Changes in Calcium Transport inMammalian Sperm Mitochondria andPlasma Membranes Caused by 780

nm IrradiationRachel Lubart, PhD,1 Harry Friedmann, PhD,1 Michael Sinyakov, PhD,2

Natalie Cohen, MSc,2 and Haim Breitbart, PhD2*

1Department of Physics and Chemistry, Bar-Ilan University, Ramat Gan, Israel2Department of Life Sciences, Bar-Ilan University, Ramat Gan, Israel

Background and Objective: Regulation of intracellular Ca2+ con-centrations are very important in control of sperm motility andacrosome reaction. It was shown previously that low-power la-sers in the visible and near-infrared range alter Ca2+ uptake bysperm cells. In the present work the effect of a 780 nm diodelaser on Ca2+ uptake by sperm mitochondria and isolatedplasma membrane vesicles is investigated.Study Design/Materials and Methods: Digitonin-treated sperma-tozoa and plasma membrane vesicles were irradiated with a780-nm diode laser at various powers and energy doses, andCa2+ uptake was measured by the filtration method.Results: It was found that 780-nm irradiation inhibits Ca2+ up-take by the mitochondria but stimulates Ca2+ binding by spermplasma membrane vesicles. The effect of light on Ca2+ uptake byplasma membrane vesicles in the absence of ATP was muchlarger than that measured in the presence of ATP. Addition ofCa2+ ionophore decreased the Ca2+ uptake by the irradiatedmembranes in the presence of ATP but enhanced it significantlyin the absence of ATP.Conclusion: 780 nm light inhibits Ca2+ uptake by sperm mito-chondria and enhances Ca2+ binding to sperm plasma mem-branes. Lasers Surg. Med. 21:493–499, 1997.© 1997 Wiley-Liss, Inc.

Key words: Ca2+ binding; spermatozoa; plasma membrane vesicles; 780 nm diodelaser

INTRODUCTION

Intracellular calcium plays a vital role in cellproliferation, and in mammalian spermatozoa ithas a pivotal role in control of sperm motility [1]and acrosome reaction [2]. Therefore, the alter-ation in calcium levels in response to exposure tolight may have considerable biological and clinicalsignificance.

It was found that irradiating various cellswith certain wavelengths at specified energydoses in the visible and in the far-red range re-sulted in acceleration of the Ca2+ uptake by thecells [3,4].

Light in the visible and far-red range can be

absorbed by mitochondrial enzymes [5]. There-fore, it was suggested [6,7] that HeNe laser radia-tion activates the redox reactions in the respira-tory chain by exciting mitochondrial porphyrinsor cytochromes. This activation could lead tochanges in [Ca2+]i in the cell. We have fixed ourattention on the effect of light on Ca2+ transportin sperm cells. The systems which regulate intra-cellular Ca2+ concentration in spermatozoa in-

*Correspondence to: Haim Breitbart, Department of Life Sci-ences, Bar-Ilan University, Ramat-Gan 52900, Israel.E-mail: breith@ashur.cc.biu.ac.il

Accepted 24 July 1997

Lasers in Surgery and Medicine 21:493–499 (1997)

© 1997 Wiley-Liss, Inc.

volve the mitochondria, [8,9], the plasma mem-brane ATP-dependent Ca2+ pump [10,11], theNa+/Ca2+ antiport [12,13], and the Ca2+ channel[14]. In a previous work [3], we measured Ca2+

uptake by sperm mitochondria irradiated withHeNe (633 nm) light and found acceleration orinhibition of Ca2+ uptake, depending on the en-ergy doses of the light. Irradiation of isolatedplasma membrane vesicles with HeNe laser didnot change their Ca2+ uptake ability.

In the present work we have examined Ca2+

uptake by sperm mitochondria and isolatedplasma membrane vesicles irradiated with 780nm light. At powers and energy doses used in ourexperiments, we found that Ca2+ uptake was in-hibited in the mitochondria and increased in theplasma membrane vesicles. In the case of isolatedplasma membrane vesicles we have investigatedthe nature of this Ca2+ uptake in the presence andabsence of ATP and Ca2+-specific ionophoreA23187. We found that the elevation in Ca2+ up-take by the plasma membrane was a consequenceof Ca2+ binding to the membrane after irradia-tion.

