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Mass production of basal bodies in paraspermiogenesis of Tubificinae (Annelida, Oligochaeta)
No full textThe oligochaete annelids, belonging to the subfamily Tubificinae, produce two types of spermatozoa: eusperm (the fertilising ones) and
parasperm, protecting and carrying the eusperm. The pathway for the production of the two types is common until the onset of meiosis, but
then a regular meiosis produces eusperm, whereas parasperm is generated through a peculiar mechanism of nuclear fragmentation giving
rise to an irregular, but very high, number of paraspermatozoa. Since every parasperm has its own flagellum, this entails the necessity of
producing a very high number of basal bodies. In the present paper, we describe how basal bodies are generated through a mechanism
similar to that producing the basal bodies in ciliated epithelia, but never observed up to now during the genesis of a uniflagellated cell. Basal
bodies form in close proximity to a precursor structure called deuterosome, which originates de novo in the cytoplasm from fibrogranular
material. The various stages of centriologenesis are positive to anti-centrin antibodies and, observed by electron microscopy, correspond
closely to the ones described for ciliated epithelia. However, once formed, the basal bodies migrate to their final position and produce the parasperm flagellum
The spermatozeugmata of Tubifex tubifex (Annelida, Clitellata, Tubificinae) studied by different technical approaches
Full text linkOligochaete annelids belonging to the subfamilies Tubificinae and Limnodriloidinae are characterized, inter alia, by the production of two different spcnn types, named eusperm and paraspenn [I]. In Tubifex tuhifex the two sperm types differ in nearly all their characters,like nuclear size [(about 30 ~ in the euspenn, but 3 11m in the paraspeml); the amount of nuclear
o A (about eight times more in the eusperm than in the parasperm); the extreme reduction
of the acrosome in the parasperm; the size of the mitochondria (about double in the
paraspeml with respect to the eusperm); and the tail (the cell membrane is separated from the axoneme by a consistent space in the parasperm, and the space is filled by y glycogen, whereas in the euspcrm the space is reduced and has pglycogen granules)]. We have studied the production of the two speml types, and we found that, starting from similar spermatocytes, euspermatooza are produced through a regular meiotic process, whereas the paraspermatozoa are produced through a peculiar process of nuclear fragmentation which, produces a very high number of aneuploid parasperm [2].
When fully mature, the two types of spenn are transmitted to the partner during copulation, and both reach the spemlathecae. There, the two sperm types are grouped to fonn typical bundles, called spennatozeugmata, rod-shaped structures, up to 2 mm long, already known
since 1861 [3]. The spennatozeugmata have an outer layer, the cortex, formed by tightly
packed parasperm, and an inner core, the axial cylinder, formed by paralelly arranged
euspcnn. The spennatozeugmata are stored in the spermathccae up to the moment of
fertilization, when they move towards the spennathccal duct at the edge of which they are broken, thus delivering the eusperm [3]. Thus, the spermatozeugmata perfonn different
functions, accompanied by particular features:
they hold together a certain number of fertilizing sperm "ready for use". This is
probably accomplished by an impressive complex of cell junctions fonned mainly by
septate junctions, followed by scalarifonn junctions connecting paraspenn tails [3]
they are able to move with a metachronal wave fomled by the distal extremities of
the paraspenn, thus carrying the fertilizing eusperm [3].
We have studied T. whifex spermatozeugmata using different techniques: TEM, SEM, freeze
fracture, high speed video microscopy. confocal microscopy and immunogold labeling.
