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

Changes in Calcium Transport in

Mammalian Sperm Mitochondria and
Plasma Membranes Caused by 780
nm Irradiation
Rachel Lubart, PhD,1 Harry Friedmann, PhD,1 Michael Sinyakov, PhD,2
Natalie Cohen, MSc,2 and Haim Breitbart, PhD 2*
1
Department of Physics and Chemistry, Bar-Ilan University, Ramat Gan, Israel
2
Department 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 and
acrosome reaction. It was shown previously that low-power la-
sers 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 sperma-
tozoa 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+ up-
take 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 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 diode
laser

INTRODUCTION absorbed by mitochondrial enzymes [5]. There-

fore, it was suggested [6,7] that HeNe laser radia-
Intracellular calcium plays a vital role in cell tion activates the redox reactions in the respira-
proliferation, and in mammalian spermatozoa it tory chain by exciting mitochondrial porphyrins
has a pivotal role in control of sperm motility [1] or cytochromes. This activation could lead to
and acrosome reaction [2]. Therefore, the alter- changes in [Ca2+]i in the cell. We have fixed our
ation in calcium levels in response to exposure to attention on the effect of light on Ca2+ transport
light may have considerable biological and clinical in sperm cells. The systems which regulate intra-
significance. cellular Ca2+ concentration in spermatozoa in-
It was found that irradiating various cells
with certain wavelengths at specified energy
doses in the visible and in the far-red range re- *Correspondence to: Haim Breitbart, Department of Life Sci-
sulted in acceleration of the Ca2+ uptake by the ences, Bar-Ilan University, Ramat-Gan 52900, Israel.
cells [3,4]. E-mail: breith@ashur.cc.biu.ac.il
Light in the visible and far-red range can be Accepted 24 July 1997

© 1997 Wiley-Liss, Inc.

494 Lubart et al.
volve the mitochondria, [8,9], the plasma mem-
brane ATP-dependent Ca2+ pump [10,11], the
Na+/Ca2+ antiport [12,13], and the Ca2+ channel
[14]. In a previous work [3], we measured Ca2+
uptake by sperm mitochondria irradiated with
HeNe (633 nm) light and found acceleration or
inhibition of Ca2+ uptake, depending on the en-
ergy doses of the light. Irradiation of isolated
plasma membrane vesicles with HeNe laser did
not change their Ca2+ uptake ability.
In the present work we have examined Ca2+
uptake by sperm mitochondria and isolated
plasma membrane vesicles irradiated with 780
nm light. At powers and energy doses used in our
experiments, we found that Ca2+ uptake was in-
hibited in the mitochondria and increased in the
plasma membrane vesicles. In the case of isolated
plasma membrane vesicles we have investigated
the nature of this Ca2+ uptake in the presence and
absence of ATP and Ca 2+ -specific ionophore
A23187. We found that the elevation in Ca2+ up-
take by the plasma membrane was a consequence
of Ca2+ binding to the membrane after irradia-
tion.

