Regulation of MBK-2/DYRK by CDK-1 and the pseudophosphatases EGG-4 and EGG-5 during the oocyte-to-embryo transition.

DYRKs are kinases that self-activate in vitro by autophosphorylation of a YTY motif in the kinase domain, but their regulation in vivo is not well understood. In C. elegans zygotes, MBK-2/DYRK phosphorylates oocyte proteins at the end of the meiotic divisions to promote the oocyte-to-embryo transition. Here we demonstrate that MBK-2 is under both positive and negative regulation during the transition. MBK-2 is activated during oocyte maturation by CDK-1-dependent phosphorylation of serine 68, a residue outside of the kinase domain required for full activity in vivo. The pseudotyrosine phosphatases EGG-4 and EGG-5 sequester activated MBK-2 until the meiotic divisions by binding to the YTY motif and inhibiting MBK-2's kinase activity directly, using a mixed-inhibition mechanism that does not involve tyrosine dephosphorylation. Our findings link cell-cycle progression to MBK-2/DYRK activation and the oocyte-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.

Caenorhabditis elegans sperm membrane protein interactome.

The interaction and organization of proteins in the sperm membrane are important for all aspects of sperm function. We have determined the interactions between 12 known mutationally defined and cloned sperm membrane proteins in a model system for reproduction, the nematode Caenorhabditis elegans. Identification of the interactions between sperm membrane proteins will improve our understanding of and ability to characterize defects in sperm function. To identify interacting proteins, we conducted a split-ubiquitin membrane yeast two-hybrid analysis of gene products identified through genetic screens that are necessary for sperm function and predicted to encode transmembrane proteins. Our analysis revealed novel interactions between sperm membrane proteins known to have roles in spermatogenesis, spermiogenesis, and fertilization. For example, we found that a protein known to play a role in sperm function during fertilization, SPE-38 (a predicted four pass transmembrane protein), interacts with proteins necessary for spermiogenesis and spermatogenesis and could serve as a central organizing protein in the plasma membrane. These novel interaction pairings will provide the foundation for investigating previously unrealized membrane protein interactions during spermatogenesis, spermiogenesis, and sperm function during fertilization.

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.

The sperm surface localization of the TRP-3/SPE-41 Ca2+ -permeable channel depends on SPE-38 function in Caenorhabditis elegans.

Despite undergoing normal development and acquiring normal morphology and motility, mutations in spe-38 or trp-3/spe-41 cause identical phenotypes in Caenorhabditis elegans-mutant sperm fail to fertilize oocytes despite direct contact. SPE-38 is a novel, four-pass transmembrane protein and TRP-3/SPE-41 is a Ca(2+)-permeable channel. Localization of both of these proteins is confined to the membranous organelles (MOs) in undifferentiated spermatids. In mature spermatozoa, SPE-38 is localized to the pseudopod and TRP-3/SPE-41 is localized to the whole plasma membrane. Here we show that the dynamic redistribution of TRP-3/SPE-41 from MOs to the plasma membrane is dependent on SPE-38. In spe-38 mutant spermatozoa, TRP-3/SPE-41 is trapped within the MOs and fails to reach the cell surface despite MO fusion with the plasma membrane. Split-ubiquitin yeast-two-hybrid analyses revealed that the cell surface localization of TRP-3/SPE-41 is likely regulated by SPE-38 through a direct protein-protein interaction mechanism. We have identified sequences that influence the physical interaction between SPE-38 and TRP-3/SPE-41, and show that these sequences in SPE-38 are required for fertility in transgenic animals. Despite the mislocalization of TRP-3/SPE-41 in spe-38 mutant spermatozoa, ionomycin or thapsigargin induced influx of Ca(2+) remains unperturbed. This work reveals a new paradigm for the regulated surface localization of a Ca(2+)-permeable channel.

Fertilization.

Fertilization-the fusion of gametes to produce a new organism-is the culmination of a multitude of intricately regulated cellular processes. In Caenorhabditis elegans, fertilization is highly efficient. Sperm become fertilization competent after undergoing a maturation process during which they become motile, and the plasma membrane protein composition is reorganized in preparation for interaction with the oocyte. The highly specialized gametes begin their interactions by signaling to one another to ensure that fertilization occurs when they meet. The oocyte releases prostaglandin signals to help guide the sperm to the site of fertilization, and sperm secrete a protein called major sperm protein (MSP) to trigger oocyte maturation and ovulation. Upon meeting one another in the spermatheca, the sperm and oocyte fuse in a specific and tightly regulated process. Recent studies are providing new insights into the molecular basis of this fusion process. After fertilization, the oocyte must quickly transition from the relative quiescence of oogenesis to a phase of rapid development during the cleavage divisions of early embryogenesis. In addition, the fertilized oocyte must prevent other sperm from fusing with it as well as produce an eggshell for protection during external development. This chapter will review the nature and regulation of the various cellular processes of fertilization, including the development of fertilization competence, gamete signaling, sperm-oocyte fusion, the oocyte to embryo transition, and production of an eggshell to protect the developing embryo.

