Polyphosphoinositide breakdown and subsequent exocytosis in the Ca2+/ionophore-induced acrosome reaction of mammalian spermatozoa.

An investigation was made of the modifications in phospholipids that occur during the exocytotic event known as the 'sperm acrosome reaction'. Phospholipids were prelabelled with 32P, and exocytosis was induced with Ca2+ and the ionophore A23187. When incubated with [32P]Pi in various media suitable for supporting sperm survival or fertilization in vitro, spermatozoa from all five species examined (ram, boar, guinea pig, mouse and human) incorporated 32P rapidly into the components of the phosphoinositide cycle. There were differences both between species and between media with respect to the actual rate of incorporation of label, and also between species with respect to other phospholipids labelled. Treatment of spermatozoa with Ca2+ and A23187 to induce the acrosome reaction resulted in a rapid breakdown of phosphatidylinositol 4, 5-bisphosphate and phosphatidylinositol 4-phosphate, which was complete within 3 min; there was also a great increase in labelling of phosphatidate. Occurrence of acrosome reactions in the sperm population was only observed after 5-10 min and reached a maximum response of greater than 90% after more than 30 min. The phosphoinositide breakdown was related to subsequent exocytosis: after EGTA/ionophore treatment, neither inositide breakdown nor exocytosis took place; however, later addition of Ca2+ resulted in immediate inositide breakdown, and exocytosis followed, with a delay relative to Ca2+ addition exactly similar to that following standard Ca2+/ionophore treatment. Neomycin inhibited both inositide breakdown and subsequent exocytosis provided it was added together with Ca2+ and ionophore; however, if the drug was added 3 min after Ca2+ and ionophore (by which time inositide breakdown was already complete), exocytosis was not inhibited. Ca2+ seemed to have several consecutive roles in the acrosome reaction. Low (micromolar) levels of free Ca2+ were needed both for phosphoinositide breakdown and for an event downstream of this breakdown; no other bivalent cation could substitute for Ca2+ in either event, and inositide breakdown was actually inhibited by Mg2+. In addition, millimolar levels of Ca2+ were needed for later stages of exocytosis, although this requirement could be satisfied by Sr2+. We conclude that breakdown of polyphosphoinositides is an essential early process after Ca2+ entry in the chain of events that lead to exocytosis in the mammalian sperm acrosome reaction.     

Effect of Ca2+ channel antagonists on the acrosome reaction of guinea pig and golden hamster spermatozoa

The effects of Ca²⁺ channel antagonists on the motility and acrosome reaction of guinea pig spermatozoa were examined by incubating the spermatozoa continuously in Ca²⁺-containing capacitating media with 10^?6 M to 10^?4 M antagonist. Antagonists tested were four voltage-gated Ca²⁺ channel antagonists (verapamil, nifedipine, nimodipine, and FR–34235) and two ligand-gated channel antagonists (NaNO₂ and Na-nitroprusside). None of these antagonists could block the acrosome reaction. Instead, three antagonists (verapamil, nimodipine, and FR-34235, each at 10^?4 M) accelerated the onset of the acrosome reaction with a subsequent decrease in sperm motility. Nifedipine and Na-nitroprusside at the same concentration caused a complete loss of sperm motility by 4 hr of incubation with no substantial effect on the rate of acrosome reaction. The detrimental effect of antagonists on the motility of spermatozoa appears to be due to a direct, Ca²⁺-independent, membrane-perturbing action of the reagents. The acrosome reaction was not inhibited when guinea pig spermatozoa were precapacitated in Ca²⁺-free medium (with a low concentration of lysolecithin) in the continuous presence of antagonists. An acceleration of the onset of the acrosome reaction by verapamil (10^?4 M) was also demonstrated in the golden hamster. These results may be interpreted as indicating that the entry of extracellular Ca²⁺ into spermatozoa, which triggers the acrosome reaction of guinea pig and hamster spermatozoa, is not mediated by Ca²⁺ channels. This is in marked contrast with the case reported in invertebrate spermatozoa. Possible mechanisms by which some of the antagonists stimulate the acrosome reaction and affect the motility of mammalian spermatozoa are discussed.     

Occurrence of Only One Form of Glutamine Synthetase in the Green Alga Monoraphidium braunii

Anion-exchange chromatography of crude extracts from the green alga Monoraphidium braunii yielded two glutamine synthetase (GS) activities. The ratio of activities was markedly different when crude extracts were subjected to various processing conditions but was not influenced by environmental factors of cell cultures. However, high performance liquid chromatography anion-exchange chromatograms showed only one GS if the crude extracts were processed immediately after cell disruption. Moreover, standard chromatography of crude extracts obtained in the absence of dithioerythritol, a reductant generally used in disruption buffers, yielded a single activity peak. Enzyme samples from the two activities obtained in the presence of dithioerythritol were purified for physicochemical characterization and antibody production. Both enzyme samples exhibited similar reactions to different inactivating agents and were undistinguishable by size-exclusion chromatography and native polyacrylamide gel electrophoresis. Additionally, the two GS preparations showed absolute antigenic identity as demonstrated by immunodiffusion and immunoblotting experiments. Immunocytochemistry of M. braunii cryosections evidenced a chloroplast-specific distribution of the enzyme, which rules out the existence of a cytoplasmic counterpart. All these results support the proposal that M. braunii possesses only one form of GS.     