MATERIALS AND METHODS

Frozen ejaculated bull sperm cells (from Fig. 1. A schematic representation of the irradiated sample.
Hasherut Artificial Insemination Center) were
thawed at 37°C in medium comprising 150 mM
NaCl, 10 mM histidine (pH 7.4). The cells were zoa the Ca2+ uptake was measured while the cells
washed by three centrifugations at 600g, at 25°C were transferred to 96-well culture plate at room
for 10 min. The washed cells were resuspended in temperature and the light was defocused on each
buffer containing 110 mM NaCl, 5 mM KCl, and well. As we preferred to measure Ca2+ uptake into
10 mM sodium morpholinopropanesulfonate plasma membrane vesicles at 37°C and not at
(MOPS), pH 7.4. room temperature, we had to transfer the light
through a fiber into a rotating tube immersed in a
Irradiation 37°C water bath [15]. In such a set-up, time of
A schematic representation of the irradia- exposure of the cells to the light beam is un-
tion set-up is given in Figure 1. The light source known, so the results can only be qualitatively
was a 780 ± 5-nm, 25-mW diode laser (Lasotron- compared with those obtained previously [3] on
ics). An optical multimode fiber made of silica intact sperm cells.
glass was connected to the laser and was inserted Digitonin treatment. Since it is impossible
into a rotating tube, at 37°C, containing 2–5 ml to isolate coupled mitochondria from spermato-
cell suspension of 3 × 108 cells/ml or 30 mg prot./ml zoa, we have decided to follow calcium transport
of membranes. The light power at the end of the into permeabilized sperm. Under these condi-
fiber ranged from 3 mW to 25 mW. (Neutral den- tions, the Ca2+ uptake is completely abolished by
sity filters were used to reduce the intensity of the the mitochondrial uncoupler CCCP [2,7] (carbonyl
laser.) The irradiation time was 1–20 minutes. All cyanide para trifluoro methoxy phenyl hydra-
light intensities were measured with an Ophir zone), indicating that the transported Ca2+ is ac-
(model PD2-A) power meter. cumulated in the mitochondria. Thus, we could
The experimental set-up in this work differs test the irradiation effect on mitochondrial Ca2+
from that used in our previous experiments on transport. A suspension of 4 × 108 cells/ml in 5-ml
intact spermatozoa [3]. With the intact spermato- buffer A was mixed with 65.5 ml of 14.27 mM dig-
 Ca2+ Transport Changes Induced by Irradiation 495
itonin in dimethylsulfoxide. When motility was calcium. At various time intervals (up to 12 min)
completely stopped, the suspension was centri- 0.1 ml was removed and immediately vacuum-
fuged at 4°C at 600g. The pellet was resuspended filtered on GF/C filters. The cells trapped on the
with buffer M composed of 250 mM mannitol, 70 filter were washed three times with 5 ml of solu-
mM sucrose, and 10 mM TEA-Hepes (pH 4 7.4), tion composed of 150 mM NaCl, 10 mM Tris (pH
centrifuged as above, and then suspended in cold 7.4), and 2 mM EGTA. The dry filters were
buffer M and kept on ice. counted in scintillation vials with 5 ml Aquasol
Preparation of sperm plasma mem- (DuPont). Calcium uptake was calculated as nmol
branes. The sperm plasma membranes were pre- Ca2+/108 cells.
pared as previously described by us [10,11]. The Calcium uptake by isolated plasma
sperm cells were pelleted by centrifugation at membranes. Ca 2+ uptake by spermatozoa
1,500g for 10 min; then the cells were washed four plasma membrane vesicles was measured in a 0.8
times in buffer NKM (110 mM NaCl, 5 mM KCl, ml medium containing 18 mM histidine/18 mM
and 10 mM MOPS, pH 7.4). The washed cells imidazole buffer (pH 6.8), 0 1M KCl, 3 mM MgCl2,
