phenotypes.3,4 Yet, the molecular basis of sperm-egg interaction is not well understood. In a forward genetic screen for fertility mutants in Caenorhabditis elegans, we identified spe-51. Mutant worms make sperm that are unable to fertilize the oocyte but otherwise normal by all available measurements. The spe-51 gene encodes a secreted protein that includes an immunoglobulin (Ig)-like domain and a hydrophobic sequence of amino acids. The SPE-51 protein acts cell autonomously and localizes to the surface of the spermatozoa. We further show that the gene product of the mammalian sperm function gene Sof1 is likewise secreted. This is the first example of a secreted protein required for the interactions between the sperm and egg with genetic validation for a specific function in fertilization in C. elegans (also see spe-365). This is also the first experimental evidence that mammalian SOF1 is secreted. Our analyses of these genes begin to build a paradigm for sperm-secreted or reproductive-tract-secreted proteins that coat the sperm surface and influence their survival, motility, and/or the ability to fertilize the egg.

Isolation, profiling, and tracking of extracellular vesicle cargo in Caenorhabditis elegans.

Extracellular vesicles (EVs) may mediate intercellular communication by carrying protein and RNA cargo. The composition, biology, and roles of EVs in physiology and pathology have been primarily studied in the context of biofluids and in cultured mammalian cells. The experimental tractability of C. elegans makes for a powerful in vivo animal system to identify and study EV cargo from its cellular source. We developed an innovative method to label, track, and profile EVs using genetically encoded, fluorescent-tagged EV cargo and conducted a large-scale isolation and proteomic profiling. Nucleic acid binding proteins (∼200) are overrepresented in our dataset. By integrating our EV proteomic dataset with single-cell transcriptomic data, we identified and validated ciliary EV cargo: CD9-like tetraspanin (TSP-6), ectonucleotide pyrophosphatase/phosphodiesterase (ENPP-1), minichromosome maintenance protein (MCM-3), and double-stranded RNA transporter SID-2. C. elegans EVs also harbor RNA, suggesting that EVs may play a role in extracellular RNA-based communication.

Maternal Proteins That Are Phosphoregulated upon Egg Activation Include Crucial Factors for Oogenesis, Egg Activation and Embryogenesis in Drosophila melanogaster.

Egg activation is essential for the successful transition from a mature oocyte to a developmentally competent egg. It consists of a series of events including the resumption and completion of meiosis, initiation of translation of some maternal mRNAs and destruction of others, and changes to the vitelline envelope. This major change of cell state is accompanied by large scale alteration in the oocyte's phosphoproteome. We hypothesize that the cohort of proteins that are subject to phosphoregulation during egg activation are functionally important for processes before, during, or soon after this transition, potentially uniquely or as proteins carrying out essential cellular functions like those they do in other (somatic) cells. In this study, we used germline-specific RNAi to examine the function of 189 maternal proteins that are phosphoregulated during egg activation in Drosophila melanogaster We identified 53 genes whose knockdown reduced or abolished egg production and caused a range of defects in ovarian morphology, as well as 51 genes whose knockdown led to significant impairment or abolishment of the egg hatchability. We observed different stages of developmental arrest in the embryos and various defects in spindle morphology and aberrant centrosome activities in the early arrested embryos. Our results, validated by the detection of multiple genes with previously-documented maternal effect phenotypes among the proteins we tested, revealed 15 genes with newly discovered roles in egg activation and early embryogenesis in Drosophila. Given that protein phosphoregulation is a conserved characteristic of this developmental transition, we suggest that the phosphoregulated proteins may provide a rich pool of candidates for the identification of important players in the egg-to-embryo transition.

The zinc transporter ZIPT-7.1 regulates sperm activation in nematodes.

