Light-mediated activation reveals a key role for protein kinase A and sarcoma protein kinase in the development of sperm hyper-activated motility.

BACKGROUND: Hyper-activated motility (HAM) is part of the sperm capacitation process, which is necessary for fertilization. In this study, we investigated the effect of visible light on sperm motility and hyperactivation and evaluated pathways mediating these effects. METHODS: Human sperm (1 × 107 cells/ml) in capacitation media were irradiated for 3 min with 40 mW/cm² visible light (400-800 nm with maximum energy at 600 nm). Sperm motility was assessed and analyzed by computer-assisted sperm analysis. The involvement of sperm capacitation factors was investigated as follows. The generation of reactive oxygen species (ROS) was measured using 20,70-dichlorofluorescein diacetate. Protein kinase A (PKA) and sarcoma protein kinase (Src) activity were measured using western blot analysis and inhibited using 50 µM H89 and 10 µM PP2, respectively. Soluble adenlyl cyclase was inhibited using 20 µM 2-OH-Estradiol. The intracellular concentration of free Ca(2+) was assessed using the fluorescent calcium indicator, Fluo-4/AM. Sperm DNA fragmentation was determined using the sperm chromatin dispersion test. RESULTS: Light irradiation of human sperm caused a significant increase in hyper-HAM but not total motility. The production of ROS and activation of soluble adenylyl cyclase and PKA mediated the effect of light on HAM. Light irradiation also activated Src, and inhibition of Src significantly reduced the effect of light on HAM. Light irradiation caused a rapid increase in intracellular Ca²âº concentration and the increase in HAM was significantly reduced when voltage-dependent-Ca²âº-channel activity was blocked or when Ca²âº-deficient medium was used. CONCLUSIONS: Light irradiation of human sperm for a short time causes a significant increase in HAM in a mechanism mediated by ROS production, activation of PKA, Src and Ca²âº influx.

Influence of visible light and ultraviolet irradiation on motility and fertility of mammalian and fish sperm.

OBJECTIVE: The effects of visible light irradiation on sperm motility, fertility, and reactive oxygen species (ROS) formation were investigated and compared in ram and fish (tilapia). BACKGROUND DATA: Low-energy visible light has previously been found to modulate various processes in different biological systems. In the literature, it is accepted that the first step following visible light irradiation is the formation of ROS by endogenous cellular photosensitizers. METHODS: Sperm of ram and tilapia were irradiated with various light sources (400-800 nm white light, 660 nm red light, 360 nm blue light, 294 nm UV), and their motility and fertility rates were measured. The amount of ROS generated by irradiation was estimated using electron paramagnetic resonance (EPR) technique. RESULTS: Sperm taken from tilapia showed higher motility and fertility following red and white light irradiation. In contrast, the motility and fertility of ram sperm were slightly increased only by red light. A negative effect on motility and fertility of sperm of both species was obtained following irradiation with UV and blue light. The amount of ROS produced in irradiated tilapia sperm was much higher than that of ram sperm. CONCLUSIONS: The results show that different wavelengths differentially affect tilapia and ram sperm motility and fertilization. The difference in response to the various light sources might be explained by the different amounts of ROS formation by ram and tilapia, which are in agreement with the physiology of fertilization appropriate to each of these species. Based on these results, it is suggested that in vitro fertilization in mammals should be performed in darkness or at least under red light.

Stimulation of reactive oxygen species production by an antidepressant visible light source.

BACKGROUND: The mechanism by which visible light stimulates chronobiological phase-shifting or antidepressant effects in humans is unknown. METHODS: Normal human NIH/3T3 nonpigmented fibroblasts were irradiated with a visible light source (SunRay) used in the treatment of winter seasonal depression. Electron spin resonance was assessed before and after 10 min of illumination at 2 mW/cm(2) (illuminance of 3700 lux), with and without the presence of 5 microL of 0.0214 mg/mL vitamin C. RESULTS: The fibroblasts showed evidence of production of reactive oxygen species after 10 min of irradiation. CONCLUSIONS: These in vitro data establish that an antidepressant source of visible light is capable of inducing the production of reactive oxygen species in skin. Such species may participate in signal transduction pathways leading to mood changes.

Light irradiation of mouse spermatozoa: stimulation of in vitro fertilization and calcium signals.

