p15, A nuclear-associated protein of human sperm: Identification of four variants and their occurrence in normal and abnormal seminal cells

A basic protein of apparent molecular weight 15,000 d (p15) has been identified as a tissue-specific species-unique component of spermatogenic cells of human semen. Cytoimmunochemical study with a monoclonal antibody indicates that p15 resides in a perinuclear space in morphologically normal spermatozoa and differs in distribution and stat in abnormal seminal cell and nonnucleated bodeis. Biochemical analysis indicateds that p15 occurs as four variiants, differentially migratory on acetic acid/urea gel and differnetially extractable by NaCl in reducing solution. By correlation of the cytologic and biochemical data, we propose that variant 1 is the unmodified form of p15; in the normal progression of spermiogenesis p15 is modified to variants 2 and 3 and in the absence of the normal progression is unmodified or aberrantly modified to variant 4. The association of molecular abnormality in p15 with morphologically abnormal sperm suggests that p15 may play a role in sperm-head shaping.     

Immunogold labelling of xylans and arabinoxylans in the plant cell walls of maize stem

Summary— Polyclonal antibodies against 4-O-methyl-glucuronoxylan and α L-1-3 arabinofuranosyl poly-β-d-1-4-xylopyranosyl were raised from rabbits. An immunocytochemical technique was used to localize xylans and arabinoxylans in the plant cell walls of the apical internode of two maize lines of different digestibility. The sclerenchyma, fibres and xylem (lignified tissues) and the parenchyma (non-lignified tissue) were studied. The arabinoxylans were more heavily labelled than the xylans in the lignified tissues of the less digestible maize whereas in the more digestible line the labelling of the two polysaccharides was similar. The xylans and arabinoxylans were localized in the secondary cell wall. In both maize lines, labelling increased from the base upwards of the apical internode, reflecting the changes in growth stage.     

Importance of actin cytoskeleton behaviour during preservation of carrot cell suspensions in liquid nitrogen

Summary Conventional methods for preservation of suspended, highly vacuolated, plant cells in liquid nitrogen (LN) usually involve equilibration in molar concentrations of cryoprotective additives, followed by slow cooling to an intermediate subzero temperature (–40 °C), before quenching in LN. Cryomicroscopy was used to monitor the reversible protoplasmic shrinkage of cryoprotected carrot cells, caused by freeze-induced dehydration. Behaviour of actin filaments was analyzed by fluorescence microscopy after labelling with rhodarnine-conjugated phalloidin, in relation to the type of pretreatment and to survival and regrowth ability after preservation at — 196 °C. Loading with dimethylsulphoxide (Me₂SO, 5%) resulted in high survival rates (70%) and regrowth. After thawing, the actin filament (MF) abundance was reduced, but the structure and distribution of the remaining MFs seemed undisturbed. Higher Me₂SO concentrations caused further reduction of MFs, which appeared fragmented after thawing. MFs were maintained by pretreatment with 0.5 M sorbitol alone but carrot cells did not survive at — 196 °C. The same pretreatment, followed by incubation with cytochalasin D (10 M), which greatly reduced MFs, enabled plasmolyzed carrot cells to survive preservation in liquid nitrogen. Thus, after both Me₂SO and sorbitol plus cytochalasin D pretreatments, partial disruption of actin filaments seemed to accompany (Me₂SO) or promote (sorbitol plus cytochalasin D) freezing tolerance at extremely low temperatures.Abbreviations CD cytochalasin D - FDA fluorescein diacetate - LN liquid nitrogen - MF actin filament - Me₂SO dimethylsulphoxide     

Distribution of filamentous actin in and around spermatids and in spermatozoa of Australian conilurine rodents.

