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.

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.

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.

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.

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 (~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.

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.

The Caenorhabditis elegans spe-9 class genes, which show specific or predominant expression in the male germline, are indispensable for fertilization [ 1 and 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 and 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 and 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 and 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.

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 supportmajor spermprotein (MSP)- based cytoskeletal assembly in the spermatid that releases it, thereby facilitating sperm motility acquisition. Second, As-SRP-1 released froman activated sperminhibits, 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.

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 ~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.