MATERIALS AND METHODS

Frozen ejaculated bull sperm cells (fromHasherut Artificial Insemination Center) werethawed at 37°C in medium comprising 150 mMNaCl, 10 mM histidine (pH 7.4). The cells werewashed by three centrifugations at 600g, at 25°Cfor 10 min. The washed cells were resuspended inbuffer containing 110 mM NaCl, 5 mM KCl, and10 mM sodium morpholinopropanesulfonate(MOPS), pH 7.4.

Irradiation

A schematic representation of the irradia-tion set-up is given in Figure 1. The light sourcewas a 780 ± 5-nm, 25-mW diode laser (Lasotron-ics). An optical multimode fiber made of silicaglass was connected to the laser and was insertedinto a rotating tube, at 37°C, containing 2–5 mlcell suspension of 3 × 108 cells/ml or 30 mg prot./mlof membranes. The light power at the end of thefiber ranged from 3 mW to 25 mW. (Neutral den-sity filters were used to reduce the intensity of thelaser.) The irradiation time was 1–20 minutes. Alllight intensities were measured with an Ophir(model PD2-A) power meter.

The experimental set-up in this work differsfrom that used in our previous experiments onintact spermatozoa [3]. With the intact spermato-

zoa the Ca2+ uptake was measured while the cellswere transferred to 96-well culture plate at roomtemperature and the light was defocused on eachwell. As we preferred to measure Ca2+ uptake intoplasma membrane vesicles at 37°C and not atroom temperature, we had to transfer the lightthrough a fiber into a rotating tube immersed in a37°C water bath [15]. In such a set-up, time ofexposure of the cells to the light beam is un-known, so the results can only be qualitativelycompared with those obtained previously [3] onintact sperm cells.

Digitonin treatment. Since it is impossibleto isolate coupled mitochondria from spermato-zoa, we have decided to follow calcium transportinto permeabilized sperm. Under these condi-tions, the Ca2+ uptake is completely abolished bythe mitochondrial uncoupler CCCP [2,7] (carbonylcyanide para trifluoro methoxy phenyl hydra-zone), indicating that the transported Ca2+ is ac-cumulated in the mitochondria. Thus, we couldtest the irradiation effect on mitochondrial Ca2+

transport. A suspension of 4 × 108 cells/ml in 5-mlbuffer A was mixed with 65.5 ml of 14.27 mM dig-

Fig. 1. A schematic representation of the irradiated sample.

494 Lubart et al.

itonin in dimethylsulfoxide. When motility wascompletely stopped, the suspension was centri-fuged at 4°C at 600g. The pellet was resuspendedwith buffer M composed of 250 mM mannitol, 70mM sucrose, and 10 mM TEA-Hepes (pH 4 7.4),centrifuged as above, and then suspended in coldbuffer M and kept on ice.

Preparation of sperm plasma mem-branes. The sperm plasma membranes were pre-pared as previously described by us [10,11]. Thesperm cells were pelleted by centrifugation at1,500g for 10 min; then the cells were washed fourtimes in buffer NKM (110 mM NaCl, 5 mM KCl,and 10 mM MOPS, pH 7.4). The washed cellswere resuspended in hypotonic medium (10 mMhistidine (pH 7.4)/0.5 mM EDTA) and disruptedby using the Ultraturrax homogenizer (Janke andKunkel K6 IKA WERK Typ. TP18-10) in the fol-lowing way: 10 s low rate, 3 s high, 7 s low, 3 shigh, and 7 s low rate. Low and high rates repre-sent 3,000 and 14,000 rpm, respectively. The sus-pension was centrifuged at 3,000g for 10 min, andthe supernatant was removed and centrifuged at6,000g for 10 min. The supernatant was removedand centrifuged at 35,000g for 30 min; then thepellet was resuspended in the hypotonic medium.The suspension was layered on a discontinuoussucrose gradient composed of 0.5, 1.0, and 1.5 Msucrose solutions prepared in 10 mM histidine(pH 7.4). The tubes were centrifuged at 30,000rpm for 18 h at 4°C using a SW 41 rotor, in Spinco(Beckman) ultracentrifuge. The membrane frac-tion located just above the 1.5 M sucrose layerwas removed and diluted with 10 mM histidine(pH 7.4)/0.1 mM EDTA at 4°C. The protein con-centration was determined by the method ofLowry et al. [16] using bovine serum albumin asthe standard of reference. The membranes werestored at −20°C prior to analysis. These mem-branes showed a 45-fold enrichment of the plasmamembrane marker (Na+ + K+) ATPase and lessthan 4% of the mitochondrial marker cytochromec oxidase–specific activity found in whole cell ho-mogenates. When examined by transmission elec-tron microscopy, the membranes were vesicular,and mitochondria were not identified [see ref.10,11].