The distribution of filamentous actin around the maturing sperm head and in spermatozoa of four species of Australian conilurine rodents was investigated at the light and electron microscopic levels. Similar results were obtained for all the species studied. Mechanically isolated spermatids had NBD-phallacidin-positive longitudinal bands of fluorescence over the dorsolateral surface and, in late spermatids, bands of bright fluorescence passed perpendicularly from the dorsal convex to ventral concave surface. TEM observations indicated that these regions corresponded to filaments of ectoplasmic specializations and granular filamentous material around the tubulobulbar complexes, respectively. In testicular and cauda spermatozoa NBD-phallacidin fluorescent material was present in the two ventral processes that extended from the upper concave surface of the sperm head; also fainter material occurred along the concave border and as a dorsocaudal spur. Its distribution was identical for testicular and cauda spermatozoa. TEM of late spermatids showed that in the ventral process closest to the apical hook there were between 170 and 245 filaments, which attached to the inner surface of the postacrosomal dense lamina; in the more caudal ventral process about 70 filaments occurred. No filaments were, however, visible in the mature spermatozoon but, after immunocytochemical labelling for actin, deposition of gold particles was evident over ventral processes of both late spermatids and cauda spermatozoa. Within the female tract these ventral processes made contact with the zona matrix and were taken into the egg cytoplasm unchanged in morphology. The possible functional significance of the filamentous actin in these structures is discussed.     

Structural modifications of astrocytes in the hippocampus after experimental cerebral ischemia in gerbils

We studied reactions of astrocytes in the CA1 hippocampal zone of the mongolian gerbil (Meriones unguiculatus) after experimental short-lasting (7 min) cerebral ischemia resulting from bilateral occlusion of the carotid arteries. Immunocytochemical staining of hippocampal sections with antibodies against an astrocytes marker, glial fibrillary acidic protein (GFAP), was used. We measured the density of labelled cells in the layers of the CA1 zone at different time intervals (from 1 to 30 days) after cerebral ischemization. The number of labelled astrocytes within this period increased, and the dynamics of their density in different layers demonstrated significant dissimilarities. The earliest manifestations of reactive astrogliosis were observed in the hilus. The greatest rise in the number of astrocytes was found in the str. lacunosum-moleculare and str. moleculare on the 7th day, while in the str. pyramidale the maximum was reached only on the 14th day, which corresponded to the period of the highest intensity of delayed postischemic neuronal death. Thus, the intensity of morphological changes of the neurons and the level of reactivity of the astrocytes demonstrate a rather clear correlation; this fact can be one of the aspects of the dynamics of postischemic damage to the hippocampal neurons. Neirofiziologiya/Neurophysiology, Vol. 37, Nos. 5/6, pp. 410–415, September–December, 2005.     

Immunogold Cytochemistry Identifies Specialized Membrane Domains for Monocarboxylate Transport in the Central Nervous System

An efficient exchange of lactate between different cell types (such as astrocytes and neurones) would require that lactate transporters are expressed in contiguous parts of the respective plasma membranes. To settle this issue we explored the subcellular expression pattern of monocarboxylate transporters (MCTs) by use of selective antibodies and high resolution immunogold cytochemistry. We investigated whether the membrane domains containing MCT1, MCT2 and MCT4 are spatially related to each other and to other membrane domains, i.e. those containing glutamate receptors. We used retina and cerebellum as a model for our investigations. We found that MCT1 was localized in the apical membrane of pigment epithelial cells and in the photoreceptor inner segment membrane in the retina. In the brain MCT1 was present in endothelial cells. MCT2 was localized in the postsynaptic membrane of parallel fiber-Purkinje cell synapses and MCT4 was situated in the membrane of glial cells in the cerebellum.     

Microtubule distribution during fertilization in the rabbit.

The distribution of microtubules was studied during fertilization of the rabbit oocyte by immunofluorescence microscopy after staining with an anti-alpha-tubulin antibody. In ovulated oocytes, microtubules were found exclusively in the meiotic spindle. At fertilization, the paternal centrosome generated sperm astral microtubules. During pronuclear development, the sperm aster increased in size, and microtubules extended from the male pronucleus to the egg center and towards the female pronucleus. These observations indicate that microtubules emanating from the sperm centrosome were involved in the movements leading to the union of the male and female pronuclei. At late pronuclear stage, microtubules surrounded the adjacent pronuclei. The mitotic spindle that emerged from the perinuclear microtubules contained broad anastral poles.     

