As Caenorhabditis elegans hermaphrodites age, sperm become depleted, ovulation arrests, and oocytes accumulate in the gonad arm. Large ribonucleoprotein (RNP) foci form in these arrested oocytes that contain RNA-binding proteins and translationally masked maternal mRNAs. Within 65 min of mating, the RNP foci dissociate and fertilization proceeds. The majority of arrested oocytes with foci result in viable embryos upon fertilization, suggesting that foci are not deleterious to oocyte function. We have determined that foci formation is not strictly a function of aging, and the somatic, ceh-18, branch of the major sperm protein pathway regulates the formation and dissociation of oocyte foci. Our hypothesis for the function of oocyte RNP foci is similar to the RNA-related functions of processing bodies (P bodies) and stress granules; here, we show three orthologs of P body proteins, DCP-2, CAR-1 and CGH-1, and two markers of stress granules, poly (A) binding protein (PABP) and TIA-1, appear to be present in the oocyte RNP foci. Our results are the first in vivo demonstration linking components of P bodies and stress granules in the germ line of a metazoan. Furthermore, our data demonstrate that formation of oocyte RNP foci is inducible in non-arrested oocytes by heat shock, osmotic stress, or anoxia, similar to the induction of stress granules in mammalian cells and P bodies in yeast. These data suggest commonalities between oocytes undergoing delayed fertilization and cells that are stressed environmentally, as to how they modulate mRNAs and regulate translation.

The germ cells, and germ cell precursors, in the nematode Caenorhabditis elegans contain distinctive granules called P granules. During early embryogenesis, P granules are segregated asymmetrically into those blastomeres that eventually produce the germ line. Because of the correlation between P granule distribution and the development of the germ line, P granules are widely thought to function in some aspect of germ line specification or differentiation. Most of the analysis of P granule structure and localization has focused on the early embryo, when P granules are located in the cytoplasm. However, during most of development P granules are associated with germ cell nuclei. We report here an ultrastructural analysis of the nuclear-associated P granules in the germ cells of the adult hermaphrodite gonad. We show that P granules are tightly associated with nuclear pores and that the positions of certain structures within the P granules correspond to the positions of pores on the nuclear envelope. We present immunocytochemical and ultrastructural data suggesting that P granules can associate, or remain associated, with pore-like structures even after they detach from the nuclear envelope during oogenesis. Finally, we show that nuclear-associated P granules in the gonad contain RNA, complementing previous studies showing that cytoplasmic P granules in embryos contain RNA.

Protective cellular responses to stress and aging in the germ line are essential for perpetuation of a species; however, relatively few studies have focused on how germ cells respond to stress and aging. We have previously shown that large ribonucleoprotein (RNP) complexes assemble in oocytes of Caenorhabditis during extended meiotic arrest or after environmental stress. Here we explore the regulation of these dynamic RNPs and demonstrate their assembly is coordinated with dramatic, nuclear membrane blebbing in oocytes. Our ultrastructural analyses reveal distinct changes in the endoplasmic reticulum, and the first evidence for the assembly of stacked annulate lamellae in Caenorhabditis. We further show several nucleoporins are required for the complete assembly of RNP granules, and a disruption in RNP granule assembly coupled with a low frequency of nuclear blebbing in arrested oocytes negatively impacts embryonic viability. Our observations support a model where nuclear membrane blebbing is required to increase the trafficking of nucleoporins to the cell cortex in stressed or meiotically-arrested cells and to facilitate the recruitment of RNA and protein components of RNPs into large complexes. These new insights may have general implications for better understanding how germ cells preserve their integrity when fertilization is delayed and how cells respond to stress.

In a variety of cell types in plants, animals, and fungi, ribonucleoprotein (RNP) complexes play critical roles in regulating RNA metabolism. These RNP granules include processing bodies and stress granules that are found broadly across cell types, as well as RNP granules unique to the germline, such as P granules, polar granules, sponge bodies, and germinal granules. This review focuses on RNP granules localized in oocytes of the major model systems, Caenorhabditis elegans, Drosophila, Xenopus, mouse, and zebrafish. The signature families of proteins within oocyte RNPs include Vasa and other RNA-binding proteins, decapping activators and enzymes, Argonaute family proteins, and translation initiation complex proteins. This review describes the many recent insights into the dynamics and functions of RNP granules, including their roles in mRNA degradation, mRNA localization, translational regulation, and fertility. The roles of the cytoskeleton and cell organelles in regulating RNP granule assembly are also discussed.

