Membrane-associated cytoplasmic granules carrying the Argonaute protein WAGO-3 enable paternal epigenetic inheritance in Caenorhabditis elegans.

Epigenetic inheritance describes the transmission of gene regulatory information across generations without altering DNA sequences, enabling offspring to adapt to environmental conditions. Small RNAs have been implicated in this, through both the oocyte and the sperm. However, as much of the cellular content is extruded during spermatogenesis, it is unclear whether cytoplasmic small RNAs can contribute to epigenetic inheritance through sperm. Here we identify a sperm-specific germ granule, termed the paternal epigenetic inheritance (PEI) granule, that mediates paternal epigenetic inheritance by retaining the cytoplasmic Argonaute protein WAGO-3 during spermatogenesis in Caenorhabditis elegans. We identify the PEI granule proteins PEI-1 and PEI-2, which have distinct functions in this process: granule formation, Argonaute selectivity and subcellular localization. We show that PEI granule segregation is coupled to the transport of sperm-specific secretory vesicles through PEI-2 in an S-palmitoylation-dependent manner. PEI-like proteins are found in humans, suggesting that the identified mechanism may be conserved.

MIB-1 Is Required for Spermatogenesis and Facilitates LIN-12 and GLP-1 Activity in Caenorhabditis elegans.

Covalent attachment of ubiquitin to substrate proteins changes their function or marks them for proteolysis, and the specificity of ubiquitin attachment is mediated by the numerous E3 ligases encoded by animals. Mind Bomb is an essential E3 ligase during Notch pathway signaling in insects and vertebrates. While Caenorhabditis elegans encodes a Mind Bomb homolog (mib-1), it has never been recovered in the extensive Notch suppressor/enhancer screens that have identified numerous pathway components. Here, we show that C. elegans mib-1 null mutants have a spermatogenesis-defective phenotype that results in a heterogeneous mixture of arrested spermatocytes, defective spermatids, and motility-impaired spermatozoa. mib-1 mutants also have chromosome segregation defects during meiosis, molecular null mutants are intrinsically temperature-sensitive, and many mib-1 spermatids contain large amounts of tubulin. These phenotypic features are similar to the endogenous RNA intereference (RNAi) mutants, but mib-1 mutants do not affect RNAi. MIB-1 protein is expressed throughout the germ line with peak expression in spermatocytes followed by segregation into the residual body during spermatid formation. C. elegans mib-1 expression, while upregulated during spermatogenesis, also occurs somatically, including in vulva precursor cells. Here, we show that mib-1 mutants suppress both lin-12 and glp-1 (C. elegans Notch) gain-of-function mutants, restoring anchor cell formation and a functional vulva to the former and partly restoring oocyte production to the latter. However, suppressed hermaphrodites are only observed when grown at 25°, and they are self-sterile. This probably explains why mib-1 was not previously recovered as a Notch pathway component in suppressor/enhancer selection experiments.

Spermatogenesis.

Most organisms consist of two cell lineages - somatic cells and germ cells. The former are required for the current generation, and the latter create offspring. Male and female germ cells are usually produced during spermatogenesis and oogenesis, which take place in the testis and the ovary, respectively. Spermatogenesis involves the differentiation of spermatogonial stem cells into spermatocytes via mitotic cell division and the production of haploid spermatids from the tetraploid primary spermatocytes via meiotic cell division. Spermatids subsequently give rise to spermatozoa in the final phase of spermatogenesis, called spermiogenesis. These fundamental steps, where mitotic proliferation precedes meiosis during spermatogenesis, are observed in a wide variety of organisms. However, developing a comprehensive understanding of the cell biology and genetics of spermatogenesis is difficult for most species because it occurs within a complex testicular environment characterized by the intimate association of developing sperm with accessory cells. In this Primer, we summarize the processes of spermatogenesis occurring in two pivotal model animals - mouse and Caenorhabditis elegans - and compare them to consider which important features might be evolutionarily conserved.

Gamete interactions require transmembranous immunoglobulin-like proteins with conserved roles during evolution.

