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.

A motor independent requirement for dynein light chain in Caenorhabditis elegans meiotic synapsis.

The dynein motor complex is thought to aid in homolog pairing in many organisms by moving chromosomes within the nuclear periphery to promote and test homologous interactions. This precedes synaptonemal complex (SC) formation during homolog synapsis, which stabilizes homolog proximity during recombination. We observed that depletion of the dynein light chain (DLC-1) in Caenorhabditis elegans irreversibly prevents synapsis, causing an increase in off-chromatin formation of SC protein foci with increasing temperature. This requirement for DLC-1 is independent of its function in dynein motors, as SYP protein foci do not form with depletion of other dynein motor components. In contrast to normal SC-related structures, foci formed with DLC-1 depletion are resistant to dissolution with 1,6-hexanediol, similar to aggregates of SC proteins formed in high growth temperatures. Dynein light chains have been shown to act as hub proteins that interact with other proteins through a conserved binding motif. We identified a similar DLC-1 binding motif in the C. elegans SC protein SYP-2, and mutation of the putative motif causes meiosis defects that are exacerbated by elevated temperatures. We propose that DLC-1 acts as a pre-synapsis chaperone-like factor for SYP proteins to help regulate their self-association prior to the signals for SC assembly, a role that is revealed by its increased essentiality at elevated temperatures.

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.

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.

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.

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.

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.