Irradiation of mouse spermatozoa by 630 nm He-Ne laser was found to enhance the intracellular calcium levels and fertilizing potential of these cells. The effect of light on calcium transport and on fertilization rate was abrogated in the absence of Ca2+ during the irradiation time, indicating that the effect of light is Ca2+ dependent. The stimulatory effect of light on Ca2+ uptake was abolished in the presence of a voltage-dependent Ca(2+)-channel inhibitor nifedipine, indicating the involvement of a plasma membrane voltage-dependent Ca2+ channel. Furthermore, the stimulatory effect of light was completely inhibited by the mitochondrial uncoupler FCCP, indicating that laser irradiation might affect the mitochondrial Ca2+ transport mechanisms. A causal association between laser irradiation, reactive oxygen species (ROS) generation and sperm function was indicated by studies with ROS scavengers, superoxide dismutase (SOD) and catalase, and exogenous hydrogen peroxide. The SOD treatment, which enhanced H2O2 production, resulted in increased Ca2+ uptake and enhanced fertilization rate. On the other hand, catalase, which decomposes H2O2, impaired the light-induced stimulation in Ca2+ uptake and the fertilization rate. Taken together, the data suggest that H2O2 might be involved in the irradiation effects, and indeed laser irradiation enhances the production of H2O2 by spermatozoa. These results indicate that the effect of 630 nm He-Ne laser irradiation is mediated through the generation of H2O2 by the spermatozoa and that this effect plays a significant role in the augmentation of the sperm cells' capability to fertilize metaphase II-arrested eggs in vitro.

Effect of light on calcium transport in bull sperm cells.

The effect of light on calcium transport was studied. Bull sperm cells were irradiated with an He-Ne (630 mm) laser and a 780 nm diode laser at various energy doses, and 45Ca2+ uptake was measured by the filtration technique. It was found that there is an accelerated Ca2+ transport in the irradiated cells, which means that laser light can stimulate Ca2+ exchange through the cell membrane. This may cause transient changes in the cytoplasmic Ca2+ concentration which, in spermatozoa, has a regulatory role in control of motility and acrosome reaction, and in other cells can trigger mitosis.

Effects of visible and near-infrared lasers on cell cultures.

The effect of 360, 632 and 780 nm light on NIH fibroblast cells was examined. Mitosis counts of irradiated cells at various energy doses were taken. Scanning electron micrographs of these cells were studied. It is suggested that low-level laser therapy in the visible and in the near-infrared region is due to cell respiration stimulation by either the endogenous porphyrins in the cell, or by the cytochromes.

Changes in calcium transport in mammalian sperm mitochondria and plasma membrane irradiated at 633 nm (HeNe laser).

The effect of light on calcium transport in mammalian sperm mitochondria and plasma membrane was studied. Digitonine-treated spermatozoa and plasma membrane vesicles were irradiated with an HeNe laser at various powers and energy doses and Ca2+ uptake was measured by the filtration method. It was found that there is an accelerated Ca2+ uptake by the mitochondria after low power HeNe irradiation and inhibition after high power. The flux of Ca2+ from the mitochondria was also examined and was found to be unaffected by the HeNe light. The ATP-dependent Ca2+ uptake by the bovine plasma membrane vesicles was not changed by the HeNe irradiation.

A possible mechanism for the bactericidal effect of visible light.

Visible light at high intensity was found to kill bacteria while low-power light in the visible and near infrared region enhances bacterial proliferation. The present review summarizes evidence demonstrating that the mechanism of visible light- bacteria interaction involves reactive oxygen species (ROS) generation. The ROS are photo induced by bacterial endogenous photosensitizers. Phototoxic effects were found to involve induction of high amounts of reactive oxygen species (ROS) by the bacteria while low amounts of ROS may promote their proliferation. Intense blue light, preferably at 415nm, is better than red light for bacteria killing.

New methods of treatment of severely injured sciatic nerve and spinal cord. An experimental study.

It has been demonstrated that low-energy laser irradiation (LELI) applied simultaneously to the injured sciatic nerve and the corresponding segment of the spinal cord, accelerates the process of regeneration of the injured peripheral nerve. A beneficial influence of LELI was also observed when it was applied to the spinal cord following transection and implantation of a segment of an autologous sciatic nerve, but further studies are necessary to evaluate if real regeneration or only earlier distal cord automatism occurred. Both methods are proposed for treatment of peripheral nerve lesions (PNS) and spinal cord injuries.

The in-vivo-nerve response to direct low-energy-laser irradiation.

To study the in-vivo effects of direct Low-Energy Laser Irradiation (LELI) on the Peripheral Nervous System, the sciatic nerve in rats was surgically exposed, crushed and then subjected to the direct irradiation of either of two continuous-wave HeNe lasers--0.3 mW or 17 mW. We found that the 0.3 mW laser significantly boosts the electrical activity in both the injured and non-injured nerves. The temperature changes of the nerve were measured during irradiation, and no thermal effect was detected. These findings could have direct therapeutic applications in various surgical situations.

