Effect of Milling on the Mechanical Properties of Chopped SiC Fiber-Reinforced ZrB2.

This work aims at studying the effect of the milling conditions on the microstructure and mechanical properties of a ZrB2-5 vol% Si3N4 matrix reinforced with chopped Hi-Nicalon SiC fibers. Several composites were obtained using different milling conditions in terms of time, speed and type of milling media. The composites were prepared from commercial powders, ball milled, dried and shaped, and hot pressed at 1720 °C. Their relative bulk densities achieved values as high as 99%. For each material the fiber length distribution, the extent of reacted fiber area and matrix mean grain size were evaluated in order to ascertain the effects of milling time, milling speed and type of milling media. While the fracture toughness and hardness were statistically the same independently of the milling conditions, the flexural strength changed. From the results obtained, the best milling conditions for optimized mechanical properties were determined.

Angiotensin II stimulates contraction and growth of testicular peritubular myoid cells in vitro.

Seminiferous tubule contraction, an important step in the regulation of spermatogenesis and testicular sperm output, is regulated by several agonists. In the present paper, we investigated whether angiotensin II (Ang II) may have a place among them. In binding experiments performed to assess the presence of specific receptors in rat peritubular myoid cells (TPMC), binding of (125)I-Ang II to TPMC was saturable in a time-dependent manner. Competition binding experiments performed with Losartan and PD 123319 showed that Losartan was able to inhibit the binding of (125)I-Ang II, whereas PD 123319 was ineffective. Ang II induced a dose-dependent rise in intracellular Ca(2+). Depletion of intracellular calcium stores by thapsygargin resulted in a lower rise of intracellular calcium, and the L-type voltage-operated calcium channel (VOCC-L) blocker verapamil abolished the Ca(2+) influx in rat TPMC. Altogether, these findings indicate that the Ang II-induced increase in [Ca(2+)](i) involves both extracellular influx and Ca(2+) release from intracellular stores. Ang II induced a dose-dependent TPMC contraction, and Losartan and not PD 123319 inhibited the response. Ang II-induced contraction was inhibited by adrenomedullin, previously shown to antagonize endothelin 1-provoked contraction in those cells. Ang II elicited (3)H-thymidine DNA incorporation and proliferation in a dose-dependent manner in TPMC. Losartan and both MAPK inhibitor PD 98059 and tyrosine kinase inhibitor AG18 were able to inhibit Ang II-induced (3)H-thymidine uptake and cell proliferation. In conclusion, the present study documents that angiotensin II, the active mediator of the tissue and circulating renin-angiotensin system present in the mammalian testis, induces contraction, growth and rise in intracellular calcium in rat peritubular myoid cells via angiotensin II type 1 receptors, and suggests that Ang II is involved in the paracrine regulation of the seminiferous tubule function.

Polychlorobiphenyls inhibit skeletal muscle differentiation in culture.

Polychlorinated biphenyls (PCBs) are ubiquitous and persistent pollutants whose role in developmental toxicity is of great concern. The observation that the offspring of PCB-exposed mothers (both in humans and rodents) display reduced body mass prompted us to investigate the effects of commercial mixtures of PCB congeners (Aroclor 1232, 1254, and 1262) on differentiation of both a myogenic cell line and primary myogenic cell cultures. The fusion of L6 myoblasts into multinucleated myotubes and the increase of creatine kinase (CK) activity were dose-dependently inhibited by Aroclor 1254 at concentrations (0.1-4 microg/ml) that caused no effect on cell density. Ultrastructural analysis demonstrated that Aroclor 1254 also prevented the accumulation of contractile filaments while inducing hypertrophy of the smooth endoplasmic reticulum and appearance of membrane-filled autophagosomes. Half-maximal inhibition (IC50) of CK activity accumulation occurred at 0.01 microg/ml for Aroclor 1262, 2 microg/ml for Aroclor 1254, and 8 microg/ml for Aroclor 1232. Aroclor-dependent inhibition of myogenic differentiation was also shown by the reduced expression and nuclear accumulation of beta-galactosidase in primary cultures of fetal myoblasts from transgenic mice expressing this reporter gene under the control of the myosin light chain promoter. These data show that skeletal muscle differentiation is specifically impaired by PCBs and may explain the reported depression of body mass growth in PCB-exposed offspring at birth. Furthermore, myogenic cell cultures are highly sensitive to PCBs and allow the detection of biological effects of environmental levels of these pollutants.

