Purification and Characterization of a Sperm Motility Inhibiting Factor from Caprine Epididymal Plasma

Several studies have been reported on the occurrence of sperm motility inhibiting factors in the male reproductive fluids of different mammalian species, but these proteins have not been adequately purified and characterized. A novel sperm motility inhibiting factor (MIF-II) has been purified from caprine epididymal plasma (EP) by Hydroxylapatite gel adsorption chromatography, DEAE-Cellulose ion-exchange chromatography and chromatofocusing. The MIF-II has been purified to apparent homogeneity and the molecular weight estimated by Sephacryl S-300 gel filtration is 160 kDa. MIF-II is a dimeric protein, made up of two subunits each having a molecular mass of 80 kDa as shown by SDS-PAGE. The isoelectric point of MIF-II is 5.1 as determined by chromatofocusing and isoelectric focusing. It is a heat labile protein and maximal active at the pH 6.9 to 7.5. The sperm motility inhibiting protein factor at 2 µg/ml (12.5 nM) level showed maximal motility-inhibiting activity. The observation that the epididymal plasma factor lowered the intracellular cAMP level of spermatozoa in a concentration-dependent manner suggests that it may block the motility of caprine cauda spermatozoa by interfering the cAMP dependent motility function. The results revealed that the purified protein factor has the potential of sperm motility inhibition and may serve as a vaginal contraceptive. The antibody raised against the MIF-II has the potential for enhancement of forward motility of cauda-spermatozoa. This antibody may thus be useful for solving some of the problems of male infertility due to low sperm motility

Spin Biochemistry Modulates Reactive Oxygen Species (ROS) Production by Radio Frequency Magnetic Fields

The effects of weak magnetic fields on the biological production of reactive oxygen species (ROS) from intracellular superoxide (O2 N2) and extracellular hydrogen peroxide (H2O2) were investigated in vitro with rat pulmonary arterial smooth muscle cells (rPASMC). A decrease in O2 N2 and an increase in H2O2 concentrations were observed in the presence of a 7 MHz radio frequency (RF) at 10 mTRMS and static 45 mT magnetic fields. We propose that O2 N2 and H2O2 production in some metabolic processes occur through singlet-triplet modulation of semiquinone flavin (FADHN) enzymes and O2 N2 spin-correlated radical pairs. Spin-radical pair products are modulated by the 7 MHz RF magnetic fields that presumably decouple flavin hyperfine interactions during spin coherence. RF flavin hyperfine decoupling results in an increase of H2O2 singlet state products, which creates cellular oxidative stress and acts as a secondary messenger that affects cellular proliferation. This study demonstrates the interplay between O2 N2 and H2O2 production when influenced by RF magnetic fields and underscores the subtle effects of low-frequency magnetic fields on oxidative metabolism, ROS signaling, and cellular growth

RF magnetic fields increase H₂O₂ production in rPASMC independently of Paraquat and DPI.

<p>(<b>A</b>) The Paraquat control (200 μM) slightly decreases H₂O₂ production, while DPI (10 μM) slightly increases H₂O₂ compared to the SMF control cells (<b>B</b>) RF magnetic fields show no preferential effects on Paraquat/DPI-induced H₂O₂ production. RF magnetic field overall enhances H₂O₂ production in rPASMC by 30 to 40% as seen by (B) RF vs. (A) SMF control. Data are a representation of three independent experiments.</p

Inhibition of Cellular Proliferation and Enhancement of Hydrogen Peroxide Production in Fibrosarcoma Cell Line by Weak Radio Frequency Magnetic Fields