Sperm activation is a fascinating example of cell differentiation, in which immotile spermatids undergo a rapid and dramatic transition to become mature, motile sperm. Because the sperm nucleus is transcriptionally silent, this transition does not involve transcriptional changes. Although Caenorhabditis elegans is a leading model for studies of sperm activation, the mechanisms by which signaling pathways induce this transformation remain poorly characterized. Here we show that a conserved transmembrane zinc transporter, ZIPT-7.1, regulates the induction of sperm activation in Caenorhabditis nematodes. The zipt-7.1 mutant hermaphrodites cannot self-fertilize, and males reproduce poorly, because mutant spermatids are defective in responding to activating signals. The zipt-7.1 gene is expressed in the germ line and functions in germ cells to promote sperm activation. When expressed in mammalian cells, ZIPT-7.1 mediates zinc transport with high specificity and is predominantly located on internal membranes. Finally, genetic epistasis places zipt-7.1 at the end of the spe-8 sperm activation pathway, and ZIPT-7.1 binds SPE-4, a presenilin that regulates sperm activation. Based on these results, we propose a new model for sperm activation. In spermatids, inactive ZIPT-7.1 is localized to the membranous organelles, which contain higher levels of zinc than the cytoplasm. When sperm activation is triggered, ZIPT-7.1 activity increases, releasing zinc from internal stores. The resulting increase in cytoplasmic zinc promotes the phenotypic changes characteristic of activation. Thus, zinc signaling is a key step in the signal transduction process that mediates sperm activation, and we have identified a zinc transporter that is central to this activation process.

spe-43 is required for sperm activation in C. elegans.

Successful fertilization requires that sperm are activated prior to contacting an oocyte. In C. elegans, this activation process, called spermiogenesis, transforms round immobile spermatids into motile, fertilization-competent spermatozoa. We describe the phenotypic and genetic characterization of spe-43, a new component of the spe-8 pathway, which is required for spermiogenesis in hermaphrodites; spe-43 hermaphrodites are self-sterile, while spe-43 males show wild-type fertility. When exposed to Pronase to activate sperm in vitro, spe-43 spermatids form long rigid spikes radiating outward from the cell periphery instead of forming a motile pseudopod, indicating that spermiogenesis initiates but is not completed. Using a combination of recombinant and deletion mapping and whole genome sequencing, we identified F09E8.1 as spe-43. SPE-43 is predicted to exist in two isoforms; one isoform appears to be a single-pass transmembrane protein while the other is predicted to be a secreted protein. SPE-43 can bind to other known sperm proteins, including SPE-4 and SPE-29, which are known to impact spermiogenesis. In summary, we have identified a membrane protein that is present in C. elegans sperm and is required for sperm activation via the hermaphrodite activation signal.

Marriage shrines and worms impacting our understanding of mammalian fertilization.

Genetic approaches in C. elegans are complementing the biochemical and antibody based strategies traditionally used to study the molecular underpinnings of fertilization in other organisms. A pair of worm studies, one based on forward genetics and one based on reverse genetics, converge on the sperm immunoglobulin superfamily molecule SPE-45. Loss of spe-45 function leads to the production of sperm that cannot fertilize wild-type eggs. This is a strikingly similar phenotype as those seen in mice lacking the immunoglobulin superfamily protein Izumo1. This work sets the stage for leveraging the power of the C. elegans model system to learn more about Izumo-like molecular function but also for the discovery of additional deeply conserved components of fertility pathways.

The molecular complexity of fertilization: Introducing the concept of a fertilization synapse.

The details of sperm-egg interactions remain a relative mystery despite many decades of research. As new molecular complexities are being discovered, we need to revise the framework in which we think about fertilization. As such, we propose that fertilization involves the formation of a synapse between the sperm and egg. A cellular synapse is a structure that mediates cell adhesion, signaling, and secretion through specialized zones of interaction and polarity. In this review, we draw parallels between the immune synapse and fertilization, and argue that we should consider sperm-egg recognition, binding, and fusion in the context of a "fertilization synapse." Mol. Reprod. Dev. 83: 376-386, 2016. © 2016 Wiley Periodicals, Inc.

Forward Genetics Identifies a Requirement for the Izumo-like Immunoglobulin Superfamily spe-45 Gene in Caenorhabditis elegans Fertilization.