Vesicle-mediated phosphatidylcholine reapposition to the plasma membrane following hormone-induced phospholipase D activation.

Phospholipase D (PLD) activation involved in signal transduction may lead to the hydrolysis of conspicuous amounts of phosphatidylcholine (PC). This study shows that PLD activation significantly alters the plasma membrane (PM) environment and the membrane exchange dynamics. PC-PLD activation in vasopressin (AVP)-stimulated L6 myogenic cells was accompanied by increased exocytosis and decreased membrane fluidity, as shown by transmission EM and fluorescence spectroscopy of trimethylammonium-diphenyl-hexatriene. AVP-induced exocytosis appeared to be brefeldin A-insensitive. PLD inhibition by Zn(2+) and PC de novo synthesis inhibition by hexadecylphosphocholine abolished AVP-induced vesicle traffic. Upon AVP stimulation, metabolically labeled PC decreased in PM, then transiently increased in microsomes, and returned to the prestimulus level in the PM within 5 min, a phenomenon requiring PC neosynthesis and microtubule functionality. Vesicle traffic with similar features was also observed after endothelin-1-induced PC-PLD activation in rat peritubular myoid cells. These results indicate that, in nonsecretory cells, exocytosis coupled to PC de novo synthesis restores PM-PC, conspicuously consumed during PLD-mediated signal transduction.

Surface remodeling associated with vasopressin-induced membrane traffic in L6 myogenic cells.

The plasma membrane is dynamically remodeled as a function of the cell cycle, motility and membrane traffic. We have previously shown that arg8-vasopressin (AVP) stimulation of L6 myoblasts induces the activation of phosholipase D during the first minutes of stimulation, and the differentiation of 1,6 myoblasts as a long term effect. We now report that AVP also induces two types of morphological responses in L6 cells within a few minutes of stimulation: exocytosis, apparent as uncoated pits, and the generation of membrane projections and reffles. Thus, such an experimental model is suitable for the study of hormone-induced morphological surface modifications and their regulatory mechanisms. In L6 cells, AVP-induced projection generation depends on the integrity of microfilaments, intermediate filaments, and microtubules. Moreover, projection generation and exocytosis appear to be independently regulated phenomena: in fact, inhibition of the de novo synthesis of phosphatidylcholine inhibits membrane traffic but fails to block projection appearance. Conversely, the latter phenomenon, unlike exocytosis, is mediated by PI3-kinase signaling. Thus, AVP induces two early, independently regulated morphological modifications in L6 cells: exocytosis, involved in plasma membrane phospholipid turnover, and membrane projections, likely involved in cell migration.

Neuronal glycolytic pathway impairment induced by HIV envelope glycoprotein gp120.

Neurological impairment is a common feature of Acquired Immunodeficiency Syndrome (AIDS); functional alterations have been reported both in central and peripheral nervous system and the Human Immunodeficiency Virus (HIV) envelope glycoprotein gp120 has been proposed as a neurotoxin acting through a calcium-dependent mechanism. On the other hand it has been reported that gp120 treatment also induce about a 20% decrease in the cerebral glucose utilization and in the cellular ATP levels. The reported observations were performed on experimental system where also non-neuronal cells where present; in order to evaluate whether a direct interaction between HIV proteins and neuronal cells takes place, we used a neuroblastoma cultures where only neuronal cells are present. We analysed the effects of gp120 on the N18TG2 neuroblastoma clone. Treatments were performed both on growing and confluent cultures. Short time treatment with gp120 of confluent cultures causes a 25% reduction in the level of neuron-specific enolase, resulting in a similar decrease of oxygen consumption. Long time exposure of growing cells also causes a reduction in cell survival. Furthermore, using a membrane-specific fluorescent probe we observed that gp120 produces an increase of membrane trafficking. These observations suggest a direct interaction between the viral envelope protein and neuronal cells, which results in an alteration of glycolytic metabolism. This alteration may be related to the neurologic impairments observed in AIDS patients.

A novel aspect of lindane testicular toxicity: in vitro effects on peritubular myoid cells.

