Metaphase to anaphase (mat) transition-defective mutants in Caenorhabditis elegans.

The metaphase to anaphase transition is a critical stage of the eukaryotic cell cycle, and, thus, it is highly regulated. Errors during this transition can lead to chromosome segregation defects and death of the organism. In genetic screens for temperature-sensitive maternal effect embryonic lethal (Mel) mutants, we have identified 32 mutants in the nematode Caenorhabditis elegans in which fertilized embryos arrest as one-cell embryos. In these mutant embryos, the oocyte chromosomes arrest in metaphase of meiosis I without transitioning to anaphase or producing polar bodies. An additional block in M phase exit is evidenced by the failure to form pronuclei and the persistence of phosphohistone H3 and MPM-2 antibody staining. Spermatocyte meiosis is also perturbed; primary spermatocytes arrest in metaphase of meiosis I and fail to produce secondary spermatocytes. Analogous mitotic defects cause M phase delays in mitotic germline proliferation. We have named this class of mutants "mat" for metaphase to anaphase transition defective. These mutants, representing six different complementation groups, all map near genes that encode subunits of the anaphase promoting complex or cyclosome, and, here, we show that one of the genes, emb-27, encodes the C. elegans CDC16 ortholog.

Anucleate Caenorhabditis elegans sperm can crawl, fertilize oocytes and direct anterior-posterior polarization of the 1-cell embryo.

It has long been appreciated that spermiogenesis, the cellular transformation of sessile spermatids into motile spermatozoa, occurs in the absence of new DNA transcription. However, few studies have addressed whether the physical presence of a sperm nucleus is required either during spermiogenesis or for subsequent sperm functions during egg activation and early zygotic development. To determine the role of the sperm nucleus in these processes, we analyzed two C. elegans mutants whose spermatids lack DNA. Here we show that these anucleate sperm not only differentiate into mature functional spermatozoa, but they also crawl toward and fertilize oocytes. Furthermore, we show that these anucleate sperm induce both normal egg activation and anterior-posterior polarity in the 1-cell C. elegans embryo. The latter finding demonstrates for the first time that although the anterior-posterior embryonic axis in C. elegans is specified by sperm, the sperm pronucleus itself is not required. Also unaffected is the completion of oocyte meiosis, formation of an impermeable eggshell, migration of the oocyte pronucleus, and the separation and expansion of the sperm-contributed centrosomes. Our investigation of these mutants confirms that, in C. elegans, neither the sperm chromatin mass nor a sperm pronucleus is required for spermiogenesis, proper egg activation, or the induction of anterior-posterior polarity.

Expression patterns and transcript processing of ftt-1 and ftt-2, two C. elegans 14-3-3 homologues.

A wide diversity of biological functions have been attributed to the highly conserved and ubiquitous 14-3-3 protein family. Yet how much of this diversity is inherent in the basic structure of 14-3-3 and how much is due to isoform specific functions is not yet fully understood. Here, two Caenorhabditis elegans 14-3-3 isoforms whose protein sequences are 90% similar were found to differ significantly in both their genomic structure and expression patterns. The two genes, ftt-1 (IV) (fourteen-three-three) and ftt-2 (X), differ in both the position and sequence of their introns. Since the various intron/exon boundaries respect neither functional nor structural protein motifs, the introns appear to be relatively recent evolutionary additions. ftt-1(IV) encodes three germline enhanced transcripts, two of which are related through the differential use of alternative poly(A) addition sites. RNA in situ hybridization studies reveal high levels of ftt-1 throughout the gonad with particularly high levels in the distal arm. In contrast, ftt-2 (X) encodes a single transcript which is expressed somatically. In embryos, high levels of ftt-1 transcripts appear to be maternally supplied, whereas ftt-2 is expressed as an early zygotic transcript whose expression pattern later localizes to the posterior region of post-proliferative embryos. These expression pattern differences between ftt-1 and ftt-2 suggest that these two 14-3-3 isoforms perform distinct biological roles within the worm.

Molecular evolution of the 14-3-3 protein family.