CRISPR/Cas9 Mediated Fluorescent Tagging of Caenorhabditis elegans SPE-38 Reveals a Complete Localization Pattern in Live Spermatozoa.

The Caenorhabditis elegans spe-38 gene encodes a four-pass transmembrane molecule that is required in sperm for fertilization. In previous work, the localization of the SPE-38 protein was examined using polyclonal antibodies on spermatids and mature amoeboid spermatozoa. SPE-38 is localized to unfused membranous organelles (MOs) in nonmotile spermatids. Different fixation conditions revealed that SPE-38 either localized to fused MOs and the cell body plasma membrane or the pseudopod plasma membrane of mature sperm. To address this localization paradox in mature sperm, CRISPR/Cas9 genome editing was used to tag endogenous SPE-38 with fluorescent wrmScarlet-I. Homozygous male and hermaphrodite worms encoding SPE-38::wrmScarlet-I were fertile indicating the fluorescent tag does not interfere with SPE-38 function during sperm activation or fertilization. We found that SPE-38::wrmScarlet-I localized to MOs in spermatids consistent with previous antibody localization. In mature and motile spermatozoa we found SPE-38::wrmScarlet-I in fused MOs, the cell body plasma membrane, and the pseudopod plasma membrane. We conclude that the localization pattern observed with SPE-38::wrmScarlet-I represents the complete distribution of SPE-38 in mature spermatozoa and this localization pattern is consistent with a hypothesized role of SPE-38 directly in sperm-egg binding and/or fusion.

Where are all the egg genes?

Complementary forward and reverse genetic approaches in several model systems have resulted in a recent burst of fertilization gene discovery. The number of genetically validated gamete surface molecules have more than doubled in the last few years. All the genetically validated sperm fertilization genes encode transmembrane or secreted molecules. Curiously, the discovery of genes that encode oocyte molecules have fallen behind that of sperm genes. This review discusses potential experimental biases and inherent biological reasons that could slow egg fertilization gene discovery. Finally, we shed light on current strategies to identify genes that may result in further identification of egg fertilization genes.

Identification and sequencing of temperature sensitive alleles of the Anaphase Promoting Complex component mat-3 in C. elegans.

The Anaphase Promoting Complex (APC) regulates the transition from metaphase to anaphase during cell division and is important to prevent defects in chromosome segregation. In a recent temperature sensitive genetic screen looking for further genes involved in fertilization, we isolated a new temperature sensitive allele of mat-3 (as49) . We also sequenced a previously identified mat-3 ( or344 ) allele that did not previously have an annotated sequence. We determined that the as49 allele was an Alanine to Threonine (A451T) mutation in the sixth exon and the or344 mutation was a Leucine to Phenylalanine (L474F) mutation in the seventh exon. These locations of the mutant alleles are consistent with other previously annotated alleles that displayed the same metaphase to anaphase transition defect phenotype and further reinforce the importance of the tetratricopeptide repeats to mediate protein interactions.

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.

Fertilization: what we can learn from worms.

Infertility and development of contraceptive methods have profound societal affects; however, the genetic mechanisms underlying this are still largely unknown. Here, we describe how using the small worm Caenorhabditis elegans has helped us to discover the genes involved in these processes. Nobel Laureate Sydney Brenner established the nematode worm C. elegans as a genetic model system with a powerful ability to discover genes in many biological pathways through mutagenesis. In this tradition, many labs have been using the substantial genetic tools established by Brenner and the 'worm' research community to discover genes required for uniting sperm and egg. Our understanding of the molecular underpinnings of the fertilization synapse between sperm and egg rivals that of any organism. Genes have been discovered in worms that share homology and mutant phenotypes with mammals. We provide an overview of the state of our understanding of fertilization in worms as well as exciting future directions and challenges.

Meiotic maturation: receptor trafficking is the key.

Meiotic maturation and ovulation rates in Caenorhabditis elegans are regulated by a sperm-released gradient of major sperm protein (MSP). Recent work has provided insights into the modulation of the MSP signal by the trafficking of its receptor in oocytes.

The molecular underpinnings of fertility: Genetic approaches in Caenorhabditis elegans.