C. elegans spe-9 class genes are male germline-enriched in their expression and indispensable during sperm-oocyte fusion. Identification of mammalian orthologs that exhibit similar functions to these C. elegans genes has been a challenge. The mouse Izumo1 gene encodes a sperm-specific, immunoglobulin (Ig)-like transmembrane (TM) protein that is required for gamete fusion. We recently identified the C. elegans spe-45 gene, which shows male germline-enriched expression and encodes an Ig-like TM protein. spe-45 mutant worms produced otherwise normal spermatozoa that cannot fuse with oocytes, causing essentially the same phenotype as that seen in the Izumo1-knockout mice. By counting the number of self-sperm in the spermatheca of spe-45 hermaphrodites, it was found that this gene might be involved in sperm guidance from the uterus into the spermatheca, as well as gamete fusion. Moreover, we discovered that SPE-45 and IZUMO1 share certain functions for gamete fusion, which are presumably related to binding with cis- and/or trans-partners. Intriguingly, various organisms have Ig-like TM proteins that act during gamete interactions, indicating the wide-spread utility of Ig-like domains during fertilization.

The Immunoglobulin-like Gene spe-45 Acts during Fertilization in Caenorhabditis elegans like the Mouse Izumo1 Gene.

The Caenorhabditis elegans spe-9 class genes, which show specific or predominant expression in the male germline, are indispensable for fertilization [1, 2]. However, due to the rapid evolution of genes involved in reproduction, we do not currently know if there are spe-9 class genes in mammals that play similar roles during fertilization to those found in C. elegans. In mice, the Izumo1 gene encodes a sperm-specific transmembrane (TM) protein with a single immunoglobulin (Ig)-like domain that is absolutely required for gamete fusion [3, 4]. In this study, we hypothesized that C. elegans has a new member of the spe-9 class genes coding for an IZUMO1-like protein. We screened C. elegans microarray data [5, 6] to identify male germline-enriched genes that encode membrane proteins with Ig-like domains. A deletion (tm3715) in one such gene (F28D1.8) caused hermaphrodites to show a male germline-dependent self-sterility, so we have named it spe-45. Mutant spe-45 worms seemed to normally undergo spermatogenesis (spermatid production by meiosis) and spermiogenesis (spermatid activation into actively motile spermatozoa). spe-45 mutant spermatozoa, however, could not complete gamete fusion, which is a characteristic of all spe-9 class mutants [1, 2]. Moreover, spe-45 self-sterile worms were rescued by a transgene expressing chimeric SPE-45 protein in which its Ig-like domain was replaced by the Ig-like domain from mouse IZUMO1. Hence, C. elegans SPE-45 and mouse IZUMO1 appear to have retained a common function(s) that is required during fertilization.

Role of posttranslational modifications in C. elegans and ascaris spermatogenesis and sperm function.

Generally, spermatogenesis and sperm function involve widespread posttranslational modification of regulatory proteins in many different species. Nematode spermatogenesis has been studied in detail, mostly by genetic/molecular genetic techniques in the free-living Caenorhabditis elegans and by biochemistry/cell biology in the pig parasite Ascaris suum. Like other nematodes, both of these species produce sperm that use a form of amoeboid motility termed crawling, and many aspects of spermatogenesis are likely to be similar in both species. Consequently, work in these two nematode species has been largely complementary. Work in C. elegans has identified a number of spermatogenesis-defective genes and, so far, 12 encode enzymes that are implicated as catalysts of posttranslational protein modification. Crawling motility involves extension of a single pseudopod and this process is powered by a unique cytoskeleton composed of Major Sperm Protein (MSP) and accessory proteins, instead of the more widely observed actin. In Ascaris, pseudopod extension and crawling motility can be reconstituted in vitro, and biochemical studies have begun to reveal how posttranslational protein modifications, including phosphorylation, dephosphorylation and proteolysis, participate in these processes.

Vps33b pathogenic mutations preferentially affect VIPAS39/SPE-39-positive endosomes.