The in vitro effects of the insecticide lindane have been investigated in rat testis peritubular myoid cells (PMCs). Upon PMC exposure to lindane, polarity increase and decrease of dipole dynamics were seen at the membrane level (EC50 20 microM), leading to a partial dissipation of the membrane intrinsic dipole potential. The initial membrane depolarization was increased by Cl- efflux and limited by Ca(2+)-activated repolarizing currents. Concomitantly, lindane produced an increase in [Ca2+]i (EC50 125 microM) resulting from both Ca2+ release from an inositol 1,4,5-trisphosphate-sensitive intracellular store and a voltage-dependent Ca2+ influx from the extracellular medium. Of particular interest from a toxicologic point of view, insecticide concentrations well below those effective in altering ion homeostasis potently inhibited both [Ca2+]i increase and contraction induced by the natural agonists vasopressin and endothelin-1 (IC50s < 10 microM). These data demonstrate that PMCs are highly susceptible to lindane and suggest that the insecticide may exert testicular toxicity by interfering with hormone-regulated PMC function.

Effects of organochlorine xenobiotics on human spermatozoa.

The organochlorine insecticide lindane is a widely distributed environmental pollutant belonging to the growing family of endocrine disrupting chemicals (EDCs). Lindane intercalates into the sperm membrane and alters the molecular dynamics of the bilayer. In the present paper, preliminary data are reported showing that doses of lindane as low as those found in the female genital tract secretions inhibit the sperm cytological responsiveness to progesterone, the physiological agonist which stimulates the onset of acrosome reactions at the site of fertilization. The hypothesis is put forth that even background levels of lindane may exert antifertility effects independently on the health status of either the male and female reproductive organs.

Rapid decline of fertility in a case of adrenoleukodystrophy.

Adrenoleukodystrophy/adrenomyeloneuropathy (ALD/AMN) is a group of genetically determined peroxisomal disorders associated with progressive central demyelination, primary adrenal cortical insufficiency (Addison's disease) and, frequently, primary hypogonadism. Recently, testicular dysfunction was described in ALD/AMN patients but no information on sperm characteristics was provided. In this paper we studied the reproductive function of a patient with adult cerebral ALD, focusing our attention on sperm characteristics. At the time of diagnosis the patient was 22 years old, had high plasma C26 and C24 very-long-chain fatty acid (VLCFA) concentrations and adrenal insufficiency. Plasma testosterone concentration was in the normal range. The patient was prescribed a low-fat diet and 'Lorenzo's oil', which led to normalization of plasma VLCFA concentrations within 3 months of therapy. Semen analysis showed normal sperm count, gross morphological alterations and reduced motility. Electron microscopy analysis of sperm cells showed pathological changes in the head, the plasma membrane and the nucleus in 60% of the spermatozoa examined. However, isolated motile spermatozoa showed normal molecular dynamics of phospholipid bilayer surface and physiological responsiveness to progesterone. At the 12 months follow-up, the patient became azoospermic and testicular histology showed arrested maturation. To our knowledge, this is the first description of sperm alterations in a post-pubertal ALD patient, in which severe impairment of spermatogenesis and rapid progression to azoospermia occurred despite normalization of plasma VLCFA concentrations.

Partition of the organochlorine insecticide lindane into the human sperm surface induces membrane depolarization and Ca2+ influx.

The effects of the insecticide lindane (the gamma-isomer of 1,2,3,4,5,6-hexachlorocyclohexane) on membrane potential, cytosolic free Ca2+ concentration ([Ca2+]i) and surface biophysical properties were studied in human spermatozoa. The insecticide induces rapid, transient and reproducible membrane depolarization and opening of voltage-dependent Ca2+ channels leading to an increase in [Ca2+]i. In contrast with the effect in somatic cells, lindane did not affect gamma-aminobutyric acid receptor-linked Cl- currents. Ca2+ and K+ currents were found to drive lindane-induced membrane depolarization and repolarization respectively, whereas Na+ and Cl- fluxes appear not to have a role in the phenomenon. The insecticide was still able to produce membrane depolarization both in the combined absence of extracellular Ca2+ and Na+ and in high-K+ buffer, suggesting that lindane alters the membrane dipole potential. In agreement with this, Laurodan and Prodan fluorescence spectroscopy revealed that lindane partition into the sperm plasma membrane lowers water molecular dynamics in the uppermost region of the membrane external leaflet, probably as the result of reordering of water dipoles. We propose that the first effect of lindane partitioning into the sperm plasma membrane is a change in the membrane dipole potential, which results in the activation of membrane-located Ca2+-influx pathways.

Insulin binding and fluid-phase endocytosis stimulation in the mouse neuroblastoma cell line 41A3.