Members of the highly conserved and ubiquitous 14-3-3 protein family modulate a wide variety of cellular processes. To determine the evolutionary relationships among specific 14-3-3 proteins in different plant, animal, and fungal species and to initiate a predictive analysis of isoform-specific differences in light of the latest functional and structural studies of 14-3-3, multiple alignments were constructed from forty-six 14-3-3 sequences retrieved from the GenBank and SwissProt databases and a newly identified second 14-3-3 gene from Caenorhabditis elegans. The alignment revealed five highly conserved sequence blocks. Blocks 2-5 correlate well with the alpha helices 3, 5, 7, and 9 which form the proposed internal binding domain in the three-dimensional structure model of the functioning dimer. Amino acid differences within the functional and structural domains of plant and animal 14-3-3 proteins were identified which may account for functional diversity amongst isoforms. Protein phylogenic trees were constructed using both the maximum parsimony and neighbor joining methods of the PHYLIP(3.5c) package; 14-3-3 proteins from Entamoeba histolytica, an amitochondrial protozoa, were employed as an outgroup in our analysis. Epsilon isoforms from the animal lineage form a distinct grouping in both trees, which suggests an early divergence from the other animal isoforms. Epsilons were found to be more similar to yeast and plant isoforms than other animal isoforms at numerous amino acid positions, and thus epsilon may have retained functional characteristics of the ancestral protein. The known invertebrate proteins group with the nonepsilon mammalian isoforms. Most of the current 14-3-3 isoform diversity probably arose through independent duplication events after the divergence of the major eukaryotic kingdoms. Divergence of the seven mammalian isoforms beta, zeta, gamma, eta, epsilon, tau, and sigma (stratifin/HME1) occurred before the divergence of mammalian and perhaps before the divergence of vertebrate species. A possible ancestral 14-3-3 sequence is proposed.

An ECM-bound, PDGF-like growth factor and a TGF-alpha-like growth factor are required for gastrulation and spiculogenesis in the Lytechinus embryo.

Growth factors and the extracellular matrix have been shown to fulfill vital developmental roles in many embryonic systems. Our hypothesis is that a developmental role played by the extracellular matrix in sea urchins may be the binding of a PDGF-like growth factor to promote signaling activity. We report here that anti-human PDGF-B antibodies and anti-human TGF-alpha antibodies immunoprecipitated specific proteins isolated from Lytechinus embryos. Addition of these antibodies to Lytechinus embryos inhibited gastrulation and spiculogenesis. The embryos are sensitive to the antibodies from the four-cell through the hatching blastula stages, which suggests that the TGF-alpha-like and PDGF-like ligands are required for the early differentiation of the gut and spicules. We present evidence that the PDGF-like growth factor depends on the extracellular matrix for signaling activity. Synthetic peptides representing the heparan sulfate proteoglycan binding sequence on human PDGF-B were added to Lytechinus embryo cultures to compete for binding sites with the endogenous PDGF-like growth factor. The experimental peptide inhibited gastrulation and caused radially arranged multiple spicules to form. Development was unaffected by a control peptide. These studies support our hypothesis and suggest that TGF-alpha-like and PDGF-like growth factors induce signaling events required for sea urchin gastrulation and spiculogenesis and suggest that an extracellular matrix-associated PDGF-like growth factor is involved in differentiation along the oral-aboral axis.

Isolation and sequence analysis of a Caenorhabditis elegans cDNA which encodes a 14-3-3 homologue.

We report the cloning of the Caenorhabditis elegans homologue of a 14-3-3 protein-encoding gene which is located within the unc-22 gene cluster on chromosome IV. Sequence analysis reveals that the cDNA-encoded product is 78% identical to both the Drosophila melanogaster and bovine 14-3-3 proteins. Our cDNA hybridizes to at least three major transcripts of 1.5, 1.35 and 0.9 kb, which are all more abundant in fertile hermaphrodites than in those lacking germ cells.

A sperm-supplied product essential for initiation of normal embryogenesis in Caenorhabditis elegans is encoded by the paternal-effect embryonic-lethal gene, spe-11.

Loss-of-function mutations in the spe-11 gene in Caenorhabditis elegans result in a paternal-effect embryonic-lethal phenotype: fertilization of wild-type oocytes by sperm from homozygous spe-11 mutant males leads to abnormal zygotic development, whereas oocytes from homozygous spe-11 hermaphrodites when fertilized by wild-type sperm develop normally. Embryos fertilized by sperm from homozygous spe-11 worms fail to complete meiosis and show defects in eggshell formation, mitotic spindle orientation, and cytokinesis. Genetic analysis suggests that the spe-11 gene is expressed before the completion of spermatogenesis and that the wild-type locus encodes a product that is present in sperm and participates, directly or indirectly, in initiating the correct program of early events in C. elegans embryos. Such an ontogenetic role of the spe-11+ gene product in early embryogenesis distinguishes spe-11 mutations from the two paternal-effect mutations identified in Drosophila, ms(3)K81 and pal, which primarily affect chromosome behavior. Analysis of spe-11 provides the first step toward genetic dissection of the functions of the sperm in early embryogenesis in C. elegans.

Mutations that disrupt the morphogenesis and localization of a sperm-specific organelle in Caenorhabditis elegans.