Mutations in Vps33 isoforms cause pigment dilution in mice (Vps33a, buff) and Drosophila (car) and the neurogenic arthrogryposis, renal dysfunction and cholestasis syndrome in humans (ARC1, VPS33B). The later disease is also caused by mutations in VIPAS39, (Vps33b interacting protein, apical-basolateral polarity regulator, SPE-39 homolog; ARC2), a protein that interacts with the HOmotypic fusion and Protein Sorting (HOPS) complex, a tether necessary for endosome-lysosome traffic. These syndromes offer insight into fundamental endosome traffic processes unique to metazoans. However, the molecular and cellular mechanisms underlying these mutant phenotypes remain poorly understood. Here we investigate interactions of wild-type and disease-causing mutations in VIPAS39/SPE-39 and Vps33b by yeast two hybrid, immunoprecipitation and quantitative fluorescent microscopy. We find that although few mutations prevent interaction between VIPAS39/SPE-39 and Vps33b, some mutants fragment VIPAS39/SPE-39-positive endosomes, but all mutants alter the subcellular localization of Vps33b to VIPAS39/SPE-39-positive endosomes. Our data suggest that the ARC syndrome may result through impaired VIPAS39/SPE-39 and Vps33b-dependent endosomal maturation or fusion.

Developmental genetics of secretory vesicle acidification during Caenorhabditis elegans spermatogenesis.

Secretory vesicles are used during spermatogenesis to deliver proteins to the cell surface. In Caenorhabditis elegans, secretory membranous organelles (MO) fuse with the plasma membrane to transform spermatids into fertilization-competent spermatozoa. We show that, like the acrosomal vesicle of mammalian sperm, MOs undergo acidification during development. Treatment of spermatids with the V-ATPase inhibitor bafilomycin blocks both MO acidification and formation of functional spermatozoa. There are several spermatogenesis-defective mutants that cause defects in MO morphogenesis, including spe-5. We determined that spe-5, which is on chromosome I, encodes one of two V-ATPase B paralogous subunits. The spe-5 null mutant is viable but sterile because it forms arrested, multi-nucleate spermatocytes. Immunofluorescence with a SPE-5-specific monoclonal antibody shows that SPE-5 expression begins in spermatocytes and is found in all subsequent stages of spermatogenesis. Most SPE-5 is discarded into the residual body during spermatid budding, but a small amount remains in budded spermatids where it localizes to MOs as a discrete dot. The other V-ATPase B subunit is encoded by vha-12, which is located on the X chromosome. Usually, spe-5 mutants are self-sterile in a wild-type vha-12 background. However, an extrachromosomal transgene containing wild-type vha-12 driven by its own promoter allows spe-5 mutant hermaphrodites to produce progeny, indicating that VHA-12 can at least partially substitute for SPE-5. Others have shown that the X chromosome is transcriptionally silent in the male germline, so expression of the autosomally located spe-5 gene ensures that a V-ATPase B subunit is present during spermatogenesis.

Nematode sperm maturation triggered by protease involves sperm-secreted serine protease inhibitor (Serpin).

Spermiogenesis is a series of poorly understood morphological, physiological and biochemical processes that occur during the transition of immotile spermatids into motile, fertilization-competent spermatozoa. Here, we identified a Serpin (serine protease inhibitor) family protein (As_SRP-1) that is secreted from spermatids during nematode Ascaris suum spermiogenesis (also called sperm activation) and we showed that As_SRP-1 has two major functions. First, As_SRP-1 functions in cis to support major sperm protein (MSP)-based cytoskeletal assembly in the spermatid that releases it, thereby facilitating sperm motility acquisition. Second, As_SRP-1 released from an activated sperm inhibits, in trans, the activation of surrounding spermatids by inhibiting vas deferens-derived As_TRY-5, a trypsin-like serine protease necessary for sperm activation. Because vesicular exocytosis is necessary to create fertilization-competent sperm in many animal species, components released during this process might be more important modulators of the physiology and behavior of surrounding sperm than was previously appreciated.

Metazoan cell biology of the HOPS tethering complex.

Membrane fusion with vacuoles, the lysosome equivalent of the yeast Saccharomyces cerevisiae, is among the best understood membrane fusion events. Our precise understanding of this fusion machinery stems from powerful genetics and elegant in vitro reconstitution assays. Central to vacuolar membrane fusion is the multi-subunit tether the HO motypic fusion and Protein Sorting (HOPS) complex, a complex of proteins that organizes other necessary components of the fusion machinery. We lack a similarly detailed molecular understanding of membrane fusion with lysosomes or lysosome-related organelles in metazoans. However, it is likely that fundamental principles of how rabs, SNAREs and HOPS tethers work to fuse membranes with lysosomes and related organelles are conserved between Saccharomyces cerevisiae and metazoans. Here, we discuss emerging differences in the coat-dependent mechanisms that govern HOPS complex subcellular distribution between Saccharomyces cerevisiae and metazoans. These differences reside upstream of the membrane fusion event. We propose that the differences in how coats segregate class C Vps/HOPS tethers to organelles and domains of metazoan cells are adaptations to complex architectures that characterize metazoan cells such as those of neuronal and epithelial tissues.

