SACY-1 DEAD-Box Helicase Links the Somatic Control of Oocyte Meiotic Maturation to the Sperm-to-Oocyte Switch and Gamete Maintenance in Caenorhabditis elegans

In sexually reproducing animals, oocytes arrest at diplotene or diakinesis and resume meiosis (meiotic maturation) in response to hormones. In Caenorhabditis elegans, major sperm protein triggers meiotic resumption through a mechanism involving somatic Gα_s–adenylate cyclase signaling and soma-to-germline gap-junctional communication. Using genetic mosaic analysis, we show that the major effector of Gα_s–adenylate cyclase signaling, protein kinase A (PKA), is required in gonadal sheath cells for oocyte meiotic maturation and dispensable in the germ line. This result rules out a model in which cyclic nucleotides must transit through sheath-oocyte gap junctions to activate PKA in the germ line, as proposed in vertebrate systems. We conducted a genetic screen to identify regulators of oocyte meiotic maturation functioning downstream of Gα_s–adenylate cyclase–PKA signaling. We molecularly identified 10 regulatory loci, which include essential and nonessential factors. sacy-1, which encodes a highly conserved DEAD-box helicase, is an essential germline factor that negatively regulates meiotic maturation. SACY-1 is a multifunctional protein that establishes a mechanistic link connecting the somatic control of meiotic maturation to germline sex determination and gamete maintenance. Modulatory factors include multiple subunits of a CoREST-like complex and the TWK-1 two-pore potassium channel. These factors are not absolutely required for meiotic maturation or its negative regulation in the absence of sperm, but function cumulatively to enable somatic control of meiotic maturation. This work provides insights into the genetic control of meiotic maturation signaling in C. elegans, and the conserved factors identified here might inform analysis in other systems through either homology or analogy.     

Regulated trafficking of the MSP/Eph receptor during oocyte meiotic maturation in C. elegans

BACKGROUND: In C. elegans, a sperm-sensing mechanism regulates oocyte meiotic maturation and ovulation, tightly coordinating sperm availability and embryo production; sperm release the major sperm protein (MSP) signal to trigger meiotic resumption. Meiotic arrest depends on the parallel function of the oocyte VAB-1 MSP/Eph receptor and somatic G protein signaling. MSP promotes meiotic maturation by antagonizing Eph receptor signaling and counteracting inhibitory inputs from the gonadal sheath cells. RESULTS: Here, we present evidence suggesting that in the absence of the MSP ligand, the VAB-1 Eph receptor inhibits meiotic maturation while either in or in transit to the endocytic-recycling compartment. VAB-1::GFP localization to the RAB-11-positive endocytic-recycling compartment is independent of ephrins but is antagonized by MSP signaling. Two negative regulators of oocyte meiotic maturation, DAB-1/Disabled and RAN-1, interact with the VAB-1 receptor and are required for its accumulation in the endocytic-recycling compartment in the absence of MSP or sperm (hereafter referred to as MSP/sperm). Inactivation of the endosomal recycling regulators rme-1 or rab-11.1 causes a vab-1-dependent reduction in the meiotic-maturation rate in the presence of MSP/sperm. Further, we show that Galpha(s) signaling in the gonadal sheath cells, which is required for meiotic maturation in the presence of MSP/sperm, affects VAB-1::GFP trafficking in oocytes. CONCLUSIONS: Regulated endocytic trafficking of the VAB-1 MSP/Eph receptor contributes to the control of oocyte meiotic maturation in C. elegans. Eph receptor trafficking in other systems may be influenced by the conserved proteins DAB-1/Disabled and RAN-1 and by crosstalk with G protein signaling in neighboring cells.     

Trafficking of GFP-tagged Delta F508-CFTR to the plasma membrane in a polarized epithelial cell line

The F508 mutationreduces the amount of cystic fibrosis transmembrane conductanceregulator (CFTR) expressed in the plasma membrane of epithelial cells.However, a reduced temperature, butyrate compounds, and "chemicalchaperones" allow F508-CFTR to traffic to the plasma membrane andincrease Cl permeability in heterologous and nonpolarizedcells. Because trafficking is affected by the polarized state ofepithelial cells and is cell-type dependent, our goal was to determinewhether these maneuvers induce F508-CFTR trafficking to the apicalplasma membrane in polarized epithelial cells. To this end, wegenerated and characterized a line of polarized Madin-Darby caninekidney (MDCK) cells stably expressing F508-CFTR tagged with greenfluorescent protein (GFP). A reduced temperature, glycerol, butyrate,or DMSO had no effect on 8-(4-chlorophenylthio)-cAMP(CPT-cAMP)-stimulated transepithelial Cl secretion acrosspolarized monolayers. However, when the basolateral membrane waspermeabilized, butyrate, but not the other experimental maneuvers,increased the CPT-cAMP-stimulated Cl current across theapical plasma membrane. Thus butyrate increased the amount offunctional F508-CFTR in the apical plasma membrane. Butyrate failedto stimulate transepithelial Cl secretion because ofinhibitory effects on Cl uptake across the basolateralmembrane. These observations suggest that studies on heterologous andnonpolarized cells should be interpreted cautiously. The GFP tag onF508-CFTR will allow investigation of F508-CFTR trafficking inliving, polarized MDCK epithelial cells in real time.

