Rosella Colonna

Extremely low frequency electromagnetic fields (ELF‐EMFs) induce in vitro angiogenesis process in human endothelial cells

Effects of extremely low frequency (ELF) electromagnetic fields (EMFs) on activation of angiogenesis were analysed using cultured umbilical human vein endothelial cells (HUVECs). The cultures were exposed to a sinusoidal EMF to intensity of 1 mT, 50 Hz for up to 12 h. EMFs increased the degree of endothelial cell proliferation and tubule formation, coupled by an acceleration in the process of wound healing. Since this process is physiologically accompanied by a large modification in the structural organization of actin and focal adhesions, we analyzed the rearrangement of some cytoskeleton elements demonstrating a major reorganization of the fibres and of the focal adhesion complexes after EMF exposure. Finally, Western blot analysis revealed a significant increase in phosphorylation as well as the overall expression of VEGF receptor 2 (KDR/Flk-1) suggesting that EMFs may modulate in vitro some endothelial functions correlated to angiogenesis through signal transduction pathways dependent on VEGF.

Involvement of mitochondrial activity in mediating ELF-EMF stimulatory effect on human sperm motility

It has recently been reported that the exposure of human spermatozoa to an extremely low frequency (ELF) electromagnetic field (EMF) with a square waveform of 5 mT amplitude and frequency of 50 Hz improves sperm motility. The functional relationship between the energy metabolism and the enhancement of human sperm motility induced by ELF-EMF was investigated. Sperm exposure to ELF-EMF resulted in a progressive and significant increase of mitochondrial membrane potential and levels of ATP, ADP and NAD(+) that was associated with a progressive and significant increase in the sperm kinematic parameters. No significant effects were detected on other parameters such as ATP/ADP ratio and energy charge. When carbamoyl cyanide m-chlorophenylhydrazone (CICCP) was applied to inhibit the oxidative phosphorylation in the mitochondria, the values of energy parameters and motility in the sperm incubated in the presence of glucose and exposed to ELF-EMF did not change, thus indicating that the glycolysis was not involved in mediating ELF-EMF stimulatory effect on motility. By contrast, when pyruvate and lactate were provided instead of glucose, the energy status and motility increased significantly in ELF-EMF-treated sperm. Under these culture conditions, the inhibition of glycolitic metabolism by 2-deoxy-D-glucose (DOG) again resulted in increased values of energy and kinematic parameters, indicating that gluconeogenesis was not involved in producing glucose for use in glycolysis. We concluded that the key role in mediating the stimulatory effects exerted by ELF-EMF on human sperm motility is played by mitochondrial oxidative phosphorylation rather than glycolysis.

Mammalian oocyte growth in vitro is stimulated by soluble factor(s) produced by preantral granulosa cells and by Sertoli cells

We have evaluated the possibility that mouse oocyte growth in vitro could be achieved under the influence of soluble compound(s) released by different somatic cell types. For this purpose, zona‐free denuded oocytes from 12‐day‐old mice were cultured on monolayers of NIH‐3T3 fibroblasts, which are able to establish gap junctional communications with them, in the presence or absence of media conditioned by preantral granulosa cells or by Sertoli cells, plated at increasing concentrations from 0.3–1 × 106 ml−1 cells. After 3 days, no increase in vitellus diameter was recorded from fibroblast‐coupled oocytes maintained in culture medium or in the presence of media conditioned by 0.3 × 106 ml−1 Sertoli cells. By contrast, increasing proportions of coupled oocytes grew, provided the continuous presence of media conditioned by 0.5 or 1 × 106 ml−1 Sertoli cells, or by 0.3, 0.5, and 1 × 106 ml−1 preantral granulosa cells. Since the ligand of c‐kit, the growth factor KL, promotes the growth in vitro of oocytes cultured in follicles from 8‐day‐old mice, an antibody against mouse KL was used to evaluate whether in our culture conditions KL might also be responsible for the growth of oocytes from 12‐day‐old mice. No inhibition of growth was evident in oocytes cultured directly on preantral granulosa or Sertoli‐cell monolayers. Furthermore, the growth of fibroblast‐coupled oocytes cultured in media conditioned by preantral granulosa cells was not significantly affected by the presence of this antibody during culture. By contrast, a high percentage of oocytes cultured on fibroblasts in the presence of media conditioned by Sertoli cells showed a significant inhibition of growth and no metabolic cooperativity. It was concluded that, besides KL, other bioactive factor(s) released by either preantral granulosa or Sertoli cells can induce a significant stimulation of mouse oocyte growth in vitro.