were resuspended in hypotonic medium (10 mM 0.18 mM CaCl2 (10 mM free Ca), 1 mCi 45CaCl2,
histidine (pH 7.4)/0.5 mM EDTA) and disrupted 0.2 mM EGTA, and 240 mg plasma membrane
by using the Ultraturrax homogenizer (Janke and protein. In some experiments, 10 mM A23187 Ca-
Kunkel K6 IKA WERK Typ. TP18-10) in the fol- specific ionophore (CalBiochem) was added. After
lowing way: 10 s low rate, 3 s high, 7 s low, 3 s a 5-min preincubation at 37°C, Na-ATP was
high, and 7 s low rate. Low and high rates repre- added to achieve a final concentration of 2 mM
sent 3,000 and 14,000 rpm, respectively. The sus- ATP. Part of the experiments were done without
pension was centrifuged at 3,000g for 10 min, and exogenous ATP. The reaction mixture was incu-
the supernatant was removed and centrifuged at bated at 37°C and irradiated with 780 nm light.
6,000g for 10 min. The supernatant was removed At appropriate time intervals, samples (100 ml)
and centrifuged at 35,000g for 30 min; then the were removed and vacuum filtered on 0.45-mm
pellet was resuspended in the hypotonic medium. millipore filters. The membrane vesicles trapped
The suspension was layered on a discontinuous on the filter were washed with cold water and
sucrose gradient composed of 0.5, 1.0, and 1.5 M placed in scintillation vials for measurement of
sucrose solutions prepared in 10 mM histidine the b radioactivity.
(pH 7.4). The tubes were centrifuged at 30,000 The experiments were carried out in tripli-
rpm for 18 h at 4°C using a SW 41 rotor, in Spinco cate for a single preparation of ejaculated sperm
(Beckman) ultracentrifuge. The membrane frac- and at least three different preparations, each
tion located just above the 1.5 M sucrose layer taken from a different animal, were used. The
was removed and diluted with 10 mM histidine percentage of Ca2+ uptake of each sample relative
(pH 7.4)/0.1 mM EDTA at 4°C. The protein con- to the maximum Ca2+ uptake of a non-irradiated
centration was determined by the method of sample in the absence of ATP was calculated. The
Lowry et al. [16] using bovine serum albumin as results of at least three preparations were ana-
the standard of reference. The membranes were lyzed statistically by Student’s t-test and analysis
stored at −20°C prior to analysis. These mem- of variance.
branes showed a 45-fold enrichment of the plasma
membrane marker (Na+ + K+) ATPase and less
RESULTS
than 4% of the mitochondrial marker cytochrome
c oxidase–specific activity found in whole cell ho- Exposure of sperm mitochondria (permeabi-
mogenates. When examined by transmission elec- lized cells) to 780-nm light at 4 mW and 9 mW at
tron microscopy, the membranes were vesicular, various energy doses, 0.2–6.0 J, resulted in Ca2+
and mitochondria were not identified [see ref. uptake inhibition (Fig. 2). We used permeabilized
10,11]. sperm cells because it is difficult to isolate coupled
Calcium uptake by permeabilized cells. sperm mitochondria. Their separation requires
Uptake of 45Ca2+ was determined by the filtration the breakage of disulfide bridges [17]. Digitonin
technique. Cells (3 × 108 ml) were incubated in a treatment disrupts the sperm plasma membrane
medium containing 10 mM lactate, 0.5 mM phos- while leaving the mitochondria functionally in-
phate, 0.2 mM CaCl2, and 0.5 mCi 45CaCl2. The tact [18]. When digitonin-treated cells were
cells were irradiated with 780-nm light (4 mW, 9 washed free of phosphate and mitochondrial sub-
mW, 24 mW) immediately after the addition of strate, little Ca2+ uptake occurred. However, a
496 Lubart et al.