Clathrin-dependent mechanisms modulate the subcellular distribution of class C Vps/HOPS tether subunits in polarized and nonpolarized cells.

Coats define the composition of carriers budding from organelles. In addition, coats interact with membrane tethers required for vesicular fusion. The yeast AP-3 (Adaptor Protein Complex 3) coat and the class C Vps/HOPS (HOmotypic fusion and Protein Sorting) tether follow this model as their interaction occurs at the carrier fusion step. Here we show that mammalian Vps class C/HOPS subunits and clathrin interact and that acute perturbation of clathrin function disrupts the endosomal distribution of Vps class C/HOPS tethers in HEK293T and polarized neuronal cells. Vps class C/HOPS subunits and clathrin exist in complex with either AP-3 or hepatocyte growth factor receptor substrate (Hrs). Moreover, Vps class C/HOPS proteins cofractionate with clathrin-coated vesicles, which are devoid of Hrs. Expression of FK506 binding protein (FKBP)-clathrin light chain chimeras, to inhibit clathrin membrane association dynamics, increased Vps class C/HOPS subunit content in rab5 endosomal compartments. Additionally, Vps class C/HOPS subunits were concentrated at tips of neuronal processes, and their delivery was impaired by expression of FKBP-clathrin chimeras and AP20187 incubation. These data support a model in which Vps class C/HOPS subunits incorporate into clathrin-coated endosomal domains and carriers in mammalian cells. We propose that vesicular (AP-3) and nonvesicular (Hrs) clathrin mechanisms segregate class C Vps/HOPS tethers to organelles and domains of mammalian cells bearing complex architectures.

Spermatogenesis-defective (spe) mutants of the nematode Caenorhabditis elegans provide clues to solve the puzzle of male germline functions during reproduction.

In most species, each sex produces gametes, usually either sperm or oocytes, from its germline during gametogenesis. The sperm and oocyte subsequently fuse together during fertilization to create the next generation. This review focuses on spermatogenesis and the roles of sperm during fertilization in the nematode Caenorhabditis elegans, where suitable mutants are readily obtained. So far, 186 mutants defective in the C. elegans male germline functions have been isolated, and many of these mutations are alleles for one of the approximately 60 spermatogenesis-defective (spe) genes. Many cloned spe genes are expressed specifically in the male germline, where they play roles during spermatogenesis (spermatid production), spermiogenesis (spermatid activation into spermatozoa), and/or fertilization. Moreover, several spe genes are orthologs of mammalian genes, suggesting that the reproductive processes of the C. elegans and the mammalian male germlines might share common pathways at the molecular level.

The CIL-1 PI 5-phosphatase localizes TRP Polycystins to cilia and activates sperm in C. elegans.

BACKGROUND: C. elegans male sexual behaviors include chemotaxis and response to hermaphrodites, backing, turning, vulva location, spicule insertion, and sperm transfer, culminating in cross-fertilization of hermaphrodite oocytes with male sperm. The LOV-1 and PKD-2 transient receptor potential polycystin (TRPP) complex localizes to ciliated endings of C. elegans male-specific sensory neurons and mediates several aspects of male mating behavior. TRPP complex ciliary localization and sensory function are evolutionarily conserved. A genetic screen for C. elegans mutants with PKD-2 ciliary localization (Cil) defects led to the isolation of a mutation in the cil-1 gene. RESULTS: Here, we report that a phosphoinositide (PI) 5-phosphatase, CIL-1, regulates TRPP complex ciliary receptor localization and sperm activation. cil-1 does not regulate the localization of other ciliary proteins, including intraflagellar transport (IFT) components, sensory receptors, or other TRP channels in different cell types. Rather, cil-1 specifically controls TRPP complex trafficking in male-specific sensory neurons and does so in a cell-autonomous fashion. In these cells, cil-1 is required for normal PI(3)P distribution, indicating that a balance between PI(3,5)P2 and PI(3)P is important for TRPP localization. cil-1 mutants are infertile because of sperm activation and motility defects. In sperm, the CIL-1 5-phosphatase and a wortmannin-sensitive PI 3-kinase act antagonistically to regulate the conversion of sessile spermatids into motile spermatozoa, implicating PI(3,4,5)P3 signaling in nematode sperm activation. CONCLUSION: Our studies identify the CIL-1 5-phosphatase as a key regulator of PI metabolism in cell types that are important in several aspects of male reproductive biology.

