1. Systems Biology in Reproductive Medicine, 56:303–310, 2010
Copyright & Informa Healthcare USA, Inc.
ISSN: 1939-6368 print/1939-6376 online
DOI: 10.3109/19396368.2010.492447
Research Article
Venom of the Chilean Latrodectus mactans
Alters Bovine Spermatozoa Calcium and
Function by Blocking the TEA-sensitive
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K+ Current
Patricia Navarrete
Centro de neurociencia y Biologıa´ The morphology and size of spermatozoa make it difficult to study the
´
de peptidos-BIOREN, Departamento
´
de Ciencias Preclınicas, Facultad de
functional properties of the plasma membrane, however, some studies have
Medicina, Universidad de La revealed the presence of a number of ion channels in this cell. We measured
Frontera, Chile the calcium (Ca++ ) influx induced by depolarization of the plasma membrane
and by venom isolated from the Chilean black widow spider (Latrodectus
For personal use only.
´ ´
Ataulfo Martınez-Torres
´
Laboratorio Neurobiologıa Molecular mactans), and functional changes in the presence of either high potassium or
y Celular II, Departamento de total venom. Our results indicate that the venom increased the Ca++ influx,
´
Neurobiologıa Molecular y Celular,
with an EC50 of 6.1 mg/mL and triggering the acrosome reaction in 43.26% of
´
Instituto de Neurobiologıa, Campus
´ ´
Juriquilla-Queretaro, UNAM, Mexico the cells. The application of potassium (10 mM K + ) or total venom (10 mg/
mL) did not affect the morphology or DNA stability of the sperm. The effects
´ ´ ´
Raul Sanchez Gutierrez,
Fernando Romero Mejıa, and´ induced by high K + and venom suggest that direct blocking of K + currents
Jorge Parodi alters the passive properties of the plasma membrane, leading to the entry of
Centro de neurociencia y Biologıa´ Ca++ . These results show the importance of functional changes induced by
´
de peptidos-BIOREN, Departamento
depolarizing the spermatozoa and by venom. This venom possesses one or
´
de Ciencias Preclınicas, Facultad de
Medicina, Universidad de La more molecules that may be used as pharmacological tools for studies on
Frontera, Chile spermatozoa and have potential applications in reproductive biotechnology.
KEYWORDS calcium, ionic currents, Latrodectus venom, membrane potential,
Abbreviations: Ca++ : Calcium; spermatozoa
CATsper: Ca++ channels; FITC:
Fluorescein isothiocyanate; Ca++ :
i
Intracellular Ca++ ; L. mactans:
Latrodectus mactans; PBS: Phosphate
salie buffer solution; VDCCs: Voltage-
dependent calcium channels.
INTRODUCTION
Received 26 November 2009; There is strong experimental evidence that mature mammalian sperma-
accepted 24 March 2010. tozoa have several ionic-conductances, including those driven by voltage
Address correspondence to dependent K + channels [Darszon et al. 1996; Labarca et al. 1995; Nuccitelli
Jorge Parodi, Centro de
´ ´
Neurociencias y Biologıa de Peptidos, and Ferguson 1994]. In addition, recent reports in which a whole cell patch-
´ ´
Nucleo cientıfico BIOREN, clamp was used to study spermatozoa ion-conductance have described the
Universidad de la Frontera,
Montevideo 0870, Temuco, Chile. functional properties of Ca++ channels (CATsper), which are key compo-
E-mail: jparodi@ufro.cl nents in the capacitation process [Darszon et al. 2005; Wennemuth et al.
303

2. 2000]. Recently other reports have shown a K + has been also suggested that Ca++ entry and
channel sensitive to TEA in bovine spermatozoa spermatozoa capacitation are triggered by changes
[Marconi et al. 2008] which is important for the in intracellular pH and in transient plasma membrane
control of membrane potential [Gutman et al. 2003]. depolarization [Fraire-Zamora and Gonzalez-Martinez
Mammalian spermatozoa acquire the functional 2004; Neri-Vidaurri Pdel et al. 2006]; this is similar to
capacity to fertilize an egg during their trajectory the series of events that occur in somatic cells [Baker
along the female genital tract [Boni et al. 2007]. et al. 1973]. Nevertheless, the detailed mechanism
In this process the plasma membrane potential is leading to spermatozoa capacitation has yet to be
suddenly hyperpolarized by the activation of pH- fully understood.
sensitive K + -channels, leading to an increase in Ca++ In a previous report we described that the venom of
permeability [Kumar et al. 2000; Linares-Hernandez the Chilean black widow spider Latrodectus mactans
et al. 1998; Shi and Ma 1998]. However, this increase (L. mactans) increased the Ca++ concentration of the
i
of intracellular Ca++ (Ca++ ) is not the sole mecha-
i spermatozoa [Romero et al. 2007]. Furthermore, this
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nism by which the sperm is hyperpolarized, and it venom is known to block the TEA-sensitive K +
For personal use only.
FIGURE 1 Venom-induced rise of intracellular Ca++ without morphological effect. Sample images of spermatozoa in different conditions.
A shows spermatozoa in control (a), high potassium (10 mM) (b), and venom (10 mg/mL) (c). B shows Fluo-4 AM loaded spermatozoa
in control (a), high potassium (b), and venom (c), calibration bar ¼ 5 mm. C shows the images in B as plots from fluorometric records of
spermatozoa in control, potassium, and venom; the dots are mean7E.R. of 3 different measurements. The images are representative of
5 different experiments. AFU: arbitrary fluorescent unit.
304 P. Navarrete et al.