Distribution of actin microfilaments in frog skin epithelial granular cells

Summary— Microfilaments were localised by immunofluorescence and immunogold cytochemistry to examine their distribution in granular cells of the isolated frog skin epithelium. Strongly fluorescent bundles of actin were observed beneath the plasma membrane with little evidence for actin in the central regions. Higher resolution offered by cytochemistry revealed that bundles of actin filaments comprised a substantial portion of the cortical cytoskeleton. Quantitative analysis of the frequency of gold label revealed an extremely rich array of filaments beneath the apical membrane of granular cells, with markedly less babel along the basolateral membrane and in the central cytoplasm. Treating cells with cytochalasin B or arginine vasopressin caused an apparent disruption of the apical actin fibres, concurrent with a decrease in gold label density. Assumably these signs are indicative of depolymerization of the filaments. Although the significance of this distribution is unknown, the apical polarisation of actin is consistent with a role in regulating the Na⁺ permeability of the apical membrane. The data are discussed in relation to possible roles of the cytoskeleton in the regulation of transepithelial sodium transport by vasopressin.     

Protrusion and actin assembly are coupled to the organization of lamellar contractile structures

Directed cell migration requires continuous cycles of protrusion of the leading edge and contraction to pull up the cell rear. How these spatially distributed processes are coordinated to maintain a state of persistent protrusion remains unknown. During wound healing responses of epithelial sheets, cells along the wound edge display two distinct morphologies: ‘leader cells’ exhibit persistent edge protrusions, while the greater majority of ‘follower cells’ randomly cycle between protrusion and retraction. Here, we exploit the heterogeneity in cell morphodynamic behaviors to deduce the requirements in terms of cytoskeleton dynamics for persistent and sporadic protrusion events. We used quantitative Fluorescent Speckle Microscopy (qFSM) to compare rates of F-actin assembly and flow relative to the local protrusion and retraction dynamics of the leading edge. Persistently protruding cells are characterized by contractile actomyosin structures that align with the direction of migration, with converging F-actin flows interpenetrating over a wide band in the lamella. Conversely, non-persistent protruders have their actomyosin structures aligned perpendicular to the axis of migration, and are characterized by prominent F-actin retrograde flows that end into transverse arcs. Analysis of F-actin kinetics in the lamellipodia showed that leader cells have three-fold higher assembly rates when compared to followers. To further investigate a putative relationship between actomyosin contraction and F-actin assembly, myosin II was inhibited by blebbistatin. Treated cells at the wound edge adopted a homogeneously persistent protrusion behavior, with rates matching those of leader cells. Surprisingly, we found that disintegration of actomyosin structures led to a significant decrease in F-actin assembly. Our data suggests that persistent protrusion in these cells is achieved by a reduction in overall F-actin retrograde flow, with lower assembly rates now sufficient to propel forward the leading edge. Based on our data we propose that differences in the protrusion persistence of leaders and followers originate in the distinct actomyosin contraction modules that differentially regulate leading edge protrusion-promoting F-actin assembly, and retraction-promoting retrograde flow.     

Regional distribution of glutathione-related antioxidant defences in the normal rabbit aorta

In 6 normal rabbits, the aortic arch, the descending thoracic and the abdominal aorta were tested for non proteic thiol compounds, selenium-dependent and selenium-independent glutatione peroxidase, glutatione reductase, glutatione transferase and thiobarbituric acid reactive substances. The aortic arch showed the greatest content of non proteic thiol compounds and thiobarbituric acid reactive substances, associated to the highest activities of glutathione-related enzymes. However, not significant differences were detectable between aortic arch and descending thoracic aorta, except for the glutathione transferase activity (0.395 +/- 0.031 vs 0.330 +/- 0.053 U/mg protein, p less than 0.05). Furthermore, both aortic arch and descending thoracic aorta showed significantly higher values of non proteic thiol compounds (46.05 +/- 10.15% and 33 +/- 13.5%, p less than 0.05), selenium-dependent glutathione peroxidase activity (70.35 +/- 26% and 54.3 +/- 9.5%, p less than 0.05), glutathione reductase activity (25.4 +/- 7% and 18.4 +/- 4.5%, p less than 0.05) and thiobarbituric acid reactive substances (65.8 +/- 18% and 47.2 +/- 17%, p less than 0.05) with respect to the abdominal aorta. The selenium-independent glutathione peroxidase activity was not detectable. In conclusion, a biochemical gradient in glutathione-related antioxidant defences and thiobarbituric acid reactive substances proceeding from the proximal to the distal segments seems to exist in the normal rabbit aorta. These results could contribute to explain the non homogeneous distribution of experimental atherosclerosis in the rabbit aorta.     