Calcium uptake by permeabilized cells.Uptake of 45Ca2+ was determined by the filtrationtechnique. Cells (3 × 108 ml) were incubated in amedium containing 10 mM lactate, 0.5 mM phos-phate, 0.2 mM CaCl2, and 0.5 mCi 45CaCl2. Thecells were irradiated with 780-nm light (4 mW, 9mW, 24 mW) immediately after the addition of

calcium. At various time intervals (up to 12 min)0.1 ml was removed and immediately vacuum-filtered on GF/C filters. The cells trapped on thefilter were washed three times with 5 ml of solu-tion composed of 150 mM NaCl, 10 mM Tris (pH7.4), and 2 mM EGTA. The dry filters werecounted in scintillation vials with 5 ml Aquasol(DuPont). Calcium uptake was calculated as nmolCa2+/108 cells.

Calcium uptake by isolated plasmamembranes. Ca2+ uptake by spermatozoaplasma membrane vesicles was measured in a 0.8ml medium containing 18 mM histidine/18 mMimidazole buffer (pH 6.8), 0 1M KCl, 3 mM MgCl2,0.18 mM CaCl2 (10 mM free Ca), 1 mCi 45CaCl2,0.2 mM EGTA, and 240 mg plasma membraneprotein. In some experiments, 10 mM A23187 Ca-specific ionophore (CalBiochem) was added. Aftera 5-min preincubation at 37°C, Na-ATP wasadded to achieve a final concentration of 2 mMATP. Part of the experiments were done withoutexogenous ATP. The reaction mixture was incu-bated at 37°C and irradiated with 780 nm light.At appropriate time intervals, samples (100 ml)were removed and vacuum filtered on 0.45-mmmillipore filters. The membrane vesicles trappedon the filter were washed with cold water andplaced in scintillation vials for measurement ofthe b radioactivity.

The experiments were carried out in tripli-cate for a single preparation of ejaculated spermand at least three different preparations, eachtaken from a different animal, were used. Thepercentage of Ca2+ uptake of each sample relativeto the maximum Ca2+ uptake of a non-irradiatedsample in the absence of ATP was calculated. Theresults of at least three preparations were ana-lyzed statistically by Student’s t-test and analysisof variance.

RESULTS

Exposure of sperm mitochondria (permeabi-lized cells) to 780-nm light at 4 mW and 9 mW atvarious energy doses, 0.2–6.0 J, resulted in Ca2+

uptake inhibition (Fig. 2). We used permeabilizedsperm cells because it is difficult to isolate coupledsperm mitochondria. Their separation requiresthe breakage of disulfide bridges [17]. Digitonintreatment disrupts the sperm plasma membranewhile leaving the mitochondria functionally in-tact [18]. When digitonin-treated cells werewashed free of phosphate and mitochondrial sub-strate, little Ca2+ uptake occurred. However, a

Ca2+ Transport Changes Induced by Irradiation 495

high degree of Ca2+ uptake can be observed withthe addition of mitochondrial substrate and phos-phate. In this work we used lactate as a spermmitochondrial substrate. Ca2+ uptake into perme-abilized cells is above 90% inhibited by mitochon-drial uncoupler (data not shown), indicating thatmost of the absorbed Ca2+ is accumulated in themitochondria.

Irradiation of plasma membrane vesicleswith 25 mW 780-nm light at energy doses of 1.5–30 J enhanced Ca2+ uptake by the membranes(Fig. 3). A similar enhancement was achievedwith a 9-mW 780-nm laser (data not shown).Comparison of the relevant pairs, namely irradi-ated versus non-irradiated membrane specimens,reveals clear-cut statistically significant differ-ences and indicates that the effect of light in theabsence of ATP (P < .01) is much larger than thatobserved in the presence of ATP (P < .05). Ca2+

uptake in the non-irradiated control specimenswas significantly higher in the presence of ATP ascompared to that in the absence of ATP (P < .003),while the difference in Ca2+ uptake in the irradi-ated samples was not significant.