Influence of granulosa cells and of different somatic cell types on mammalian oocyte development in vitro

In mammals the ability of an oocyte to become fertilised is the result of a complex process occurring within the ovarian follicle which depends on the stagespecific expression of oocyte genes and the presence of granulosa cells (for a review see Buccione et al., 1990a). The coordinated development of germinal and somatic components of the follicle is regulated by two principal systems of interaction, based on the presence of gap junctions and on the production of paracrine factors. Gap junctions link granulosa cells to each other and to the oocyte (Anderson & Albertini, 1976), and represent a major route for the transfer of small molecules involved in oocyte metabolism (for a review see Mangia et al., 1992) and regulation of the arrest and resumption of meiosis (for a review see Eppig, 1993). The production of paracrine factors by granulosa cells has been suggested by the findings that these cells express the production of the Steel locus, the Steel factor (SLF) or kit ligand (KL; Motro et al., 1991; Manova et al., 1993), and that this factor promotes oocyte growth in vitro when used at high concentrations (Packer et al., 1994). Since KL is too large to be transmitted through gap junctions, it must necessarily be released in the extracellular environment before binding to the c-kit receptor present on oocyte membrane (Manova et al., 1990; Horie et al., 1991).

Inhibition of Angiogenesis Mediated by Extremely Low- Frequency Magnetic Fields (ELF-MFs)

The formation of new blood vessels is an essential therapeutic target in many diseases such as cancer, ischemic diseases, and chronic inflammation. In this regard, extremely low-frequency (ELF) electromagnetic fields (EMFs) seem able to inhibit vessel growth when used in a specific window of amplitude. To investigate the mechanism of anti-angiogenic action of ELF-EMFs we tested the effect of a sinusoidal magnetic field (MF) of 2 mT intensity and frequency of 50 Hz on endothelial cell models HUVEC and MS-1 measuring cell status and proliferation, motility and tubule formation ability. MS-1 cells when injected in mice determined a rapid tumor-like growth that was significantly reduced in mice inoculated with MF-exposed cells. In particular, histological analysis of tumors derived from mice inoculated with MF-exposed MS-1 cells indicated a reduction of hemangioma size, of blood-filled spaces, and in hemorrhage. In parallel, in vitro proliferation of MS-1 treated with MF was significantly inhibited. We also found that the MF-exposure down-regulated the process of proliferation, migration and formation of tubule-like structures in HUVECs. Using western blotting and immunofluorescence analysis, we collected data about the possible influence of MF on the signalling pathway activated by the vascular endothelial growth factor (VEGF). In particular, MF exposure significantly reduced the expression and activation levels of VEGFR2, suggesting a direct or indirect influence of MF on VEGF receptors placed on cellular membrane. In conclusion MF reduced, in vitro and in vivo, the ability of endothelial cells to form new vessels, most probably affecting VEGF signal transduction pathway that was less responsive to activation. These findings could not only explain the mechanism of anti-angiogenic action exerted by MFs, but also promote the possible development of new therapeutic applications for treatment of those diseases where excessive angiogenesis is involved.

Amino acid transport systems in growing mouse oocytes

Amino acid transport systems have been studied in isolated mouse oocytes throughout oogenesis. While these cells were lacking the A-transport system of Ehrlich cell (Christensen, 1975), they were found to be provided with the L- and ASC-systems of Ehrlich cell (Christensen, 1975). Since, in contrast to the A-system, the L- and ASC-systems mainly work by exchanging internal and external amino acids, we propose that their main function is to actively maintain the appropriate internal balance between different amino acids within the growing oocyte.

