Chemotaxis of capacitated rabbit spermatozoa to follicular fluid revealed by a novel directionality-based assay

Precontact communication between gametes is established by chemotaxis. Sperm chemotaxis toward factor(s) in follicular fluid (FF) has been demonstrated in humans and mice. In humans, the chemotactic responsiveness is restricted to capacitated spermatozoa. Here, we investigated whether sperm chemotaxis to factor(s) present in FF also occurs in rabbits and, if so, whether only capacitated spermatozoa are chemotactically responsive. Chemotaxis assays were performed by videomicroscopy in a Zigmond chamber. We measured chemotactic responsiveness as a function of FF dilution by means of a novel directionality-based method that considers the ratio between the distances traveled by the spermatozoa both parallel to the chemoattractant gradient and perpendicular to it. A peak of maximal response was observed at 10(-4) dilution of FF, resulting in a typical chemotactic concentration-dependent curve in which 23% of the spermatozoa were chemotactically responsive. In contrast, the percentage of cells exhibiting FF-dependent enhanced speed of swimming increased with the FF concentration, whereas the percentage of cells maintaining linear motility decreased with the FF concentration. The percentages of chemotactically responsive cells were very similar to those of capacitated spermatozoa. Depletion of the latter by stimulation of the acrosome reaction resulted in a total loss of the chemotactic response, whereas the reappearance of capacitated cells resulted in a recovery of chemotactic responsiveness. We conclude that rabbit spermatozoa, like human spermatozoa, are chemotactically responsive to FF factor(s) and acquire this responsiveness as part of the capacitation process.     

Molecular phylogenetics of Stenodermatini bat genera: congruence of data from nuclear and mitochondrial DNA

Within the tribe Stenodermatini the systematics of the complex of species allied with the genus Artibeus has generated several alternative phylogenetic hypotheses. The most recent treatment recognized four genera (Artibeus, Dermanura, Enchisthenes, and Koopmania) and suggested that the most recent common ancestor of these four genera would include the common ancestor of all other currently recognized Stenodermatini genera except Sturnira. To test this hypothesis, we examined an EcoRI-defined nuclear satellite DNA repeat and 402 bp of DNA sequence variation from the mitochondrial cytochrome b gene. Phylogenetic conclusions based on Southern blot analyses, in situ hybridization, and mitochondrial DNA sequence data indicate that Enchisthenes is not closely related to Dermanura, Artibeus, or Koopmania and that Dermanura, Artibeus, and Koopmania shared a common ancestor after diverging from the remainder of the Stenodermatini. If our conclusions are correct, then justification for recognizing Dermanura and Koopmania as generically distinct from Artibeus must be based on the magnitude of difference that distinguishes each rather than on the conclusion that to place them as congeneric with Artibeus creates a paraphyletic taxon.      

Biotinyl-l-3-(2-naphthyl)-alanine hydrazide derivatives of N-glycans: versatile solid-phase probes for carbohydrate-recognition studies

Biotinyl-oligosaccharides are a relatively new generation of saccharide probes that enable immobilization of desired oligosaccharides on streptavidin matrices for studies of carbohydrate-protein interactions. Here we describe the facile preparation of biotinyl-l-3-(2-naphthyl)- alanine hydrazide (BNAH) derivatives of oligosaccharides, containing a strong UV absorbing and fluorescent group, in which the ring of the reducing-end monosaccharide is nonreduced. We evaluate reactivities of immobilized BNAH- N -glycans with plant lectins that recognize aspects of the oligosaccharide core or outer-arms. We make some comparisons with 2-amino-6-amidobiotinyl-pyridine (BAP) derivatives obtained by reductive amination, and 6-(biotinyl)-aminocaproyl-hydrazide (BACH) derivatives which have a longer spacer-arm. N -Glycan-BNAH and-BAP derivatives have, overall, comparable reactivities with lectins which recognize N -glycan outer-arms or the trimannosyl core, but only BNAH and BACH derivatives are bound by lectins which recognize the non- reduced core. Moreover, with Pisum sativum agglutinin (PSA) which additionally requires the fucosyl- N- glycan-asparaginyl core for high affinity binding, the immobilized BNAH derivative (which is an alanine hydrazide beta-glycoside) can substitute for the natural beta- glycosylasparaginyl core, whereas the BACH derivative (aminocaproyl- hydrazide-beta-glycoside) is less effective. BNAH is a derivatization reagent of choice, therefore, for solid phase carbohydrate-binding experiments with immobilized N -glycans.      