We were able to show the metachronal wave produced by the paraspenn tails and measure its beat frequency (10 Herz). For the first time using CLSM we have documented by a TRITC phalloidin labeling the presence of a large amount of F-actin inside the cytoplasm of the paraspenn tails. Actin was also localized by means of immunogold staining under TEM. It remains to be established to what extent actin is cOJUlected to the septate junctions
SCLIP, a microtubule-destabilizing factor, interacts with RasGRF1 and inhibits its ability to promote Rac activation and neurite outgrowth
No full textRasGRF1 is a neuron-specific guanine nucleotide exchange factor for the small GTPases Ras and Rac. It is implicated in the regulation of memory formation and in the development of tolerance to drug abuse, although the mechanisms have been elucidated only in part. Here we report the isolation, by the yeast two-hybrid screen, of the microtubule-destabilizing factor SCLIP (SCG10-like protein) as a novel RasGRF1-interacting protein. This interaction requires the region spanning the Dbl-homology domain of RasGRF1, endowed with catalytic activity on Rac. In search for a possible function we found by biochemical means that SCLIP influences the signaling properties of RasGRF1, greatly reducing its ability to activate the Rac/p38 MAPK pathway, while the Ras/Erk one remains unaffected. Moreover, a potential role is suggested by transfection studies in neuronal PC12 cells in which RasGRF1 induces neurite outgrowth, and coexpression of SCLIP counteracts this effect, causing a dramatic decrease in the percentage of cells bearing neurites, which also appear significantly shortened. This study unveils a physical and functional interaction between RasGRF1 and SCLIP. We suggest that this novel interplay may have possible implications in mechanisms that regulate neuronal morphology and structural plasticity
Paraquat embryotoxicity in the Xenopus laevis cleavage phase
No full textThe high Paraquat (PQ, 1-1'-dimethyl-4,4',bipyridylium dichloride) embryotoxicity in Xenopus laevis has been shown to be due to its rapid reduction and instantaneous re-oxidation which produces a reactive oxygen species, ROS. Nevertheless, PQ did not show any effects before hatching, stage 32, which showed a resistance, in early X. laevis development, to oxidative damage. Moreover, in view of its genotoxic properties in several experimental models, we studied PQ in the X. laevis cleavage phase that, characterized by a series of rapid mitotic divisions, might be damaged by genotoxic compounds. Embryos were exposed to 20, 40, 60, and 80 mg/l PQ concentrations from stage 2 to stage 9, and then left to develop in control FETAX solution until stage 47. The 80 mg/l PQ concentration gave 19% embryo mortality at the end of the exposure time, and 16.7% larvae mortality at the end of the test; both values were statistically different from the control, 5 and 6.8% respectively. These results confirmed the high resistance in early X. laevis development to PQ oxidative damage. The malformed larva percentages in the PQ exposed groups were higher as regards the control value but did not show any concentration-response; the most frequent malformed larvae found were affected by abnormal tail flexure coupled with abnormal gut coiling. A further experiment was carried out using the same methodology, but exposing embryos only to the 80 mg/l PQ concentration. The surviving blastulae were embedded in Paraplast, then the slides were stained with 4',6-diarnidino-2-phenylindole (DA-PI) and the nuclei were examined with a confocal microscope. This new preliminary procedure did not reveal any significant presence of micronucleated micromeres in PQ exposed blastulae with respect to the control. Nevertheless, the mechanism by which PQ induced abnormal tail flexure after cleavage exposure remained unknown. PQ seemed to pass through the jelly coats and vitelline membrane, but it expressed teratogenicity between the 2nd and 3rd day. PQ might be accumulated in the embryos during the exposure, and might express teratogenicity later, but it did not seem to induce genotoxicity during the cleavage phase of X. laevis even at very high concentrations
Regulation of glutamate release by heteromeric nicotinic receptors in layer V of the secondary motor region (Fr2) in the dorsomedial shoulder of prefrontal cortex in mouse
No full textWe studied how nicotinic acetylcholine receptors (nAChRs) regulate glutamate release in the secondary motor area (Fr2) of the dorsomedial murine prefrontal cortex, in the presence of steady agonist levels. Fr2 mediates response to behavioral situations that require immediate attention and is a candidate for generating seizures in the frontal epilepsies caused by mutant nAChRs. Morphological analysis showed a peculiar chemoarchitecture and laminar distribution of pyramidal cells and interneurons. Tonic application of 5 μM nicotine on Layer V pyramidal neurons strongly increased the frequency of spontaneous glutamatergic excitatory postsynaptic currents. The effect was inhibited by 1 μM dihydro-β-erythroidine (which blocks α4-containing nAChRs) but not by 10 nM methyllicaconitine (which blocks α7-containing receptors). Excitatory postsynaptic currents s were also stimulated by 5-iodo-3-[2(S)-azetidinylmethoxy]pyridine, selective for β2-containing receptors, in a dihydro-β-erythroidine -sensitive way. We next studied the association of α4 with different populations of glutamatergic terminals, by using as markers the vesicular glutamate transporter type (VGLUT) 1 for corticocortical synapses and VGLUT2 for thalamocortical projecting fibers. Immunoblots showed higher expression of α4 in Fr2, as compared with the somatosensory cortex. Immunofluorescence showed intense VGLUT1 staining throughout the cortical layers, whereas VGLUT2 immunoreactivity displayed a more distinct laminar distribution. In Layer V, colocalization of α4 nAChR subunit with both VGLUT1 and VGLUT2 was considerably stronger in Fr2 than in somatosensory cortex. Thus, in Fr2, α4β2 nAChRs are expressed in both intrinsic and extrinsic glutamatergic terminals and give a major contribution to control glutamate release in Layer V, in the presence of tonic agonist levels
Are the peripheric cells of macula to produce its increase?