Fertilization is a conserved process in all sexually reproducing organisms whereby sperm bind and fuse with oocytes. Despite the importance of sperm-oocyte interactions in fertilization, the molecular underpinnings of this process are still not well understood. The only cognate ligand-receptor pair identified in the context of fertilization is sperm-surface Izumo and egg-surface Juno in the mouse [1]. Here we describe a genetic screening strategy to isolate fertilization mutants in Caenorhabditis elegans in order to generate a more complete inventory of molecules required for gamete interactions. From this screening strategy, we identified, cloned, and characterized spe-45, a gene that encodes an Izumo-like immunoglobulin superfamily protein. Mammalian Izumo is required for male fertility and has the same basic mutant phenotype as spe-45. Worms lacking spe-45 function produce morphologically normal and motile sperm that cannot fuse with oocytes despite direct contact in the reproductive tract. The power of this screen to identify proteins with ancient sperm functions suggests that characterization of additional mutants from our screen may reveal other deeply conserved components in fertility pathways and complement studies in other organisms.

Phospho-regulation pathways during egg activation in Drosophila melanogaster.

Egg activation is the series of events that transition a mature oocyte to an egg capable of supporting embryogenesis. Increasing evidence points toward phosphorylation as a critical regulator of these events. We used Drosophila melanogaster to investigate the relationship between known egg activation genes and phosphorylation changes that occur upon egg activation. Using the phosphorylation states of four proteins-Giant Nuclei, Young Arrest, Spindly, and Vap-33-1-as molecular markers, we showed that the egg activation genes sarah, CanB2, and cortex are required for the phospho-regulation of multiple proteins. We show that an additional egg activation gene, prage, regulates the phosphorylation state of a subset of these proteins. Finally, we show that Sarah and calcineurin are required for the Anaphase Promoting Complex/Cyclosome (APC/C)-dependent degradation of Cortex following egg activation. From these data, we present a model in which Sarah, through the activation of calcineurin, positively regulates the APC/C at the time of egg activation, which leads to a change in phosphorylation state of numerous downstream proteins.

Molecular changes during egg activation.

Egg activation is the final transition that an oocyte goes through to become a developmentally competent egg. This transition is usually triggered by a calcium-based signal that is often, but not always, initiated by fertilization. Activation encompasses a number of changes within the egg. These include changes to the egg's membranes and outer coverings to prevent polyspermy and to support the developing embryo, as well as resumption and completion of the meiotic cell cycle, mRNA polyadenylation, translation of new proteins, and the degradation of specific maternal mRNAs and proteins. The transition from an arrested, highly differentiated cell, the oocyte, to a developmentally active, totipotent cell, the activated egg or embryo, represents a complete change in cellular state. This is accomplished by altering ion concentrations and by widespread changes in both the proteome and the suite of mRNAs present in the cell. Here, we review the role of calcium and zinc in the events of egg activation, and the importance of macromolecular changes during this transition. The latter include the degradation and translation of proteins, protein posttranslational regulation through phosphorylation, and the degradation, of maternal mRNAs.

Protein phosphorylation changes reveal new candidates in the regulation of egg activation and early embryogenesis in D. melanogaster.

Egg activation is the series of events that must occur for a mature oocyte to become capable of supporting embryogenesis. These events include changes to the egg's outer coverings, the resumption and completion of meiosis, the translation of new proteins, and the degradation of specific maternal mRNAs. While we know some of the molecules that direct the initial events of egg activation, it remains unclear how multiple pathways are coordinated to change the cellular state from mature oocyte to activated egg. Using a proteomic approach we have identified new candidates for the regulation and progression of egg activation. Reasoning that phosphorylation can simultaneously and rapidly modulate the activity of many proteins, we identified proteins that are post-translationally modified during the transition from oocyte to activated egg in Drosophila melanogaster. We find that at least 311 proteins change in phosphorylation state between mature oocytes and activated eggs. These proteins fall into various functional classes related to the events of egg activation including calcium binding, proteolysis, and protein translation. Our set of candidates includes genes already associated with egg activation, as well as many genes not previously studied during this developmental period. RNAi knockdown of a subset of these genes revealed a new gene, mrityu, necessary for embryonic development past the first mitosis. Thus, by identifying phospho-modulated proteins we have produced a focused candidate set for future genetic studies to test their roles in egg activation and the initiation of embryogenesis.