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.

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.

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.

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.

Identification of calcitonin receptors in human spermatozoa.

Calcitonin (CT) is a powerful hypocalcemic hormone which regulates calcium balance in cells. The presence of CT and CT receptors has been demonstrated in many extrathyroidal tissues, including the male genital tract. CT immunoreactivity has also been found in human seminal fluid, and an inhibitory effect of salmon CT on human sperm motility in vitro was recently reported. In this study the presence of specific binding sites for synthetic salmon CT in intact human spermatozoa was investigated using [125I]salmon CT. Binding experiments demonstrated a CT-sperm interaction involving a receptor-mediated mechanism. The binding was very rapid and minimally reversible, with the maximal site saturation occurring at approximately 2 nM labeled peptide. The dissociation of the CT-receptor complex was only slightly influenced by the addition of unlabeled hormone. Increasing concentrations of unlabeled salmon, eel, and human CT produced a dose-dependent inhibition of [125I]salmon CT binding. These data fulfill the major criteria for demonstration of specific receptors for salmon CT in human spermatozoa. Owing to the key role of calcium ions in regulating sperm motility and the onset of the acrosomal reaction, CT receptors could be important in male gamete physiology.

Evidence for the presence of specific receptors for N-formyl chemotactic peptides on human spermatozoa.

Synthetic N-formylated peptides are potent chemoattractants for human spermatozoa in vitro. The specific structure-activity relations for eliciting a chemotactic response and the ability of the antagonist tertbutoxycarbonyl-phenylalanyl-leucyl-phenylalanyl-leucyl- phenylalanine (Boc-Phe-Leu-Phe-Leu-Phe) to inhibit the chemotaxis induced by these peptides strongly suggest the presence of receptors on human spermatozoa. The following studies were performed to identify specific binding sites on human spermatozoa by using [35S]-N-formyl-methionyl-leucyl-phenylalanine [( 35S]f-Met-Leu-Phe), a potent chemotactic peptide. Binding of the [35S]formyl-peptide to human spermatozoa was rapid (t1/2, 8 min) and reversible. Binding isotherms of the saturation experiments revealed a single class of high affinity, low capacity binding sites (equilibrium dissociation constant, 17.7 nM; maximal binding, 109 fmol/2 X 10(6) cells) and an average number of 60,000 receptors per cells. The biological potencies of a series of formyl peptides as chemoattractants correlated closely with their relative abilities to compete with [35S]f-Met-Leu-Phe for specific binding to human spermatozoa. These data fulfill the major criteria for demonstration of specific receptors for chemotactic peptides on human spermatozoa. It is likely that these receptor sites initiate the chemotactic response of human spermatozoa to N-formyl peptides.

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.

Histones instead of protamines in terminal germ cells of infertile, oligospermic men.

A study was carried out, by means of polyacrylamide gel electrophoresis, of the nucleoproteins of spermatozoa from infertile, oligospermic patients affected by partial idiopathic spermatidic arrest. Results in a normal group of men, in agreement with previous reports, showed the presence of protamines in mature spermatozoa. Data relative to nucleoproteins in "spermatozoa" from the infertile patients showed that, in these cells, no protamines were detectable, but histones exclusively. These findings suggest that in our patients a maturational defect of spermatogenesis exists both at the meiotic level (a DNA content double that of spermatozoa) and during spermatidic maturation (no substitution of histones by protamines). Therefore, the nuclear maturation of these terminal germ cells is that typical of that of primary spermatocytes.

High deoxyribonucleic acid content of spermatozoa from infertile, oligospermic human males.

A study has been carried out, by means of an indole microchemical technique, of the DNA content of spermatozoa from a group of 13 normal subjects and 13 patients affected by infertility, characterized by oligospermia with a clinical, hormonal, and testicular picture peculiar to "idiopathic spermatidic arrest." The DNA content of spermatozoa from the infertile, oligospermic patients was nearly double that of normal subjects. This finding can be explained tentatively on the basis of genetic and/or cytologic events occurring during meiosis.

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.

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.

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.

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.

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.

Oxygen consumption of human seminal plasma: relationships with semen characteristics.

It is possible to determine oxygen consumption rate in human seminal plasma through voltammetric measurements. The seminal fluid of the sterile patients in this investigation was characterized by an anomalous sperm motility. Also studied was a control group of proven fertile subjects. The mean value of oxygen consumption resulted greater in the reduced sperm motility group than in the control group. The polyaminoxidase system is the most important factor in oxygen consumption in seminal plasma; however, it is likely that the high toxicity of polyamine degradation products is the cause of reduced and/or anomalous sperm motility in some seminal fluids.

Arginine evaluation to verify sperm maturation in man.

A modified version of Sakaguchi's histochemical reaction is proposed for the evaluation of arginine content in spermatozoa as an index of sperm maturation. Results obtained with this procedure, compared with DNA and basic nuclear proteins determinations in the sperm cells from normal subjects and from patients with oligospermia, appear to confirm the usefulness of this technique.

Zinc uptake in human seminal spermatozoa: characterization and effects on cell membranes.

Human ejaculated spermatozoa take up zinc in a time- and concentration-dependent manner. The kinetics of 65Zn2+ uptake is suggestive of an at least partly carrier-mediated transport. The lack of effect of the respiratory chain inhibitor antimycin A could mean that mitochondrial ATP is not required as an energy source for the uptake. The failure of nonpermeant SH reagent mersalyl to modify zinc uptake indicates that functional membrane sulfhydryl groups are not involved in the process. A dose-dependent inhibition of 65Zn2+ uptake was induced by the "anticalmodulin" drug trifluoperazine, suggesting that the calcium-binding protein calmodulin could have a role in zinc transport. In in vitro experiments this cation brought about a powerful effect in protecting the spermatozoa from being damaged by hypo-osmosis.