Within the rhabditid phylogeny of nematodes, the great majority of species are gonochoristic, having evolved as obligate male/female species. In contrast, the well-studied nematode model system, Caenorhabditis elegans, is androdioecious, utilizing a hermaphroditic/male reproductive system. We have previously determined that in the arrested oocytes of old-aged C. elegans hermaphrodites with depleted sperm, large cytoplasmic ribonucleoprotein foci form. The formation of these foci is reversible, as they dissociate within 3 h after a male mates with the hermaphrodite, resupplying it with sperm. The functional significance of these oocyte foci is not known and previously has not been clear for a hermaphroditic species in which oocytes of young adults wait only approximately 23 min to be fertilized. One hypothesis is that the foci function to maintain maternal mRNAs in oocytes while fertilization is delayed. In this paper, we examine four gonochoristic rhabditid species: Caenorhabditis remanei, Caenorhabditis sp. CB5161, Caenorhabditis sp. PS1010, and Rhabditella axei DF5006. We demonstrate that in three of these four species, ovulation arrests in unmated females until mating occurs and large cytoplasmic foci develop in arrested oocytes. The oocyte foci contain nuclear pore proteins and, in C. remanei at least, the RNA-binding protein MEX-3 as well as RNA. We speculate that these foci maintain the integrity of ooctyes, possibly maintaining the stability or translational repression of maternal mRNAs in unmated females. We further speculate that their presence in oocytes of old-aged C. elegans hermaphrodites is due to conservation from an ancestral gonochoristic state.

P granules, ribonucleoprotein (RNP) complexes specific to the cytoplasmic side of the nuclear pores of C. elegans germ cells, are implicated in post-transcriptional control of maternally-transcribed mRNAs. Here we show a relationship in C. elegans of Dicer, the riboendonuclease processing enzyme of the RNA interference and microRNA pathways, with GLH-1, a germline-specific RNA helicase and a constitutive component of P granules. Based on results from GST-pull-downs and immunoprecipitations, GLH-1 binds DCR-1 and this binding does not require RNA. Both GLH-1 protein and glh-1 mRNA levels are reduced in the dcr-1(ok247) null mutant background; conversely, a reduction of DCR-1 protein is observed in the glh-1(gk100) deletion strain. Thus, in the C. elegans germline, DCR-1 and GLH-1 are interdependent. In addition, evidence indicates DCR-1 protein levels, like those of GLH-1, are likely regulated by the Jun N-terminal kinase (JNK), KGB-1. In adult germ cells, DCR-1 is found in uniformly-distributed, small puncta both throughout the cytoplasm and the nucleus, on the inner side of nuclear pores, and associated with P granules. In arrested oocytes, GLH-1 and DCR-1 re-localize to cytoplasmic and cortically-distributed RNP granules and are necessary to recruit other components to these complexes. We predict the GLH-1/DCR-1 complex may function in the transport, deposition, or regulation of maternally-transcribed mRNAs and their associated miRNAs.

SummaryLarge cytoplasmic ribonucleoprotein germ granule complexes are a common feature in germ cells. In C. elegans these are called P granules and for much of the life-cycle they associate with nuclear pore complexes in germ cells. P granules are rich in proteins that function in diverse RNA pathways. Here we report that the C. elegans homolog of the eIF4E-transporter IFET-1 is required for oogenesis but not spermatogenesis. We show that IFET-1 is required for translational repression of several maternal mRNAs in the distal gonad and functions in conjunction with the broad-scale translational regulators CGH-1, CAR-1 and PATR-1 to regulate germ cell sex determination. Furthermore we have found that IFET-1 localizes to P granules throughout the gonad and in the germ cell lineage in the embryo. Interestingly, IFET-1 is required for the normal ultrastructure of P granules and for the localization of CGH-1 and CAR-1 to P granules. Our findings suggest that IFET-1 is a key translational regulator and is required for normal P granule formation.

P granules are cytoplasmic structures of unknown function that are associated with germ nuclei in the C. elegans gonad, and are localized exclusively to germ cells, or germ cell precursors, throughout the life cycle. All the known protein components of P granules contain putative RNA-binding motifs, suggesting that RNA is involved in either the structure or function of the granules. However, no specific mRNAs have been identified within P granules in the gonad. We show here that P granules normally contain a low level of RNA, and describe conditions that increase this level. We present evidence that several, diverse mRNAs, including pos-1, mex-1, par-3, skn-1, nos-2 and gld-1 mRNA, are present at least transiently within P granules. In contrast, actin and tubulin mRNA and rRNA are either not present in P granules, or are present at relatively low levels. We show that pgl-1 and the glh (Vasa-related) gene family, which encode protein components of P granules, do not appear essential for RNA to concentrate in P granules; these proteins may instead function in events that are a prerequisite for RNAs to be transported efficiently from the nuclear surface.