Effects of Boron on Proton Transport and Membrane Properties of Sunflower (Helianthus annuus L.) Cell Microsomes

Boron deficiency and toxicity inhibit ATP-dependent H+ pumping and vanadate-sensitive ATPase activity in sunflower roots and cell suspensions. The effects of boron on H+ pumping and on passive H+ conductance, as well as on fluorescence anisotropy in KI-washed microsomes isolated from sunflower (Helianthus annuus L. cv Enano) cell suspensions, have been investigated. Boron deficiency reduced the total and vanadate-sensitive ATPase activities as well as the vanadate-sensitive ATP-dependent H+ pumping without affecting the amount of antigenic ATPase protein as measured by immunoblotting with an Arabidopsis thaliana plasma membrane anti-H+-ATPase polyclonal antibody. Kinetic studies revealed that boron deficiency reduced Vmax of vanadate-sensitive ATPase activity with little change in the apparent Km for Mg2+-ATP. Proton leakage was greater in microsomal vesicles isolated from cells grown without boron and incubated in reaction medium without added boron, and this effect was reversed by addition of boron to the reaction medium. Fluorescence anisotropy indicated that diphenyl hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene probes were immobilized to a greater extent in microsomes from cells grown without boron than in those from cells grown with 100 [mu]M H3BO3. The apparent decrease of membrane fluidity in microsomes from cells grown without boron was reversed by the addition of boron to the reaction medium. Taken together these data suggest that inhibition of H+ gradient formation in microsomes from sunflower cells grown in the absence of boron could be due to the combined effects of reduced H+-ATPase activity and increased passive conductance across the membrane, possibly resulting from increased membrane rigidity.     

Is the hook of muroid rodent's sperm related to sperm train formation?

Competition between spermatozoa of rival males to gain fertilizations has led to a wide array of modifications in sperm structure and function. Sperm cells of most muroid rodents have hook‐shaped extensions in the apical–ventral tip of the head, but the function of this structure is largely unknown. These ‘hooks’ may facilitate aggregation of spermatozoa in so‐called ‘trains’, as an adaptation to sperm competition, because sperm in trains may swim faster than free‐swimming cells. However, there is controversy regarding the role of the hook in train formation, and in relation to whether it is selected by sperm competition. We examined spermatozoa from muroid rodents with varying levels of sperm competition to assess whether (i) sperm aggregates are common in these taxa, (ii) presence of a hook relates to the formation of sperm aggregations, and (iii) formation of sperm aggregations is explained by sperm competition. Our analyses in 25 muroid species revealed that > 92% of spermatozoa swim individually in all species, with the exception of the wood mouse, Apodemus sylvaticus, which has ~50% spermatozoa swimming freely. Species with hooked spermatozoa had higher sperm competition levels and longer sperm than species whose sperm lack a hook. Neither the presence of hook nor sperm competition levels were related to the percentage of sperm in aggregations. Thus, (i) sperm aggregates in muroid rodents are an exceptional trait found only in a few species, (ii) evolution of the sperm hook is associated to sperm competition levels, but (iii) the hook is unlikely to be related to the formation of sperm aggregates. The evolutionary significance of the sperm head hook thus remains elusive, and future studies should examine potential roles of this pervasive structure in sperm's hydrodynamic efficiency and sperm–female tract interactions.     

Differential role of the Mu B protein in phage Mu integration vs. replication: mechanistic insights into two transposition pathways

The Mu B protein is an ATP-dependent DNA-binding protein and an allosteric activator of the Mu transposase. As a result of these activities, Mu B is instrumental in efficient transposition and target-site choice. We analysed in vivo the role of Mu B in the two different recombination reactions performed by phage Mu: non-replicative transposition, the pathway used during integration, and replicative transposition, the pathway used during lytic growth. Utilizing a sensitive PCR-based assay for Mu transposition, we found that Mu B is not required for integration, but enhances the rate and extent of the process. Furthermore, three different mutant versions of Mu B, Mu BC99Y, Mu BK106A, and Mu B1-294, stimulate integration to a similar level as the wild-type protein. In contrast, these mutant proteins fail to support Mu growth. This deficiency is attributable to a defect in formation of an essential intermediate for replicative transposition. Biochemical analysis of the Mu B mutant proteins reveals common features: the mutants retain the ability to stimulate transposase, but are defective in DNA binding and target DNA delivery. These data indicate that activation of transposase by Mu B is sufficient for robust non-replicative transposition. Efficient replicative transposition, however, demands that the Mu B protein not only activate transposase, but also bind and deliver the target DNA.     

Cytotoxic and antiviral activities of Colombian medicinal plant extracts of the Euphorbia genus

Forty-seven plant extracts of 10 species of the genus Euphorbia (Euphorbiaceae) used by Colombian traditional healers for the treatment of ulcers, cancers, tumors, warts, and other diseases, were tested in vitro for their potential antitumour (antiproliferative and cytotoxic) and antiherpetic activity. To evaluate the capacity of the extracts to inhibit the lytic activity of herpes simplex virus type 2 (HSV-2) and the reduction of viability of infected or uninfected cell cultures, the end-point titration technique (EPTT) and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay were used, respectively. The therapeutic index of the positive extracts for the antiviral activity was determined by calculating the ratio CC50 (50% cytotoxic concentration) over IC50 (50% inhibitory concentration of the viral effect). Five of the 47 extracts (11%) representing 3 out of 10 Euphorbia species (30%) exhibited antiherpetic action; the highest activity was found in the leaf/stem water-methanol extracts from E. cotinifolia and E. tirucalli. The therapeutic indexes of these two plant species were > 7.1; these extracts exhibited no cytotoxicity. Six extracts (13%) representing 4 plant species (40%) showed cytotoxic activity. The highest cytotoxicity was found in the dichloromethane extract obtained from E. cotinifolia leaves and the CC50 values for the most susceptible cell lines, HEp-2 and CHO, were 35.1 and 18.1 microgram/ml, respectively.