Addition of Ca2+-specific ionophore A23187

to the reaction mixture in the absence of ATP(Fig. 4) resulted in a very small decrease of Ca2+

uptake in the non-irradiated control membranepreparations (P < .003). At the same time, theionophore did not eliminate the effect of 780-nmirradiation; moreover, Ca2+ uptake in the irradi-ated membranes was additionally and signifi-cantly (P < .002) increased in the presence of theionophore.

In the presence of ATP, addition of the iono-phore (Fig. 5) drastically and significantly de-creased Ca2+ uptake in both the non-irradiatedcontrol membranes (P < .003) and the irradiatedspecimens (P < .03); in the latter case, Ca2+ up-take in the presence of ionophore dropped to thelevel of the non-irradiated control in the absenceof ionophore (see Fig. 5). The effect of light onCa2+ uptake was much higher (see Figs. 4, 5) inthe presence of the Ca2+-ionophore than in its ab-sence. In Figure 5, the light effect in the presenceof A23187 seems to be lower in comparison to la-ser alone. But, if we subtract the controls, we find6 times enhancement of Ca2+ uptake by light inthe presence of A23187, and only 1.6-fold increasein its absence.

Fig. 2. Ca2+ uptake by irradiated digitonin-treated cells at 780 nm, relative to maximum Ca2+ uptake by non-irradiateddigitonin-treated sperm cells. The time in the graph represents time of irradiation and time of Ca2+ uptake. The values are themean ± S.D of three experiments each performed in triplicate. The difference between the control and the light-treated cellsis highly significant (P < .05 for the 4-mW diode laser and P < .01 for the 9-mW laser). The 100% represents Ca2+ uptake bycontrol cells after 12 min of incubation. (100% 4 14.05 nmol Ca2+/108 cells). The Ca2+ uptake oscillation in response to 4-mWirradiation does not always appear in samples from different bulls.

496 Lubart et al.

DISCUSSION

The mechanism by which light in the visibleand far-red range interacts with the living cell isstill being debated. As intracellular Ca2+ concen-tration [Ca2+]i is responsible for various cell ac-tivities, several authors investigated the effect oflight on [Ca2+]i. Young et al. [4] found an in-creased 45Ca2+ uptake by macrophages after 660-,820-, and 870-nm irradiation. Karu [19] found anelevation of [Ca2+]i in lymphocytes after HeNe ir-radiation. We measured changes in Ca2+ uptakeby bovine sperm cells due to 633-nm and 780-nm

irradiation [3]. We found that light in the visibleand in the far-red range at a specified energy doseaccelerated Ca2+ uptake by the various cells. Inorder to better understand the mechanism bywhich light affects the cell, we measured Ca2+ up-take by HeNe-irradiated sperm mitochondria(permeabilized cells) and by isolated plasmamembranes [15]. We found that HeNe laser irra-diation at low power (0.3 mW) and low energydoses (0.06–0.2 J), produced enhanced Ca2+ up-take, whereas at higher power (10 mW) and doses(0.6–7.2 J) the uptake was inhibited. No effectwas observed when sperm plasma membranevesicles were irradiated with HeNe laser.

In the present work the influence of far-red,780-nm, laser diode radiation was examined. Asdescribed in Figure 2, 780-nm light at 0.2–6.0 J,

Fig. 3. Ca2+ uptake by irradiated (25 mW, 780 nm) plasmamembrane vesicles from sperm cells in the presence or ab-sence of ATP. The 100% represent Ca2+ uptake by non-irradiated membranes in the absence of ATP at 20 min up-take time and corresponds to the uptake of 1.48 nM Ca2+/mgmembrane protein. The time in the graph represents time ofirradiation and time of Ca2+ uptake. Each value representsthe mean of three experiments performed in triplicate. InFigures 3–5 it was possible to perform a linear fit transfor-mation of the results obtained because of the high values ofthe correlation coefficients which ranged from 0.88 to 0.98.