This study presents experimental data for the effects of weak radio frequency (RF) magnetic fields on hydrogen peroxide (H2O2) production and cellular growth rates of fibrosarcoma HT1080 cells in vitro. Cells were exposed either to 45μT static magnetic fields (SMFs)-oriented vertical to the plane of growth or to SMFs combined with weak 5 and 10MHz RF magnetic fields of 10μTRMS intensity perpendicular to the static field. Cell numbers were reduced up to 30% on Day 2 for the cells exposed to the combination of SMF and a 10MHz RF magnetic field compared with the SMF control cells. In addition, cells exposed to 10MHz RF magnetic fields for 8h increased H2O2 production by 55%. The results demonstrate an overall magnetic field-induced biological effect that shows elevated H2O2 levels with accompanying decrease in cellular growth rates.Fil: Castello, Pablo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Nevada; Estados UnidosFil: Hill, Iain. Universidad de Nevada; Estados UnidosFil: Sivo, Frank. State University of Colorado at Boulder; Estados UnidosFil: Portelli, Lucas. State University of Colorado at Boulder; Estados UnidosFil: Barnes, Frank. State University of Colorado at Boulder; Estados UnidosFil: Usselman, Robert. National Institute of Standards and Technology; Estados UnidosFil: Martino, Carlos F.. Universidad de Nevada; Estados Unidos. University Hospital RWTH Aachen; Alemani

RF electromagnetic fields and xenobiotics had significant effects on cellular detected superoxide.

<p>(<b>A</b>) RF initially decreased the amount of detected superoxide compared with SMF control by 40%. In the Paraquat (200 μM) samples, O₂^•− was initially suppressed by 60% compared with SMF control and the effects were enhanced by RF magnetic fields by 50% compared with the Paraquat control. (<b>B</b>) In the DPI (20 μM) increased O₂^•− production by 200% compared with SMF control, whereas RF DPI superoxide was decreased by 50% compared with DPI control. The data shows typical results observed in at least three independent experiments.</p

EPR signal of nitroxide free radicals that were formed by reacting cyclic hydroxylamines spin probe T-MH in the xanthine oxidase-superoxide-generating system containing xanthine oxidase 10 mU/ml, xanthine (100–400 μM), and DTPA (0.1 mM).

<p>The efficiency of TM-H spin probe was compared to peak heights of standard TEMPO nitroxide concentrations. For the same concentrations of reacted xanthine and TEMPO, TM-H peak height slope was 1/14 as large.</p

A schematic illustration demonstrates the singlet-triplet transitions that affect the kinetics of the effective intersystem crossing rate (k_ISC) in the radical pair.

<p>The sinusoid lines represent RF-induced transitions, which influence the effective intersystem crossing rate. Radical pairs that commence in the triplet state result in an increase in singlet product yield with concomitant decrease in triplet products, <i>vice versa</i>.</p

A diagram is shown that represents the experimental apparatus for magnetic field exposure.

<p>(<b>A</b>) Tri-dimensional representation of the tri-axial set used for controlling static and alternating electromagnetic fields. Square coil pairs in a Helmholtz configuration are geometrically aligned to control the static magnetic field (SMF) and to compensate for fluctuations in the ambient magnetic fields in the (1) horizontal (X) direction, (2) horizontal (Y) direction, and (3) vertical (Z) direction. This diagram also depicts the placement of a square coil in Helmholtz configuration for the generation of RF magnetic fields (4). A Faraday cage was also used in the RF experiments to surround the setup to minimize RF reflections, but it is not shown in this diagram for clarity. (<b>B</b>) This figure depicts the directions of the magnetic fields with respect to the biological samples. (1) A tri-axial set of square coils in Helmholtz configuration for SMF generation in all 3 dimensions; (2) square coils in Helmholtz configuration for RF generation in the horizontal (Y) direction; (3) an individual 6-well plate; (4) individual wells; (5) culture medium; and (6) a Faraday cage.</p

The general reaction scheme involves the spin biochemistry of an enzyme-bound reduced flavin and molecular oxygen.

<p>The RF magnetic fields modulate the interconversion rate of singlet-triplet spin correlated radical pairs. This influence disrupts ROS homeostasis, and therefore, the product distributions of H₂O₂ and O₂^•−, which were measured by separate spectroscopic techniques.</p