The genetics and cell biology of spermatogenesis in the nematode C. elegans.

Creation of mutants that affect spermatogenesis is very challenging in most experimental systems, especially mammals. The main reason this is true is because "absence of successful mating" is a negative result that can occur for a wide variety of trivial, irrelevant reasons. The C. elegans hermaphroditic mode of reproduction has unusual features that facilitate analysis of spermatogenesis. Normally, hermaphrodites are virtually 100% self-fertile and spermatogenesis defective mutants are self-sterile. A candidate spermatogenesis defective mutant will produce cross-progeny after mating to a wild type male, showing that the presence of sperm is both necessary and sufficient to restore fertility to the sterile hermaphrodite. This has allowed selection of a large number of spermatogenesis defective mutants. In this article, I will review spermatogenesis, how mutants are made and what has been learned about the identified genes and their roles during development and fertilization.

SPE-39 family proteins interact with the HOPS complex and function in lysosomal delivery.

Yeast and animal homotypic fusion and vacuole protein sorting (HOPS) complexes contain conserved subunits, but HOPS-mediated traffic in animals might require additional proteins. Here, we demonstrate that SPE-39 homologues, which are found only in animals, are present in RAB5-, RAB7-, and RAB11-positive endosomes where they play a conserved role in lysosomal delivery and probably function via their interaction with the core HOPS complex. Although Caenorhabditis elegans spe-39 mutants were initially identified as having abnormal vesicular biogenesis during spermatogenesis, we show that these mutants also have disrupted processing of endocytosed proteins in oocytes and coelomocytes. C. elegans SPE-39 interacts in vitro with both VPS33A and VPS33B, whereas RNA interference of VPS33B causes spe-39-like spermatogenesis defects. The human SPE-39 orthologue C14orf133 also interacts with VPS33 homologues and both coimmunoprecipitates and cosediments with other HOPS subunits. SPE-39 knockdown in cultured human cells altered the morphology of syntaxin 7-, syntaxin 8-, and syntaxin 13-positive endosomes. These effects occurred concomitantly with delayed mannose 6-phosphate receptor-mediated cathepsin D delivery and degradation of internalized epidermal growth factor receptors. Our findings establish that SPE-39 proteins are a previously unrecognized regulator of lysosomal delivery and that C. elegans spermatogenesis is an experimental system useful for identifying conserved regulators of metazoan lysosomal biogenesis.

spe-10 encodes a DHHC-CRD zinc-finger membrane protein required for endoplasmic reticulum/Golgi membrane morphogenesis during Caenorhabditis elegans spermatogenesis.

C. elegans spermatogenesis employs lysosome-related fibrous body-membranous organelles (FB-MOs) for transport of many cellular components. Previous work showed that spe-10 mutants contain FB-MOs that prematurely disassemble, resulting in defective transport of FB components into developing spermatids. Consequently, spe-10 spermatids are smaller than wild type and contain defective FB-MO derivatives. In this article, we show that spe-10 encodes a four-pass integral membrane protein that has a DHHC-CRD zinc-finger motif. The DHHC-CRD motif is found in a large, diverse family of proteins that have been implicated in palmitoyl transfer during protein lipidation. Seven spe-10 mutants were analyzed, including missense, nonsense, and deletion mutants. An antiserum to SPE-10 showed significant colocalization with a known marker for the FB-MOs during wild-type spermatogenesis. In contrast, the spe-10(ok1149) deletion mutant lacked detectable SPE-10 staining; this mutant lacks a spe-10 promoter and most coding sequence. The spe-10(eb64) missense mutation, which changes a conserved residue within the DHHC-CRD domain in all homologues, behaves as a null mutant. These results suggest that wild-type SPE-10 is required for the MO to properly deliver the FB to the C. elegans spermatid and the DHHC-CRD domain is essential for this function.