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

high degree of Ca2+ uptake can be observed with to the reaction mixture in the absence of ATP
the addition of mitochondrial substrate and phos- (Fig. 4) resulted in a very small decrease of Ca2+
phate. In this work we used lactate as a sperm uptake in the non-irradiated control membrane
mitochondrial substrate. Ca2+ uptake into perme- preparations (P < .003). At the same time, the
abilized cells is above 90% inhibited by mitochon- ionophore did not eliminate the effect of 780-nm
drial uncoupler (data not shown), indicating that irradiation; moreover, Ca2+ uptake in the irradi-
most of the absorbed Ca2+ is accumulated in the ated membranes was additionally and signifi-
mitochondria. cantly (P < .002) increased in the presence of the
Irradiation of plasma membrane vesicles ionophore.
with 25 mW 780-nm light at energy doses of 1.5– In the presence of ATP, addition of the iono-
30 J enhanced Ca2+ uptake by the membranes phore (Fig. 5) drastically and significantly de-
(Fig. 3). A similar enhancement was achieved creased Ca2+ uptake in both the non-irradiated
with a 9-mW 780-nm laser (data not shown). control membranes (P < .003) and the irradiated
Comparison of the relevant pairs, namely irradi- specimens (P < .03); in the latter case, Ca2+ up-
ated versus non-irradiated membrane specimens, take in the presence of ionophore dropped to the
reveals clear-cut statistically significant differ- level of the non-irradiated control in the absence
ences and indicates that the effect of light in the of ionophore (see Fig. 5). The effect of light on
absence of ATP (P < .01) is much larger than that Ca2+ uptake was much higher (see Figs. 4, 5) in
observed in the presence of ATP (P < .05). Ca2+ the presence of the Ca2+-ionophore than in its ab-
uptake in the non-irradiated control specimens sence. In Figure 5, the light effect in the presence
was significantly higher in the presence of ATP as of A23187 seems to be lower in comparison to la-
compared to that in the absence of ATP (P < .003), ser alone. But, if we subtract the controls, we find
while the difference in Ca2+ uptake in the irradi- 6 times enhancement of Ca2+ uptake by light in
ated samples was not significant. the presence of A23187, and only 1.6-fold increase
Addition of Ca2+-specific ionophore A23187 in its absence.
 Ca2+ Transport Changes Induced by Irradiation 497

