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.

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.

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.

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.

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.

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.

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.

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.

Dramatic fertility decline in aging C. elegans males is associated with mating execution deficits rather than diminished sperm quality.

Although much is known about female reproductive aging, fairly little is known about the causes of male reproductive senescence. We developed a method that facilitates culture maintenance of Caenorhabditis elegans adult males, which enabled us to measure male fertility as populations age, without profound loss of males from the growth plate. We find that the ability of males to sire progeny declines rapidly in the first half of adult lifespan and we examined potential factors that contribute towards reproductive success, including physical vigor, sperm quality, mating apparatus morphology, and mating ability. Of these, we find little evidence of general physical decline in males or changes in sperm number, morphology, or capacity for activation, at time points when reproductive senescence is markedly evident. Rather, it is the loss of efficient mating ability that correlates most strongly with reproductive senescence. Low insulin signaling can extend male ability to sire progeny later in life, although insulin impact on individual facets of mating behavior is complex. Overall, we suggest that combined modest deficits, predominantly affecting the complex mating behavior rather than sperm quality, sum up to block effective C. elegans male reproduction in middle adult life.

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.

Calcium signaling surrounding fertilization in the nematode Caenorhabditis elegans.

Calcium plays a prominent role during fertilization in many animals. This review focuses on roles of Ca(2+) during the events around fertilization in the model organism, Caenorhabditis elegans. Specifically, the role of Ca(2+) in sperm, oocytes and the surrounding somatic tissues during fertilization will be discussed, with the focus on sperm activation, meiotic maturation of oocytes, ovulation, sperm-egg interaction and fertilization.