Spermatogenesis.

Spermatogenesis creates functional sperm from an initially undifferentiated germ cell. In the nematode Caenorhabditis elegans, both males and hermaphrodites engage in spermatogenesis. The hermaphrodite germ line, like that of the male, initiates spermatogenesis during the L4 larval stage. The hermaphrodite germ line differs from that of the male because it ceases spermatogenesis and switches to oogenesis during the adult stage. Each hermaphrodite stores her sperm and uses them to fertilize her oocytes. Many mutants have been identified where hermaphrodite self-fertility is disrupted. If such a self-sterile hermaphrodite is mated to a wild-type male, mutant hermaphrodites that either lack sperm or contain defective sperm will produce outcross progeny. Easily implemented tests are then applied to identify the subset of these mutants that produce defective sperm. Currently, more than 44 genes are known that are required for normal spermatogenesis. This chapter discusses the 25 best-understood genes that affect spermatogenesis and mutants are grouped based on the cellular structure or process that is affected. C. elegans spermatozoa lack an acrosome and a flagellum, which are organelles found in the spermatozoa produced by most other species. Like other nematodes, C. elegans spermatozoa move by crawling using a single pseudopod. Wild-type spermatogenesis and its defects in mutants can be studied in vivo because the animal is transparent and in vitro because a simple, chemically defined medium that supports development has been discovered. Unlike nearly all other C. elegans cells, homogeneous sperm can be obtained in sufficient quantities to permit biochemical analyses.

The spe-42 gene is required for sperm-egg interactions during C. elegans fertilization and encodes a sperm-specific transmembrane protein.

Fertilization, the union of sperm and egg to form a new organism, is a critical process that bridges generations. Although the cytological and physiological aspects of fertilization are relatively well understood, little is known about the molecular interactions that occur between gametes. C. elegans has emerged as a powerful system for the identification of genes that are necessary for fertilization. C. elegans spe-42 mutants are sterile, producing cytologically normal spermatozoa that fail to fertilize oocytes. Indeed, male mating behavior, sperm transfer to hermaphrodites, sperm migration to the spermatheca, which is the site of fertilization and sperm competition are normal in spe-42 mutants. spe-42 mutant sperm make direct contact with oocytes in the spermatheca, suggesting that SPE-42 plays a role during sperm-egg interactions just prior to fertilization. No other obvious defects were observed in spe-42 mutant worms. Cloning and sequence analysis revealed that SPE-42 is a novel predicted 7-pass integral membrane protein with homologs in many metazoan species, suggesting that its mechanism of action could be conserved.

Dynamic localization of SPE-9 in sperm: a protein required for sperm-oocyte interactions in Caenorhabditis elegans.

BACKGROUND: Fertilization in Caenorhabditis elegans requires functional SPE-9 protein in sperm. SPE-9 is a transmembrane protein with a predicted extracellular domain that contains ten epidermal growth factor (EGF)-like motifs. The presence of these EGF-like motifs suggests that SPE-9 is likely to function in gamete adhesive and/or ligand-receptor interactions. RESULTS: We obtained specific antisera directed against different regions of SPE-9 in order to determine its subcellular localization. SPE-9 is segregated to spermatids with a pattern that is consistent with localization to the plasma membrane. During spermiogenesis, SPE-9 becomes localized to spiky projections that coalesce to form a pseudopod. This leads to an accumulation of SPE-9 on the pseudopod of mature sperm. CONCLUSIONS: The wild type localization patterns of SPE-9 provide further evidence that like the sperm of other species, C. elegans sperm have molecularly mosaic and dynamic regions. SPE-9 is redistributed by what is likely to be a novel mechanism that is very fast (approximately 5 minutes) and is coincident with dramatic rearrangements in the major sperm protein cytoskeleton. We conclude that SPE-9 ends up in a location on mature sperm where it can function during fertilization and this localization defines the sperm region required for these interactions.