Organization of actin microfilaments in the apical border of oviduct ciliated cells

Actin microfilaments were localized in quail oviduct ciliated cells using decoration with myosin subfragment S1 and immunogold labeling. These polarized epithelial cells show a well developed cytoskeleton due to the presence of numerous cilia and microvilli at their apical pole. Most S1-decorated microfilaments extend from the microvilli downward towards the upper part of the ciliary striated rootlets with which they are connected. From the microvillous roots, a few microfilaments connect the proximal part of the basal body or the basal foot associated with the basal body. Microfilament polarity is shown by S1 arrowheads pointing away from the microvillous tip to the cell body. Furthermore, short microfilaments are attached to the plasma membrane at the anchoring sites of basal bodies and run along the basal body. The polarity of these short microfilaments is directed from the basal body anchoring fibers downward to the cytoplasm. At the cell periphery, microfilaments from microvillous roots and ciliary apparatus are connected with those of the circumferential actin belt which is associated with the apical zonula adhaerens. Together with the other cytoskeletal elements, the microfilaments increase ciliary anchorage and could be involved in the coordination of ciliary beating. Moreover, microvilli surrounding the cilia probably modify ciliary beating by offering resistance to cilium bending. The presence of microvilli could explain the fact that mainly the upper part of the cilia appanars to be involved in the axonemal bending in metazoan ciliated cells.     

Changes in rat testicular adenylate cyclase activities and gonadotrophin binding during unilateral experimental cryptorchidism

Unilaterally cryptorchid rats were examined at 3, 8, 15, 22 and 28 days after operation. There was a selective decrease in the adenylate cyclase (ATP pyrophosphate--lyase (cyclizing), EC 4.6.1.1) responses to gonadotrophin stimulation in the abdominal testis. This was associated with a parallel decrease in specific FSH and LH binding. There was no reduction in the response of testicular adenylate cyclases to prostaglandin (PG) E-1 or fluoride stimulation, indicating that both the GTP binding protein (N-component) and the catalytic subunit of the adenylate cyclase complexes were intact. The reduction in FSH-responsive adenylate cyclase activity in the abdominal testis was not due to a change in the Km for adenylate cyclase activation, but was due to a reduction in maximal velocities. Unilateral cryptorchidism was also associated with a rapid decline in soluble Mn2+-dependent adenylate cyclase activity in germ cells (spermatids). By 3 days after operation there was an 82% decrease in germ cell adenylate cyclase activity. The loss of soluble Mn2+-dependent adenylate cyclase activity was associated with a parallel decrease in Sertoli cell secretion of androgen binding protein, indicating that Sertoli cell factors may be important for the maintenance of germ cell adenylate cyclase activity. The desensitization of the gonadotrophin--responsive adenylate cyclases and the loss of gonadotrophin receptors in Leydig and Sertoli cells were not due to changes in plasma gonadotrophin values because LH concentrations were within normal limits and plasma FSH was only marginally elevated in the cryptorchid rats. No significant alterations of any of these parameters were seen in the scrotal testis of unilaterally cryptorchid rats when compared to values for intact controls.     

The unique organization of filamentous actin in the medullary canal of the medulla oblongata

In the central canal, F-actin is predominantly localized in the apical region, forming a ring-like structure around the circumference of the lumen. However, an exception is found in the medulla oblongata, where the apical F-actin becomes interrupted in the ventral aspect of the canal. To clarify the precise localization of F-actin, the fluorescence signals for F-actin were converted to the peroxidase/DAB reaction products in this study by a phalloidin-based ultrastructural technique, which demonstrated that F-actin is located mainly in the microvilli and terminal webs in the ependymocytes. It is because the ventrally oriented ependymocytes do not possess well-developed microvilli or terminal web that led to a discontinuous labeling of F-actin in the medullary canal. Since spinal motions can change the shape and size of the central canal, we next examined the cytoskeletons in the medullary canal in both rats and monkeys, because these two kinds of animals show different kinematics at the atlanto-occipital articulation. Our results first demonstrated that the apical F-actin in the medullary canal is differently organized in the animals with different head-neck kinemics, which suggests that the mechanic stretching of spinal motions is capable of inducing F-actin reorganization and the subsequent cell-shape changes in the central canal.