The study of mutations that impact fertility has a catch-22. Fertility mutants are often lost since they cannot simply be propagated and maintained. This has hindered progress in understanding the genetics of fertility. In mice, several molecules are found to be required for the interactions between the sperm and egg, with JUNO and IZUMO1 being the only known receptor pair on the egg and sperm surface, respectively. In Caenorhabditis elegans, a total of 12 proteins on the sperm or oocyte have been identified to mediate gamete interactions. Majority of these genes were identified through mutants isolated from genetic screens. In this review, we summarize the several key screening strategies that led to the identification of fertility mutants in C. elegans and provide a perspective about future research using genetic approaches. Recently, advancements in new technologies such as high-throughput sequencing and Crispr-based genome editing tools have accelerated the molecular, cell biological, and mechanistic analysis of fertility genes. We review how these valuable tools advance our understanding of the molecular underpinnings of fertilization. We draw parallels of the molecular mechanisms of fertilization between worms and mammals and argue that our work in C. elegans complements fertility research in humans and other species.

Conserved sperm factors are no longer a bone of contention.

Proteins related to a molecule involved in the formation of osteoclasts in bone are required for fertilization in worms, flies and mammals.

EGG-3 regulates cell-surface and cortex rearrangements during egg activation in Caenorhabditis elegans.

Fertilization triggers egg activation and converts the egg into a developing embryo. The events of this egg-to-embryo transition typically include the resumption of meiosis, the reorganization of the cortical actin cytoskeleton, and the remodeling of the oocyte surface. The factors that regulate sperm-dependent egg-activation events are not well understood. Caenorhabditis elegans EGG-3, a member of the protein tyrosine phosphatase-like (PTPL) family, is essential for regulating cell-surface and cortex rearrangements during egg activation in response to sperm entry. Although fertilization occurred normally in egg-3 mutants, the polarized dispersal of F-actin is altered, a chitin eggshell is not formed, and no polar bodies are produced. EGG-3 is associated with the oocyte plasma membrane in a pattern that is similar to CHS-1 and MBK-2. CHS-1 is required for eggshell deposition, whereas MBK-2 is required for the degradation of maternal proteins during the egg-to-embryo transition. The localization of CHS-1 and EGG-3 are interdependent and both genes were required for the proper localization of MBK-2 in oocytes. Therefore, EGG-3 plays a central role in egg activation by influencing polarized F-actin dynamics and the localization or activity of molecules that are directly involved in executing the egg-to-embryo transition.

SLC17A6/7/8 Vesicular Glutamate Transporter Homologs in Nematodes.

Members of the superfamily of solute carrier (SLC) transmembrane proteins transport diverse substrates across distinct cellular membranes. Three SLC protein families transport distinct neurotransmitters into synaptic vesicles to enable synaptic transmission in the nervous system. Among them is the SLC17A6/7/8 family of vesicular glutamate transporters, which endows specific neuronal cell types with the ability to use glutamate as a neurotransmitter. The genome of the nematode Caenorhabditis elegans encodes three SLC17A6/7/8 family members, one of which, eat-4/VGLUT, has been shown to be involved in glutamatergic neurotransmission. Here, we describe our analysis of the two remaining, previously uncharacterized SLC17A6/7/8 family members, vglu-2 and vglu-3 These two genes directly neighbor one another and are the result of a recent gene duplication event in C. elegans, but not in other Caenorhabditis species. Compared to EAT-4, the VGLU-2 and VGLU-3 protein sequences display a more distant similarity to canonical, vertebrate VGLUT proteins. We tagged both genomic loci with gfp and detected no expression of vglu-3 at any stage of development in any cell type of both C. elegans sexes. In contrast, vglu-2::gfp is dynamically expressed in a restricted set of distinct cell types. Within the nervous system, vglu-2::gfp is exclusively expressed in a single interneuron class, AIA, where it localizes to vesicular structures in the soma, but not along the axon, suggesting that VGLU-2 may not be involved in synaptic transport of glutamate. Nevertheless, vglu-2 mutants are partly defective in the function of the AIA neuron in olfactory behavior. Outside the nervous system, VGLU-2 is expressed in collagen secreting skin cells where VGLU-2 most prominently localizes to early endosomes, and to a lesser degree to apical clathrin-coated pits, the trans-Golgi network, and late endosomes. On early endosomes, VGLU-2 colocalizes most strongly with the recycling promoting factor SNX-1, a retromer component. Loss of vglu-2 affects the permeability of the collagen-containing cuticle of the worm, and based on the function of a vertebrate VGLUT1 protein in osteoclasts, we speculate that vglu-2 may have a role in collagen trafficking in the skin. We conclude that C. elegans SLC17A6/7/8 family members have diverse functions within and outside the nervous system.

Meiotic diapause: how a sperm signal sets you free.

Major sperm protein, a cytoskeletal molecule required for the amoeboid motility of sperm in Caenorhabditis elegans, also functions as a signaling molecule that regulates the rates of meiotic maturation and ovulation. Recent work has begun to uncover new genes required for the response to this signal in both somatic and germ line cells.