No full textAfter the birth, in the inferior Vertebrates the inner ear increase continuously and progressively. How happens such increase? Our studies have performed on the inner ear of Rana esculenta by immunofluorescence techniques and by SEM observations. In order to determine the new sensory cells’ derivation, we have given a mitogenic marker, the BrdU, to label cells that were in the process of dividing. We have evidenced that the sensory cells, with morphologic characteristics of young cells, are present in all three of maculae mostly to the periphery of the sensory epithelium. The young cells are rare between the mature sensory cells of central part of the maculae. For these data we can assume that the increase of the sensory areas happens from the periphery to the middle of the maculae by forming the new sensory and supporting cells. These cells, probably, have been originated by the division of support cells, subsequently they differ in sensory cells
External morphology and muscle arrangement of Brachionus urceolaris, Floscularia ringens, Hexarthra mira and Notommata glyphura (Rotifera, Monogononta)
No full textWe studied four monogonont rotifers (Brachionus urceolaris, Floscidaria ringens, Hexarthra mira, Notommata glyphura) using two different techniques of microscopy: (1) the presence of filamentous actin was examined using phalloidin-fluorescent labelled specimens and a confocal laser scanning microscope (CLSM); (2) external morphology was investigated using a scanning electron microscope (SEM). B. urceolaris, F. ringens, and N. glyphura showed similar patterns of muscle distribution: a set of longitudinal muscles acting as head and foot retractors, and a set of circular muscles. However, the size and distribution of circular muscles differed among these species. H. mira differed from the other species in that it lacked circular muscles but possessed strong muscles that extended into each arm. The study showed that using both CLSM and SEM provides better resolution of the anatomy and external morphology of rotifers than using one of these techniques alone. This can facilitate better understanding of the complicated anatomy of these animals
Morphological and histochemical analysis of immature bovine oocytes denuded before vitrification and their meiotic competence after thawing
No full textModulation of glutamate release by nicotinic receptors in layer V of the murine prefrontal cortex
No full textBy regulating the neocortical excitability, nicotinic acetylcholine receptors (nAChRs) control vigilance and cognition. In rodents, the neocortex mainly expresses homomeric a7 and heteromeric a4ß2 nAChRs. These are expressed in both pre- and postsynaptic locations and mediate classical synaptic transmission as well as slower paracrine effects. We have studied the contribution of heteromeric nAChRs to the control of glutamate (GLU) release in layer V of the murine prefrontal cortex (PFC). Tonic application of 5 μM nicotine more than doubled the frequency of spontaneous glutamatergic excitatory postsynaptic currents recorded on pyramidal neurons in acute brain slices. The effect of nicotine was inhibited by 1 μM dihydro-ß-erythroidine (DHßE, which blocks a4-containing receptors), but not by 10 nM methyllicaconitine (MLA, which blocks a7-containing receptors). We next studied the association of a4 with different populations of glutamatergic terminals, in both PFC and somatosensory cortex. The GLU transporter type 1 (VGLUT1) mostly labels the intrinsic glutamatergic terminals or cortical afferents, whereas the type 2 transporter VGLUT2 tends to label the thalamic afferents. Immunofluorescence showed that a4 was expressed in both VGLUT1 and VGLUT2 terminals and colocalization was considerably stronger in the PFC. Expression of the a4, VGLUT1 and VGLUT2 was also tested by immunoblots, which confirmed the overall higher expression of these proteins in the PFC compared to the somatosensory cortex. Hence, in PFC, a4-containing heteromeric nAChRs are expressed in both intrinsic and extrinsic glutamatergic terminals and regulate GLU release in the presence of steady agonist levels
Gentamicin ototoxicity and recovery phenomena in the saccule of the lizard Podarcis sicula.
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