Fig. 3. Ca2+ uptake by irradiated (25 mW, 780 nm) plasma Fig. 4. Ca2+ uptake by irradiated (25 mW, 780 nm) plasma
membrane vesicles from sperm cells in the presence or ab- membrane vesicles from sperm cells ± Ca 2+ ionophore
sence of ATP. The 100% represent Ca2+ uptake by non- A23187, in the absence of ATP relative to maximum Ca2+
irradiated membranes in the absence of ATP at 20 min up- uptake by non-irradiated membranes (as designated in leg-
take time and corresponds to the uptake of 1.48 nM Ca2+/mg end to Fig. 3).
membrane protein. The time in the graph represents time of
irradiation and time of Ca2+ uptake. Each value represents
the mean of three experiments performed in triplicate. In
Figures 3–5 it was possible to perform a linear fit transfor-
irradiation [3]. We found that light in the visible
mation of the results obtained because of the high values of and in the far-red range at a specified energy dose
the correlation coefficients which ranged from 0.88 to 0.98. accelerated Ca2+ uptake by the various cells. In
order to better understand the mechanism by
DISCUSSION which light affects the cell, we measured Ca2+ up-
take by HeNe-irradiated sperm mitochondria
The mechanism by which light in the visible (permeabilized cells) and by isolated plasma
and far-red range interacts with the living cell is membranes [15]. We found that HeNe laser irra-
still being debated. As intracellular Ca2+ concen- diation at low power (0.3 mW) and low energy
tration [Ca2+]i is responsible for various cell ac- doses (0.06–0.2 J), produced enhanced Ca2+ up-
tivities, several authors investigated the effect of take, whereas at higher power (10 mW) and doses
light on [Ca2+]i. Young et al. [4] found an in- (0.6–7.2 J) the uptake was inhibited. No effect
creased 45Ca2+ uptake by macrophages after 660-, was observed when sperm plasma membrane
820-, and 870-nm irradiation. Karu [19] found an vesicles were irradiated with HeNe laser.
elevation of [Ca2+]i in lymphocytes after HeNe ir- In the present work the influence of far-red,
radiation. We measured changes in Ca2+ uptake 780-nm, laser diode radiation was examined. As
by bovine sperm cells due to 633-nm and 780-nm described in Figure 2, 780-nm light at 0.2–6.0 J,
498 Lubart et al.
(Fig. 4). This indicates that 780-nm light en-
hances Ca2+ binding to the membrane and not
Ca2+ accumulation in the intravesicular space.
The fact that A23187 stimulates the light effect
on 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 under
HeNe (633-nm) irradiation and 780-nm diode la-
ser irradiation indicates the importance of the ef-
fect of specific wavelength on calcium transport in
sperm cells.
The stimulating action of visible and far-red
light 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 the
redox activity of the RC, enhancing the mem-
brane potential (Dc) across the inner membrane
of the mitochondria and ATP production [23]. The
enhanced Dc and ATP production can modulate
[Ca2+]i by enhancing Ca2+ influx into the mito-
chondria and by increasing the activity of the
ATPase-driven pumps in the cell membrane
[3,19]. However, in light of the results reported
here, another possible mechanism should be con-
sidered, i.e., proteins may be the direct target of
low-energy laser irradiation [24,25]. We assume
that in the case of far-red light vibrational over-
Fig. 5. Ca2+ uptake by irradiated (25 mW, 780 nm) plasma tone excitation occurs, and not non-pigmented
membrane vesicles from sperm cells in the presence of ATP proteins can undergo a conformational transfor-
and in the absence and presence of the Ca2+-ionophore
A23187, relative to maximum Ca2+ uptake by non-irradiated mation to either more activated conformation or
membranes (as designated in legend to Fig. 3). to an inhibitory one. Thus, we ascribe the inhibi-
tion of Ca2+ uptake into the mitochondria irradi-
inhibits Ca2+ uptake by the mitochondria. On the ated by 780-nm light to inhibition of the enzymes
other hand, 780-nm light at 1.5–30 J enhanced of the RC [26] or the Ca2+ uniporter. On the other
Ca 2+ uptake by isolated plasma membrane hand, the 780-nm radiation affects the conforma-
vesicles (Fig. 3). The cell plasma membranes tion of proteins in the isolated plasma membrane
regulates intracellular Ca2+ concentration mainly in a way which increases the accessibility of Ca2+
by the ATP-dependent Ca2+ pump [10,11], the to its binding sites. Alteration in Ca2+ binding un-
Na+/Ca2+ exchanger [12,13], and by a voltage- doubtedly can change intracellular Ca2+ concen-
gated Ca2+ channel [14]. Isolated plasma mem- tration [27], leading to stimulatory effects. At
brane vesicles can accumulate a significant high energy doses it can create the possibility of
amount of Ca2+ in the presence of added ATP. closing divalent ion channels, thus leading to cell
Without ATP, the uptake of Ca2+ is very low (Fig. death.
3). 780 nm light stimulates the Ca2+ uptake, and Since the effect of light is much smaller in
this effect is much higher in the absence of ATP. the presence of ATP (Fig. 3), we assume that
Also, the light effect is not canceled by adding the bound ATP might protect certain membranal
Ca2+ ionophore A23187 (Ca2+/2H+ exchanger), al- proteins from undergoing light-induced conforma-
though the ATP-dependent Ca2+ accumulation tional transformation. It is well known that bind-
was completely dissipated under these conditions ing of a substrate to an enzyme active site pro-
 Ca2+ Transport Changes Induced by Irradiation 499
tects the enzyme from conformational transfor- 14. Babcock DF, Pfeiffer DR. Independent elevation of cyto-
mations. solic Ca2+ and pH of mammalian sperm by voltage de-
pendent and pH sensitive mechanisms. J Biol Chem
1987; 262:15041–15047.
ACKNOWLEDGMENTS 15. Breitbart H, Levinshal T, Cohen N, Friedmann H, Lubart
R. Changes in calcium transport in mammalian sperm
We would like to thank the Israeli Ministry mitochondria and plasma membrane irradiated at 633
of Health for their support of this research and nm (HeNe laser). J Photochem Photobiol B 1996; 34:117–
Lasotronic for providing us with the Med-140 780- 121.
nm diode laser. Many thanks to Avrille Goldreich 16. Lowry OH, Rosenbrough NH, Farr AL, Randall RJ. Pro-
tein measurement with the folin phenol reagent. J Biol
for editing this manuscript. Chem 1951; 193:265–275.
17. Bartoov B, Messer GY. Isolation of mitochondria from
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