Nematode sperm contain unusual organelles, membranous organelles, which undergo dramatic morphological changes during spermatogenesis. Early in spermatogenesis, the membranous organelle functions to transport sperm specific components to the spermatids; later, during the formation of the crawling spermatozoa, it adds new components to the cell surface as it fuses with the plasma membrane. Genetic analysis of spermatogenesis in the nematode Caenorhabditis elegans has revealed mutations that specifically disrupt the proper cellular localization and morphogenesis of this organelle. In animals homozygous for the either the known deficiency hcDf1 or the probable deficiency h12, the membranes of the membranous organelles are aberrantly covered with ribosomes. A mutation in the spermatogenesis-defective spe-10 gene causes severe defects in the morphogenesis of a fibrous body-membranous organelle complex. In both cases, these mutations also disrupt the proper localization of both nuclei and membranous organelles in haploid spermatids and spermatozoa.

Initiation of spermiogenesis in C. elegans: a pharmacological and genetic analysis.

Spermiogenesis in Caenorhabditis elegans involves the conversion of spherical, sessile spermatids into bipolar, crawling spermatozoa. In males, spermiogenesis is induced by mating, while in hermaphrodites, spermiogenesis occurs before the first oocytes are fertilized. Alternatively, spermiogenesis can be induced in vitro by treatment with monensin triethanolamine, or pronase. Treatment with the calmodulin inhibitors, trifluoperazine, chlorpromazine, or W7, also induces spermiogenesis in vitro with a half maximal effect at 20 microM. Upon initial activation, spermatids extend long, thin spikes and undergo extensive cellular movements. Eventually, a single motile pseudopod forms through the restructuring of one or more of these spikes. These transient spikes can be prolonged in vitro by removing triethanolamine as soon as the spermatids first form spikes. Spermatids from spe-8 and spe-12 spermatogenesis-defective (spe) mutants activate in vivo with male but not hermaphrodite sperm activator. In vitro, the mutant spermatids arrest spermiogenesis at the spike stage when activated with pronase, but form normal spermatozoa if subsequently or initially treated with monensin or triethanolamine. We present a model of spermiogenesis in which the mutant defects and the action of the pharmacological agents are ordered relative to one another.

Developmental genetics of chromosome I spermatogenesis-defective mutants in the nematode Caenorhabditis elegans.

Mutations affecting Caenorhabditis elegans spermatogenesis can be used to dissect the processes of meiosis and spermatozoan morphological maturation. We have obtained 23 new chromosome I mutations that affect spermatogenesis (spe mutations). These mutations, together with six previously described mutations, identify 11 complementation groups, of which six are defined by multiple alleles. These spe mutations are all recessive and cause normally self-fertile hermaphrodites to produce unfertilized oocytes that can be fertilized by wild-type male sperm. Five chromosome I mutation/deficiency heterozygotes have similar phenotypes to the homozygote showing that the probable null phenotype of these genes is defective sperm. Spermatogenesis is disrupted at different steps by mutations in these genes. The maturation of 1 degree spermatocytes is disrupted by mutations in spe-4 and spe-5. Spermatids from spe-8 and spe-12 mutants develop into normal spermatozoa in males, but not in hermaphrodites. fer-6 spermatids are abnormal, and fer-1 spermatids look normal but subsequently become abnormal spermatozoa. Mutations in five genes (fer-7, spe-9, spe-11, spe-13 and spe-15) allow formation of normal looking motile spermatozoa that appear to be defective in either sperm-spermathecal or sperm-oocyte interactions.

Circulating osteocalcin in rats is inversely responsive to changes in corticosterone.

Decreased bone formation in rats during spaceflight may be attributable to corticosteroid excess induced by environmental factors other than weightlessness that are associated with spaceflight experiments. To determine whether decreased osteocalcin, which may reflect altered bone formation rate, could be associated with corticosteroid excess, we measured serum osteocalcin in rats after injection of corticosterone or in response to various environmental stimuli. Exogenous steroid elicited a time- and dose-related decrease in serum osteocalcin, which was significant within 1 h of administration and maximally 25% below controls 1.5 h after injection of 3.3 mg corticosterone/kg body wt, the highest dose we tested. Adrenalectomy resulted in a 38% increase in osteocalcin. Exposure to environmental stressors lasting from 1.5 h to 3 wk also resulted in decreased osteocalcin levels, which showed a strong negative correlation (P less than 0.001) with serum corticosterone levels and adrenal mass after 1-3 wk of chronic cold exposure. Changes in serum osteocalcin were maximally about +/- 40% after 3 wk of chronic exposure to steroid excess or depletion. The response of osteocalcin to the well-defined adrenal hormone system implies an important role for corticosteroids in the control of serum osteocalcin.