As well as many other hormones and growth factors, insulin is known to influence several processes in the CNS; its specific effects, however, are still poorly understood. Neuroblastoma cell lines represent a useful experimental system for the analysis of the insulin-specific effect on neurons, in the absence of possible regulatory mechanisms elicited by other neuronal/glial cells and/or soluble factors. The expression and the binding properties of insulin receptors, as well as the insulin effects on both membrane fluidity and cell surface architecture, have been investigated in 41A3 mouse neuroblastoma cells, by radioligand-binding fluorescence spectroscopy and scanning electron microscopy, respectively the same cells, insulin-induced modifications on cytoskeletal organisation also have been studied. Binding studies were performed using 125I-insulin, while the cationic fluorescent probe trimethylammonium 1,6-diphenyl-1,3,5-hexatriene was used for biophysical investigations. The results presented in this paper provide evidence that insulin interacts with 41A3 neuroblastoma cells through a receptor-mediated mechanism and that, in these cells, insulin binding modifies the cell surface morphology and stimulates endocytosis.

Endothelin-1 stimulates deoxyribonucleic acid synthesis and contraction in testicular peritubular myoid cells.

In the testis, endothelin-1 (ET-1) is produced by Sertoli cells, and it has been proposed to be a paracrine factor participating in the regulation of tubular and interstitial function. The response of purified testicular peritubular myoid cells (TPMC) to ET-1 was investigated in the present study. TPMC expressed a single class of high-affinity receptors that were shown by competitive binding experiments with sarafotoxin-6c to belong to the ETA subtype. The binding of ET-1 to TPMC was followed by rapid internalization of the receptor-ligand complex. ET-1 induced a prompt rise in intracellular Ca2+ concentration that was blunted in Ca(2+)-free medium and in the presence of Mn2+ or of voltage-operated-calcium-channel (VOC) blockers, indicating that both Ca2+ mobilization from intracellular stores and extracellular Ca2+ influx were involved. Thymidine uptake was promoted by ET-1 in a time-dependent manner, and the use of cyclo[D-Asp-L-Pro-D-Val-L-Leu-D-Trp] (BQ123) reduced the incorporation of thymidine. Protein kinase C (PKC) inhibition (100 nM calphostin C) abolished the ET-1 mitogenic effect. ET-1 also promoted TPMC contraction, as evaluated in collagen lattices, in a dose-related manner, with the half-maximal response observed at 1 nM. As in the case of mitogenesis, BQ123 blunted ET-1-induced contraction. PKC inhibition abolished ET-1-induced contraction. These findings indicate that ET-1 promotes DNA synthesis and contraction of TPMC and that both effects are mediated by PKC; they suggest as well that ET-1 may have a physiological role in the interaction between Sertoli cells and TPMC.

Laurdan fluorescence spectroscopy reveals a single liquid-crystalline lipid phase and lack of thermotropic phase transitions in the plasma membrane of living human sperm.

Membrane lipid phase(s), phase coexistence, and thermotropic phase transitions have been investigated in viable human spermatozoa using Laurdan fluorescence spectroscopy. Generalized polarization (GP) values derived from Laurdan excitation and emission spectra confirm that the sperm plasma membrane is a low polar, highly rigid (liquid-ordered) structure, and give evidence that, in the range from 10 degrees C to 42 degrees C, membrane lipids are in a single liquid-crystalline phase. The absence of phase transitions in the same thermal range argues against the hypothesis that the lipid domains previously detected on the sperm surface are produced by lipid lateral phase separation. The above findings are likely accounted for by the high cholesterol to phospholipid molar ratio found in the human sperm membrane. This is the first time that membrane lipid phase and polarity have been detected and quantified in living mammalian spermatozoa.

Platelet-derived growth factor effects on purified testicular peritubular myoid cells: binding, cytosolic Ca2+ increase, mitogenic activity, and extracellular matrix production enhancement.

The response of purified rat testicular peritubular myoid cells (PMC) to platelet-derived growth factor (PDGF) was studied. Freshly isolated PMC were devoid of measurable amounts of PDGF-binding sites. However, after 1 day in culture in serum-free conditions, specific high affinity receptors were detected. The estimated binding sites per cell revealed that PMC express more receptors for PDGF-BB, followed by PDGF-AB and PDGF-AA. PDGF treatment of cultured PMC increased the cytosolic Ca2+ concentration, showing a rank order of potencies with PDGF-BB > PDGF-AB > PDGF-AA. PMC proliferation, as measured by direct cell counting, was also stimulated by all three PDGF isoforms, with the same order of potencies observed for the increase in intracellular Ca2+. This effect was inhibited by antibodies to PDGF. Moreover, PDGF treatment increased the release of type IV collagen and fibronectin, and induced the release of type V collagen and laminin. These results demonstrate that testicular PMC are induced to express functionally active PDGF receptors in response to cell culturing. These data suggest that PMC may be a target for PDGF and that PDGF-mediated effects in vivo are dependent on factors regulating the expression of the receptors. The role that PDGF may play in normal and pathological testicular processes is discussed.

