RESUMEN
Fertilization, the fusion of sperm and oocyte to form a zygote, is the first and arguably the most important cell-cell interaction event in an organism's life. Forward and reverse genetic approaches in the nematode Caenorhabditis elegans have identified many genes that are required for gametogenesis and fertilization and thus are beginning to elucidate the molecular pathways that underlie these processes. We identified an allele of the spe-49 gene in a second filial generation (F2 ) mutagenesis screen for spermatogenesis-defective (spe) mutants. Mutant worms for spe-49 produce sperm that have normal morphology, activate to form ameboid spermatozoa, and can migrate to and maintain their position in the hermaphrodite reproductive tract but fail to fertilize oocytes. This phenotype puts spe-49 in the spe-9 class of late-acting genes that function in sperm at the time of fertilization. We cloned the spe-49 gene through a combination of deficiency mapping, transgenic rescue, and genomic sequencing. spe-49 messenger RNA (mRNA) is enriched in male germ cells, and the complementary DNA (cDNA) encodes a predicted 772-amino-acid six-pass transmembrane protein that is homologous to SPE-42. Indeed, SPE-49 and SPE-42 have identical predicted membrane topology and domain structure, including a large extracellular domain with six conserved cysteine residues, a DC-STAMP domain, and a C-terminal cytoplasmic domain containing a C4-C4 RING finger motif. The presence of two SPE-42 homologs in animal genomes from worms to humans suggests that these proteins are highly conserved components of the molecular apparatus required for the sperm-oocyte recognition, binding, and fusion.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Fertilización/genética , Proteínas de la Membrana/genética , Espermatozoides/metabolismo , Secuencia de Aminoácidos/genética , Animales , Secuencia de Bases , Clonación Molecular , Masculino , ARN Mensajero/genética , Análisis de Secuencia de ADNRESUMEN
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.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Receptores Notch/metabolismo , Espermatogénesis/genética , Ubiquitina-Proteína Ligasas/genética , Alelos , Animales , Proteínas de Caenorhabditis elegans/genética , Regulación del Desarrollo de la Expresión Génica , Mutación con Pérdida de Función , Mutación , Fenotipo , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
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.
Asunto(s)
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Vesículas Secretoras/metabolismo , Espermatogénesis/genética , Secuencia de Aminoácidos , Animales , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Expresión Génica , Masculino , Datos de Secuencia Molecular , Mutación , Transporte de Proteínas , Alineación de Secuencia , Espermatozoides/metabolismo , Testículo/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
The phenomenon of phase variation between yellow and tan forms of Myxococcus xanthus has been recognized for several decades, but it is not known what role this variation may play in the ecology of myxobacteria. We confirm an earlier report that tan variants are disproportionately more numerous in the resulting spore population of a M. xanthus fruiting body than the tan vegetative cells that contributed to fruiting body formation. However, we found that tan cells may not require yellow cells for fruiting body formation or starvation-induced sporulation of tan cells. Here we report three differences between the yellow and tan variants that may play important roles in the soil ecology of M. xanthus. Specifically, the yellow variant is more capable of forming biofilms, is more sensitive to lysozyme, and is more resistant to ingestion by bacteriophagous nematodes. We also show that the myxobacterial fruiting body is more resistant to predation by worms than are dispersed M. xanthus cells.
Asunto(s)
Caenorhabditis elegans/microbiología , Myxococcus xanthus/fisiología , Animales , Biopelículas/crecimiento & desarrollo , Glicerol/farmacología , Myxococcus xanthus/efectos de los fármacos , Myxococcus xanthus/crecimiento & desarrollo , Esporas Bacterianas/efectos de los fármacosRESUMEN
BACKGROUND: The C. elegans sperm protein SPE-42, a membrane protein of unknown structure and molecular function, is required for fertilization. Sperm from worms with spe-42 mutations appear normal but are unable to fertilize eggs. Sequence analysis revealed the presence of 8 conserved cysteine residues in the C-terminal cytoplasmic domain of this protein suggesting these residues form a zinc-coordinating RING finger structure. RESULTS: We made an in silico structural model of the SPE-42 RING finger domain based on primary sequence analysis and previously reported RING structures. To test the model, we created spe-42 transgenes coding for mutations in each of the 8 cysteine residues predicted to coordinate Zn++ ions in the RING finger motif. Transgenes were crossed into a spe-42 null background and protein function was measured by counting progeny. We found that all 8 cysteines are required for protein function. We also showed that sequence differences between the C-terminal 29 and 30 amino acids in C. elegans and C. briggsae SPE-42 following the RING finger domain are not responsible for the failure of the C. briggsae SPE-42 homolog to rescue C. elegans spe-42 mutants. CONCLUSIONS: The results suggest that a bona fide RING domain is present at the C-terminus of the SPE-42 protein and that this motif is required for sperm-egg interactions during C. elegans fertilization. Our structural model of the RING domain provides a starting point for further structure-function analysis of this critical region of the protein. The C-terminal domain swap experiment suggests that the incompatibility between the C. elegans and C. briggsae SPE-42 proteins is caused by small amino acid differences outside the C-terminal domain.
Asunto(s)
Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Dominios RING Finger , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/genética , Cisteína/química , Cisteína/metabolismo , Fertilización , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Dominios RING Finger/genética , Análisis de Secuencia de Proteína , Interacciones Espermatozoide-Óvulo , Relación Estructura-Actividad , Zinc/químicaRESUMEN
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.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Retículo Endoplásmico/fisiología , Aparato de Golgi/fisiología , Proteínas de la Membrana/fisiología , Morfogénesis , Espermatogénesis/fisiología , Dedos de Zinc , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/inmunología , Membrana Celular/metabolismo , Clonación Molecular , Inmunoglobulina G/inmunología , Masculino , Datos de Secuencia Molecular , Mutación Missense , Orgánulos , Fenotipo , Conejos , Homología de Secuencia de Aminoácido , EspermátidesRESUMEN
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.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Genes de Helminto , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Interacciones Espermatozoide-Óvulo/genética , Interacciones Espermatozoide-Óvulo/fisiología , Espermatozoides/fisiología , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Clonación Molecular , ADN de Helmintos/genética , Femenino , Fertilización , Masculino , Datos de Secuencia Molecular , Mutación , Fenotipo , Homología de Secuencia de AminoácidoRESUMEN
Transcription of the mouse testis-specific lactate dehydrogenase c (mldhc) gene is limited to cells of the germinal epithelium. Cloning and analysis of the mldhc promoter revealed that a 100-bp core promoter was able to regulate testis-specific transcription in vitro and in transgenic mice. Surprisingly, expression of the reporter in transgenic testes was limited to pachytene spermatocytes, whereas native LDH-C(4) was detected in pachytene and all later germ cells. To locate additional regulatory sequence that could recapitulate the native LDH-C(4) distribution pattern, we investigated the contribution of 5' and 3' flanking sequences to the regulation of LDH-C(4) expression. We found that transcription factor YY1 binds to the mldhc promoter, that the mldhc 3' untranslated sequence does not permit a postmeiotic expression of a beta-galactosidase reporter in transgenic mice, and that native mldhc mRNA is predominately meiotic, with only a low level of postmeiotic distribution. Our results suggest that the high level of LDH-C(4) in postmeiotic cells results from mRNA and protein stability.