Spectrin and Ankyrin‐like Proteins in the Egg of Discoglossus pictus (Anura): Their Identification and Localization in the Site of Sperm Entrance versus the Rest of the Egg

The Discoglossus pictus egg has a specific site of sperm‐egg interaction, the dimple, which has a well‐defined cytoskeleton. We studied whether there are cytoskeletal and cytoskeleton‐related proteins typically involved in the polarization of plasma membrane proteins. The identity and the localization of the molecules cross‐reacting with antispectrin, antifodrin and antiankyrin antiobodies were investigated by immunofluoresecence and immunoblotting of the proteins of the dimple (D) and of the rest of the egg (dimple‐less‐egg; DLE). Two polypeptides of about 254‐and 246‐kD were detected in the D and DLE, and localized in the egg cortex. A third molecule, weakly cross‐reacting with antispectrin and antifodrin, was found in the subcortical cytoplasm. The 246‐kD polypeptide was labile in samples prepared for SDS‐PAGE; a mild prefixation of eggs prevented its dispersion. Mild fixation was also needed to retain antispectrin reactivity in cryostat sections of the DLE cortex, while this is not necessary in D. A molecule of about 204‐kD, cross‐reacting with antiankyrin, was detected in the cortex of the whole egg. These data and the finding that the concentrations of both the 254‐kD polypeptide and ankyrin are about 12‐fold higher in D than in the DLE, suggest that, in D, spectrin has a specific organization.

ELF-MF transiently increases skeletal myoblast migration: Possible role of calpain system

Abstract Purpose: Cell migration is crucial for myogenesis since it is required for the alignment and fusion of myoblast. Ca2+ signals are involved in regulating myoblast migration and an extremely low frequency (ELF) magnetic field (MF) increases intracellular calcium levels in C2C12 myoblast. This study was aimed at investigating whether ELF-MF could affect myoblast migration. As calpains contribute to the regulation of myoblast motility, the effect of ELF-MF on μ- and m-calpain was also investigated. Materials and methods: The effect of ELF-MF (1 mT; 50 Hz) on C2C12 cell motility was observed by wound-healing assay. Protein expression of calpains, calpastatin, myristoylated alanine-rich C-kinase substrate (MARCKS) and vinculin were examined by Western blot analysis. Casein zymography and immunofluorescence analysis were carried out to evaluate, respectively, activity levels of calpains and intracellular distribution of calpains, calpastatin and actin. Results: Exposure to ELF-MF resulted in a transient but significant increase of myoblast migration. This stimulatory effect was associated with a marked increase of μ- and m-calpain activity followed by the concomitant variation in their subcellular localization. No significant changes in intracellular distribution and protein levels of calpastatin were detected. Finally, a significant decrease of MARCKS expression and modifications of actin dynamics were reported. Conclusions: This study clearly outlines an involvement of calpains in ELF-MF-mediated myoblast migration.

Stage-dependent modifications of amino acid uptake by antral and metaphase II mouse oocytes

Modifications of leucine transport system of mouse oocytes have been studied throughout Graafian follicle development and oocyte maturation. In contrast to sheep oocytes (Moor and Smith, 1979), in the mouse kinetic constants and efflux rate of leucine transport system did not vary in diestrus, proestrus, and metaphase II (met II) oocytes. However, kinetics of leucine equilibration in proestrus and met II oocytes was significantly slower than that found in diestrus cells, and this may reflect a decreased availability of internal amino acids for exchange.

Gp273, the Ligand Molecule for Sperm-Egg Interaction in the Bivalve Mollusk, Unio elongatulus, Binds to and Induces Acrosome Reaction in Human Spermatozoa Through a Protein Kinase C-Dependent Pathway

Abstract In a previous article, we suggested that gp273, the ligand molecule for sperm-egg interaction in the bivalve mollusk Unio elongatulus has functional carbohydrate epitopes in common with a human zona pellucida glycoprotein, probably ZP3. We demonstrated that: 1) anti-gp273-purified immunoglobulin G (IgG), which recognizes a carbohydrate gp273 epitope including a Lewisa-like structure, interacts with a zona pellucida protein; 2) human sperm specifically bind to gp273; and 3) binding is reversed by anti-gp273 IgG. In the present study, we confirm this suggestion by demonstrating that heat-solubilized zonae pellucidae reverse gp273-human sperm binding, that gp273-binding sites are restricted to the acrosomal region, and that gp273 induces the acrosome reaction in human sperm. We also demonstrated that gp273-binding sites on human sperm function as signaling receptors because exposure of spermatozoa to this glycoprotein results in significant stimulation of protein kinase C (PKC) activity. Because the PKC inhibitor, bisindolylmaleimide I, reverses both PKC activation and the acrosome reaction, this kinase is a key component of the signal transduction pathway activated by gp273 and leading to the exocytotic event.