The small GTPase Ran is essential for virtually all nucleocytoplasmic transport events. It is hypothesized that Ran drives vectorial transport of macromolecules into and out of the nucleus via the establishment of a Ran gradient between the cytoplasm and nucleoplasm. Although Ran shuttles between the nucleus and cytoplasm, it is concentrated in the nucleus at steady state. We show that nuclear transport factor 2 (NTF2) is required to concentrate Ran in the nucleus in the budding yeast, Saccharomyces cerevisiae. To analyze the mechanism of Ran import into the nucleus by NTF2, we use mutants in a variety of nuclear transport factors along with biochemical analyses of NTF2 complexes. We find that Ran remains concentrated in the nucleus when importin-mediated protein import is disrupted and demonstrate that NTF2 does not form a stable complex with the transport receptor, importin-β. Consistent with a critical role for NTF2 in establishing and maintaining the Ran gradient, we show that NTF2 is required for early embryogenesis inCaenorhabditis elegans. Our data distinguish between two possible mechanisms for Ran import by NTF2 and demonstrate that Ran import is independent from importin-β-mediated protein import.

Formation of zygotes in internally fertilizing organisms requires a number of successful interactions between oocytes and sperm within a receptive female reproductive tract. These interactions are usually assumed to be species-specific. For most species, it is either not possible to inseminate females with sperm from a different species or not possible to observe the consequences of such an insemination because the female is opaque. Nematodes of the genus Caenorhabditis are optically transparent and prior work indicates copulation between individuals of two different species is possible. We have used a series of vital stains and other cytological methods to analyze sperm after cross-species mating. We present here a series of analyses of the postcopulatory, prefertilization interactions among three Caenorhabditis species and find that reproductive biology is conserved, to varying degrees, among all three species. This approach allows investigation into which in vivo interactions between sperm and both oocytes and the somatic gonad have been maintained during the reproductive isolation that accompanies speciation.

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.

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.

The nematode Caenorhabditis elegans is an excellent model animal to study various biological phenomena, including reproduction. In this chapter, we focus on functional roles of spermatogenesis- or sperm-defective ( spe ) genes in the C. elegans male germline during reproduction. So far, approximately 190 mutants of C. elegans that are defective in male germline functions have been isolated, and many of them carry mutated alleles for one of the perhaps 60 spe genes. Most spe genes exhibit male germline-specific expression and play roles during spermatogenesis (spermatid production), spermiogenesis (spermatid activation into sperm), or fertilization. For example, spe - 8 class genes are indispensable for hermaphroditedependent spermiogenesis. If either of the spe - 8 class genes is aberrant, spermatids from mutant hermaphrodites, but not from males, arrest at an intermediate stage during spermiogenesis. In contrast, fertilization requires spe - 9 class genes. Hermaphrodites and males of spe - 9 class mutants produce otherwise normal sperm that are incapable of fertilizing oocytes. Because C . elegans oocytes have no egg coats, spe - 9 class genes are probably required for sperm to bind to and/or fuse with the oocyte plasma membrane. Intriguingly, several spe genes are likely to be orthologues of mammalian genes, suggesting that C. elegans and mammals share some common steps during male germline functions at the molecular level.

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.

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.