New conformational constraints in isotopically (13C) enriched oligosaccharides

Multidimensional heteronuclear NMR studies have been applied to the resonance assignment and conformational analysis of 13C-enriched Neu5Acalpha2-3Galbeta1-4Glc. It is demonstrated that three-dimensional ROESY-HSQC experiments provide through-space distance restraints which cannot be observed with conventional homonuclear 1H techniques due to resonance overlap. In particular, connectivities demonstrating the existence of the "anti" conformation about the Galbeta1-4Glc glycosidic linkage are unambiguously observed. It is shown that 13C isotopic enrichment of the trisaccharide at a level >95% enables straightforward measurement of trans-glycosidic 1H-13C and 13C-13C coupling constants and a Karplus-type relation is derived for the latter. In total 15 conformational restraints were obtained for the trisaccharide in aqueous solution, all of which were in excellent agreement with theoretical parameters computed from a 5 ns molecular dynamics simulation of the glycan.      

Method for Treating and Processing Whole Chick Embryos for Autoradiography, Immunocytochemistry and other Techniques

A method is presented for In situ treatment of whole chick embryos with drugs and immunocytochemical and fixative reagents that resembles conditions “in ovo.” The chick embryo is placed in a “shell-less” culture system where it is contained by an agar ring allowing for treatment in vivo. The conceptus (embryo + membranes) is then mounted on a microporous membrane and inserted into a filter device connected to a three-way stopcock that permits fluids to be changed using syringes. The embryo is then processed in toto or after embedding and sectioning for light or electron microscopy. The proposed handling system decreases technical artifacts and changes in the topographic microanatomy produced by conventional manipulation of chick embryos. This method is useful also for directly observing and recording changes in the embryo during drug treatments and allows processing with dangerous reagents without their direct contact with the operator. It is simple, inexpensive and requires only minimal technical training.     

The replicative helicase MCM recruits cohesin acetyltransferase ESCO2 to mediate centromeric sister chromatid cohesion

Chromosome segregation depends on sister chromatid cohesion which is established by cohesin during DNA replication. Cohesive cohesin complexes become acetylated to prevent their precocious release by WAPL before cells have reached mitosis. To obtain insight into how DNA replication, cohesion establishment and cohesin acetylation are coordinated, we analysed the interaction partners of 55 human proteins implicated in these processes by mass spectrometry. This proteomic screen revealed that on chromatin the cohesin acetyltransferase ESCO2 associates with the MCM2‐7 subcomplex of the replicative Cdc45‐MCM‐GINS helicase. The analysis of ESCO2 mutants defective in MCM binding indicates that these interactions are required for proper recruitment of ESCO2 to chromatin, cohesin acetylation during DNA replication, and centromeric cohesion. We propose that MCM binding enables ESCO2 to travel with replisomes to acetylate cohesive cohesin complexes in the vicinity of replication forks so that these complexes can be protected from precocious release by WAPL. Our results also indicate that ESCO1 and ESCO2 have distinct functions in maintaining cohesion between chromosome arms and centromeres, respectively.     

Pituitary responsiveness to gonadotrophin-releasing and thyrotrophin-releasing hormones in children receiving phenobarbitone.

The effect of long-term treatment with phenobarbitone on pituitary responsiveness to gonadotrophin-releasing hormone and thyrotrophin-releasing hormone was studied in 20 boys being treated with the drug to prevent febrile convulsions. Baseline concentrations of luteinising and follicle-stimulating hormones were reduced as well as the responses of these hormones to stimulation with gonadotrophin-releasing hormone. Baseline prolactin concentrations were raised in comparison with those in normal children. The response of prolactin to thyrotrophin-releasing hormone, however, was impaired only in the children who had been receiving the drug for a long time. Phenobarbitone had no effect on the secretion of growth hormone. Further studies should be carried out to ascertain how long these effects on pituitary function last after phenobarbitone is withdrawn and whether this interference with pituitary function modifies the child''s subsequent development.     

Rapid evolution in a conserved gene family. Evolution of the actin gene family in the sea urchin genus Heliocidaris and related genera

Camarodont sea urchins possess a rapidly evolving actin gene family whose members are expressed in distinct cell lineages in a developmentally regulated fashion. Evolutionary changes in the actin gene family of echinoids include alterations in number of family members, site of expression, and gene linkage, and a dichotomy between rapidly and slowly evolving isoform-specific 3' untranslated regions. We present sequence comparisons and an analysis of the actin gene family in two congeneric sea urchins that develop in radically different modes, Heliocidaris erythrogramma and H. tuberculata. The sequences of several actin genes from the related species Lytechinus variegatus are also presented. We compare the features of the Heliocidaris and Lytechinus actin genes to those of the the actin gene families of other closely related sea urchins and discuss the nature of the evolutionary changes among sea urchin actins and their relationship to developmental mode.