Fig. 4. Ca2+ uptake by irradiated (25 mW, 780 nm) plasmamembrane vesicles from sperm cells ± Ca2+ ionophoreA23187, in the absence of ATP relative to maximum Ca2+

uptake by non-irradiated membranes (as designated in leg-end to Fig. 3).

Ca2+ Transport Changes Induced by Irradiation 497

inhibits Ca2+ uptake by the mitochondria. On theother hand, 780-nm light at 1.5–30 J enhancedCa2+ uptake by isolated plasma membranevesicles (Fig. 3). The cell plasma membranesregulates intracellular Ca2+ concentration mainlyby the ATP-dependent Ca2+ pump [10,11], theNa+/Ca2+ exchanger [12,13], and by a voltage-gated Ca2+ channel [14]. Isolated plasma mem-brane vesicles can accumulate a significantamount of Ca2+ in the presence of added ATP.Without ATP, the uptake of Ca2+ is very low (Fig.3). 780 nm light stimulates the Ca2+ uptake, andthis effect is much higher in the absence of ATP.Also, the light effect is not canceled by adding theCa2+ ionophore A23187 (Ca2+/2H+ exchanger), al-though the ATP-dependent Ca2+ accumulationwas completely dissipated under these conditions

(Fig. 4). This indicates that 780-nm light en-hances Ca2+ binding to the membrane and notCa2+ accumulation in the intravesicular space.The fact that A23187 stimulates the light effecton Ca2+ uptake indicates that Ca2+ binding to in-travesicular sites is also enhanced by the light.The observed difference in Ca2+ uptake by the mi-tochondria and the plasma membranes underHeNe (633-nm) irradiation and 780-nm diode la-ser irradiation indicates the importance of the ef-fect of specific wavelength on calcium transport insperm cells.

The stimulating action of visible and far-redlight is assumed to be a consequence of light ab-sorption by endogenous photosensitizers in the re-spiratory chain (RC) [6,19]. The electronically ex-cited photosensitizers, cytochromes, or porphy-rins produce reactive oxygen species (ROS) [20–22] which act as potent oxidizers stimulating theredox activity of the RC, enhancing the mem-brane potential (Dc) across the inner membraneof the mitochondria and ATP production [23]. Theenhanced Dc and ATP production can modulate[Ca2+]i by enhancing Ca2+ influx into the mito-chondria and by increasing the activity of theATPase-driven pumps in the cell membrane[3,19]. However, in light of the results reportedhere, another possible mechanism should be con-sidered, i.e., proteins may be the direct target oflow-energy laser irradiation [24,25]. We assumethat in the case of far-red light vibrational over-tone excitation occurs, and not non-pigmentedproteins can undergo a conformational transfor-mation to either more activated conformation orto an inhibitory one. Thus, we ascribe the inhibi-tion of Ca2+ uptake into the mitochondria irradi-ated by 780-nm light to inhibition of the enzymesof the RC [26] or the Ca2+ uniporter. On the otherhand, the 780-nm radiation affects the conforma-tion of proteins in the isolated plasma membranein a way which increases the accessibility of Ca2+

to its binding sites. Alteration in Ca2+ binding un-doubtedly can change intracellular Ca2+ concen-tration [27], leading to stimulatory effects. Athigh energy doses it can create the possibility ofclosing divalent ion channels, thus leading to celldeath.

Since the effect of light is much smaller inthe presence of ATP (Fig. 3), we assume thatbound ATP might protect certain membranalproteins from undergoing light-induced conforma-tional transformation. It is well known that bind-ing of a substrate to an enzyme active site pro-

Fig. 5. Ca2+ uptake by irradiated (25 mW, 780 nm) plasmamembrane vesicles from sperm cells in the presence of ATPand in the absence and presence of the Ca2+-ionophoreA23187, relative to maximum Ca2+ uptake by non-irradiatedmembranes (as designated in legend to Fig. 3).

498 Lubart et al.

tects the enzyme from conformational transfor-mations.

ACKNOWLEDGMENTS

We would like to thank the Israeli Ministryof Health for their support of this research andLasotronic for providing us with the Med-140 780-nm diode laser. Many thanks to Avrille Goldreichfor editing this manuscript.

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