Mechanical exfoliation and layer number identification of single crystal monoclinic CrCl3.

After the recent finding that CrI3, displays ferromagnetic order down to its monolayer, extensive studies have followed to pursue new two-dimensional (2D) magnetic materials. In this article, we report on the growth of single crystal CrCl3 in the layered monoclinic phase. The system after mechanical exfoliation exhibits stability in ambient air (the degradation occurs on a time scale at least four orders of magnitude longer than is observed for CrI3). By means of mechanical cleavage and atomic force microscopy (AFM) combined with optical identification, we demonstrate the systematic isolation of single and few layer flakes onto 270 nm and 285 nm SiO2/Si (100) substrates with lateral size larger than graphene flakes isolated with the same method. The layer number identification has been carried with statistically significant data, quantifying the optical contrast as a function of the number of layers for up to six layers. Layer dependent optical contrast data have been fitted within the Fresnel equation formalism determining the real and imaginary part of the wavelength dependent refractive index of the material. A layer dependent (532 nm) micro-Raman study has been carried out down to two layers with no detectable spectral shifts as a function of the layer number and with respect to the bulk.

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.

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.

[The role of biological monitoring in the evaluation of the risk of from chemical compounds]. / Il ruolo del monitoraggio biologico nella valutazione del rischio da composti chimici.

Over the years, the assessment of the risk to human health from occupational and environmental exposure to chemicals has become increasingly important. Exposure to chemicals, their biochemical effects and individual susceptibility can be estimated by biological monitoring carried out on potentially exposed subjects. Valid markers are needed to be effectively used within the framework of biological monitoring programs. Quality assurance, which includes all those activities necessary to provide adequate confidence that the results of laboratory test are reliable, is of the utmost importance. Among these activities the participation in external quality assessment schemes is strongly recommended. Biological monitoring has a key role also in the field of human reproduction since the level of exposure to many chemicals which are known or suspected to be reproductive toxicants can be assessed by specialized laboratories.

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.

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.

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.

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.

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.

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.

Adrenomedullin inhibits the contraction of cultured rat testicular peritubular myoid cells induced by endothelin-1.

The present study documents that adrenomedullin (AM), a vasoactive peptide originally identified in pheochromocytoma tissue and present in the testis, in vitro affects the function of testicular peritubular myoid cells (TPMC), a contractile cell type located in the seminiferous tubule wall. AM stimulated cAMP production by cultured TPMC taken from 16-day-old rats, and this effect was completely inhibited by the AM antagonist AM-(22-52) and partially by the CGRP (calcitonin gene-related peptide) antagonist CGRP-(8-37). Studies on TPMC contractile activity documented that AM inhibits TPMC contraction induced by endothelin-1 (ET-1) and that its effect is antagonized by AM-(22-52). Neutralizing AM produced by TPMC with the addition of anti-AM antibody induced a significant increase of ET-1-induced contraction. When exposed to the protein kinase A inhibitor H-89, AM inhibitory activity on ET-1-induced TPMC contraction was suppressed, whereas the nitric oxide synthase inhibitor N:(G)-nitro-L-arginine methyl esther did not modify AM activity. In conclusion, our study indicates that AM stimulates cAMP production and inhibits the contraction induced by ET-1 in TPMC in vitro, and that AM produced by TPMC has an autocrine effect. We propose that AM may have a role in the control of seminiferous tubule contraction.

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.