Insulin-sperm interaction: effects on plasma membrane and binding to acrosome.

In in vitro studies, viable, intact human spermatozoa took up free radioinsulin with an apparently non-receptor-mediated mechanism. However, when a colloidal gold-insulin complex was substituted for the radiotracer, no surface binding was visualized at the ultrastructural level. Upon sperm incubation in the presence of free insulin, a dose-dependent release of phospholipid phosphorus occurred, with a concomitant derangement of head cell membrane. After head membrane removal, spermatozoa-bound radioinsulin in a time- and concentration-dependent manner, the binding was displaceable by unlabeled insulin, and an exclusive localization of the colloidal gold-insulin complex was visualized at the acrosome level. On the basis of this evidence, both the plasma membrane and the acrosome seem to represent cytological targets for insulin.

Rat Leydig cells bind platelet-derived growth factor through specific receptors and produce platelet-derived growth factor-like molecules.

The platelet-derived growth factor (PDGF) is a major mitogen for cells of mesenchymal origin. Because Leydig cells arise from mesenchymal precursors, we tested the hypothesis that these cells might be a target for PDGF. We also investigated a possible production of a PDGF-like substance by Leydig cells in culture and the distribution of PDGF-like material in the rat testis using immunohistochemistry. PDGF was found to bind specifically to high affinity receptors on the surface of purified adult rat Leydig cells. Conditioned medium from cultured Leydig cells competed with 125I-labeled PDGF for binding to the Leydig cells. The secretion of PDGF receptor-competing activity was stimulated in a dose-dependent manner by the trophic hormone hCG. Immunohistochemical studies revealed specific staining for PDGF in the Leydig cells of adult rat testis. Taken together these observations suggest that PDGF may play a role in the local control mechanisms of testicular function.

Identification and localization of atrial natriuretic factor receptors in human spermatozoa.

Specific binding sites for atrial natriuretic factor (ANF) have been detected and localized in viable human spermatozoa through radioreceptor analysis and autoradiography, respectively. Radiotracer uptake was time and concentration dependent. Scatchard analysis of saturation data showed a single class of ANF receptors with a kd of 2.5 nM and a Bmax of 1.03 fmol/10(6) sperm 2.5 min-1, corresponding to about 620 molecules per sperm. Nonreducing SDS-PAGE analysis after covalent cross-linking of sperm bound 125I-ANF evidenced a single displaceable (i.e., specific) band with an apparent molecular weight of 135-140kD. In 125I-ANF bound spermatozoa, optical autoradiography showed an exclusive distribution of silver grains covering the midpiece region. The effects of ANF binding on ionic homeostasis and cyclic nucleotide metabolism, which modulate a number of sperm cellular processes, could make this factor play outstanding roles in gamete physiology.

Calcium uptake in human spermatozoa: characterization and mechanisms.

Basal 45Ca2+ influx was analyzed in human seminal spermatozoa using a method that allows these highly reactive cells to be easily and safely handled. The uptake was a time-dependent process, with its maximum at 400 s. The kinetics of 45Ca2+ transport was saturating as a function of extracellular Ca2+ concentration with a Km of 429 microM and a Vmax of 1.6 nmol 45Ca2+/mg protein/2.5 min. Depolarizing conditions and the calcium channel blocker verapamil did not affect the uptake; based on this, the presence of operating calcium channels in seminal spermatozoa is excluded. The independence of 45Ca2+ uptake on external concentration of both Na+ and Ca2+ suggests that Na+/Ca2+ exchange does not occur in these cells. The anticalmodulin drug trifluoperazine, the mitochondrial inhibitor antimycin A, and the SH reagents N-ethylmaleimide and mersalyl all inhibited the ion transport. A calmodulin-regulated, energy-requiring, proteinaceous Ca2+ transporter seems to be the main operating mechanism of calcium uptake in human seminal gametes.