Changes in calcium transport in mammalian sperm mitochondria and plasma membranes caused by 780 nm irradiation.

BACKGROUND AND OBJECTIVE: Regulation of intracellular Ca2+ concentrations are very important in control of sperm motility and acrosome reaction. It was shown previously that low-power lasers in the visible and near-infrared range alter Ca2+ uptake by sperm cells. In the present work the effect of a 780 nm diode laser on Ca2+ uptake by sperm mitochondria and isolated plasma membrane vesicles is investigated. STUDY DESIGN/MATERIALS AND METHODS: Digitonin-treated spermatozoa and plasma membrane vesicles were irradiated with a 780-nm diode laser at various powers and energy doses, and Ca2+ uptake was measured by the filtration method. RESULTS: It was found that 780-nm irradiation inhibits Ca2+ uptake by the mitochondria but stimulates Ca2+ binding by sperm plasma membrane vesicles. The effect of light on Ca2+ uptake by plasma membrane vesicles in the absence of ATP was much larger than that measured in the presence of ATP. Addition of Ca2+ ionophore decreased the Ca2+ uptake by the irradiated membranes in the presence of ATP but enhanced it significantly in the absence of ATP. CONCLUSION: 780 nm light inhibits Ca2+ uptake by sperm mitochondria and enhances Ca2+ binding to sperm plasma membranes.

780 nm low power diode laser irradiation stimulates proliferation of keratinocyte cultures: involvement of reactive oxygen species.

BACKGROUND AND OBJECTIVE: The purpose of this study was to determine irradiation parameters of a 780 nm low power CW diode laser (6.5 mW) leading to enhanced proliferation of cultured normal human keratinocytes (NHK). The possible role of reactive oxygen species (ROS) in this response was evaluated. STUDY DESIGN/MATERIALS AND METHODS: NHK were exposed to a single dose of 0 to 3.6 J/cm2 (0-180 sec) of irradiation. Proliferation parameters studied were: incorporation of 3H-thymidine during 6-24 hr following irradiation; percentage of dividing cells and number of cells, 24 hr and 48 hr following irradiation, respectively. RESULTS: Proliferation of NHK exposed to 0.45-0.95 J/cm2 was significantly enhanced by 1.3-1.9-folds relative to sham-irradiated controls, as inferred from parameters studied. Exposure to other energy densities was considerably less effective in enhancing proliferation parameters. Added enzymatic antioxidants, superoxide dismutase or catalase, scavenging superoxide anions and H2O2, suppressed this enhanced proliferation. Added scavengers (alpha-tocopherol acetate, scavenging lipid peroxidation, or sodium azide, histidine, mannitol, scavenging singlet oxygen, superoxide anions, and hydroxyl radicals, respectively), or N-acetyl cysteine, the thiol-reducing agent, suppressed the response, but to different extents. CONCLUSIONS: The results indicate that 780 nm low power diode laser irradiation enhanced keratinocytes proliferation in vitro, with an apparent involvement of ROS in this response, and comparably, might be used to promote their proliferation in vivo to enhance wound healing.

Low-Dose UVA Illumination of Differentiating Stromal Cells Reveals Diverse Effects on Cell-Mediated Mineralisation in Culture.

In the present study the effect of low dose UVA illumination on cell-mediated mineralisation was tested. Rat marrow stromal cells were stimulated with dexamethasone (DEX) to induce mineralisation in the cultures. Different cultures were illuminated with low UVA dose, 0.1 J/cm(2), using illumination protocols which differed either by day of exposure, for single-dose protocols, or by the number of exposure-days, for multiple-dose protocols. Different responses to UVA were induced by early (day 1) versus late (days 7-8) illumination. Single-dose illumination on days 1 or 8 decreased or increased day 21 mineralisation, respectively. The mineralisation differences were not equivalent to the differences in cell counts, and thus did not reflect selection of osteoprogenitor cells (OPC), in contrast to the effect of malonate found in previous studies. Late illumination, on day 7, resulted in a higher rhodamine 123 (Rho) retention on day 7 than the early (day 1) illumination effect on the day 7-Rho retention. The opposing effect of the early and late illumination protocols on mineralisation was also not accompanied by OPC selection. Thus, we have shown that low UVA dose modulates mineralisation together with modulation of mitochondrial Rho retention. These results suggest that low doses of UVA light may affect bone mineralisation.