     

T-type calcium currents in rat cardiomyocytes during postnatal development: contribution to hormone secretion

T-type Ca(2+) channels have been suggested to play a role in cardiac automaticity, cell growth, and cardiovascular remodeling. Although three genes encoding for a T-type Ca(2+) channel have been identified, the nature of the isoform(s) supporting the cardiac T-type Ca(2+) current (I(Ca,T)) has not yet been determined. We describe the postnatal evolution of I(Ca,T) density in freshly dissociated rat atrial and ventricular myocytes and its functional properties at peak current density in young atrial myocytes. I(Ca,T) displays a classical low activation threshold, rapid inactivation kinetics, negative steady-state inactivation, slow deactivation, and the presence of a window current. Interestingly, I(Ca,T) is poorly sensitive to Ni(2+) and insensitive to R-type current toxin SNX-482. RT-PCR experiments and comparison of functional properties with recombinant Ca(2+) channel subtypes suggest that neonatal I(Ca,T) is related to the alpha(1G)-subunit. Atrial natriuretic factor (ANF) secretion was measured using peptide radioimmunoassays in atrial tissue. Pharmacological dissection of ANF secretion indicates an important contribution of I(Ca,T) to Ca(2+) signaling during the neonatal period.     

Characterization of a microtubule assembly inhibitor from Xenopus oocytes

The dynamic properties of microtubules (MTs) are important for a wide variety of cellular processes, including cell division and morphogenesis. MT assembly and disassembly in vivo are regulated by cellular factors that influence specific parameters of MT dynamics. Here, we describe the characterization of a previously reported MT assembly inhibitor activity from Xenopus oocytes [Gard and Kirschner, 1987: J. Cell Biol. 105:2191-2201]. Video microscopy measurements reveal that the inhibitor specifically decreases the plus end growth rate of MTs and increases the critical concentration for tubulin. However, catastrophe frequency, rescue frequency, and shrinkage rates are not affected by the activity. Chromatography on Mono Q and hydroxyapatite columns has shown that the activity cofractionates with a subpopulation of tubulin. This tubulin subpopulation and the MT assembly inhibitor activity also co-migrate with a large S value (25-30S) on sucrose gradients. The high molecular weight tubulin complex and the MT assembly inhibitor activity are both developmentally regulated and disappear after oocyte maturation with progesterone.     

Sesquiterpene lactones of eupatorium anomalum and eupatorium mohrii

Several new guaianolides and the previously known heliangolide eurecurvin have been isolated from Eupatorium anomalum. Eupatorium mohrii also yielded three of the new guaianolides together with eurecurvin and a new germacradienolide. The implications of these findings are discussed.     

Are opioid peptides co-localized with vasopressin or oxytocin in the neural lobe of the rat?

Summary The content of vasopressin, oxytocin, neurophysin, leucine-enkephalin, methionine-enkephalin, dynorphin-(1–13), and -neoendorphin in the rat neurohypophysis was measured after different periods of dehydration and after depolarisation of isolated neural lobes and of neurosecretory nerve endings. The rates at which the amount of neurohypophysial hormone and opioid peptides decreased, and the changes in the ratios between the amount of vasopressin or oxytocin and opioid peptide in the neurohypophysis after dehydration and in the incubation medium after depolarization in vitro cast some doubt on, and can be explained by mechanisms other than co-localisation of the different peptides.     

Immunological similarity between the chick oviduct progesterone receptor forms A and B

A large-scale purification of the progesterone receptor from laying hens is described which yields apparently homogeneous form A and form B receptor in denatured form. The purification procedure is based initially on differential DNA affinity chromatography of both form A and B receptors. Under the conditions of preparation and activation described, progesterone receptor form B binds to DNA-cellulose even in the presence of 100 mM salt. This binding cannot be observed after thermal activation. Receptors obtained at 5% purity using conventional chromatographic purification steps were covalently cross-linked with radioactive ligand by photoaffinity labeling and purified to homogeneity using preparative gel electrophoresis systems under denaturing conditions. This material has been successfully used to generate polyclonal antibodies in rabbits. Immunoblots demonstrated a high degree of cross-reaction between anti-A antibodies and progesterone receptor form B, as well as between anti-B antibodies and progesterone receptor form A, using homogeneous as well as 5% pure receptors as probes. Implications of the immunological data and the novel DNA-binding characteristics of form B are discussed with respect to topological conformation of the progesterone receptor and the structural similarity between forms A and B.     

Juvenile hormone acid methyltransferase activity in imaginal discs of Manduca sexta prepupae

The occurrence of a peak of juvenile hormone (JH) during the prepupal period has been noted in several lepidopterans. In Manduca sexta and Hyalophora cecropia this peak is known to prevent the precocious onset of adult differentiation in imaginal tissues. However, it has previously been observed in our laboratory that corpora allata (CA) of this age are incapable of making JH owing to a lack of the terminal synthetic enzyme, juvenile hormone acid methyltransferase (JHAMT). Since the CA are required for normal pupation, it is likely that JH acid is the product released by the prepupal CA. Therefore, we analyzed whether JH acid treatment would prevent precocious adultoid differentiation in allatectomized M. sexta larvae. JH acid injections were found to be as effective as JH in normalizing pupation, and acted in a time- and dose-dependent manner. This finding led to a question of whether injected or endogenous JH acid could be methylated to JH. Homogenates of several tissues from prepupae were assayed for the presence of JHAMT. Of the tissues assayed, only imaginal discs possessed significant levels of the enzyme. These results support our previously proposed mechanism for production of the prepupal JH peak in M. sexta.