3. currents in neurons [Parodi and Romero 2008; Parodi different conditions: the upper panel shows the
et al. 2010] as well as endogenous K + currents of control, the middle panel the effect of potassium
Xenopus laevis oocytes [Parodi et al. 2008]; this current (10 mM), and the lower panel the spermatazoa
is similar to that recently described in bovine exposed to venom (10 mg/mL). In all conditions the
spermatozoa [Marconi et al. 2008]. In this study we general morphology has not changed, indicating
analyzed how either depolarization or L. mactans that the venom has no toxic effects at this concen-
venom alter the properties of the sperm cells assessed tration. Figure 1B shows spermatozoa loaded with
by microscope imaging, morphology, Ca++ dynamics
i Fluo-4, and as expected, the basal level of free Ca++ i
by fluorometry, acrosome reaction, and DNA frag- remained low but was more intense in the mid piece
mentation of bovine spermatozoa. and tail. In contrast, after incubation for 15 min with
potassium or 30 min with venom, the number of
cells exhibiting fluorescence increased considerably
RESULTS (Fig. 1Bb and 1Bc) and the fluorescent signal in the
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head of the spermatozoa increased. This experiment
Exposure to Venom Increases was consistent after 3 repetitions.
Free Ca++
i
Previous reports using conventional spectro-
Venom Dose Response
photometry showed that depolarization and venom
of L. mactans increases intracellular pH and free
Dependence of Ca++
i
Ca++ In this new follow up study we assessed the
i A recent report by our group showed an increase
changes in the Ca++ by fluorimetry and image ana-
i in Ca++ induced by a single dose of venom [Parodi
i
lysis. Figure 1A shows spermatozoa exposed to et al. 2010]. Figure 2, sample A shows fluorescence
For personal use only.
FIGURE 2 Dose-response of venom-induced rise of intracellular Ca++ . Sample traces of Fluo-4 AM loaded spermatozoa in different condi-
tions. A shows traces of spermatozoa in the presence of venom (10 mg/mL), TEA (10 mM), and high potassium (10 mM) (upper panel).
The lower panel shows the effect of two venom concentrations (5 and 10 mg/mL). B shows plots of the example traces from A, the dots
are mean7E.R. of 5 different experiments. AFU: arbitrary fluorescent unit.
Effect of Venom on Mature Spermatozoa Function 305

4. intensity traces. The upper panel shows an example a premature acrosome reaction similar to the effect
with total venom (7.5 mg/mL), TEA (100 mM), or of high potassium. Figure 3C shows a plot of percent
K + (10 mM). The lower panel shows an example live spermatozoa indicating that the venom pro-
at two concentrations of venom (5 and 10 mg/mL). motes the death of bovine spermatozoa in the same
Figure 2B indicates that venom has an EC50 of way as the effect of high potassium. This data
6.173.5 mg/mL. This low concentration is similar to indicated an effect of the venom on the acrosome
previous values which were able to generate effects reaction and viability of the bovine spermatozoa.
in other experimental models [Parodi and Romero
2008; Parodi et al. 2008].
Venom Did not Induce DNA
Fragmentation
Venom-induced Acrosome Reaction Finally we explored the possibility that the venom
Considering the effect of venom on calcium may induce DNA fragmentation in the spermatozoa.
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dynamics, we explored its ability to modulate the Figure 4A shows images of bright field and fluores-
acrosome reaction. Figure 3, example A, shows cence of fragmented DNA of spermatozoa exposed to
images of live spermatozoa with or without intact DNase I (4Aa and 4Ab) and control (4Ac and 4Ad);
acrosome (3Aa) and (3Ab), and dead spermatozoa whereas Figure 4B shows a plot of percent TUNEL
with or without intact acrosome (3Ac) and (3Ad). of spermatozoa treated with either DNase I, high
Figure 3B shows a plot of percent live spermatozoa potassium (10 mM), or venom (10 mg/mL). The results
with reacted acrosome in control, high potassium indicate that neither the venom nor high potassium
(10 mM), and venom (10 mg/mL). Venom produces produce DNA fragmentation, thus there is no indication
For personal use only.
FIGURE 3 Venom-induced acrosome reaction. The figure shows microphotography images of spermatozoa in different conditions.
A shows sample images of live spermatozoa with or without intact acrosome (a) and (b), and dead spermatozoa with or without intact
acrosome (c) and (d), calibration bar ¼ 5 mm. The images are representative of 5 individual experiments. The images in A are plots of
percent live spermatozoa with reactioned acrosome in control, high potassium (10 mM), and venom (10 mg/mL) in B and percent live
spermatozoa in C. The bar is mean7E.R. of 5 different experiments. nSignificance po 0.05.
306 P. Navarrete et al.

5. Syst Biol Reprod Med Downloaded from informahealthcare.com by University Autonoma Barcelona on 11/02/10
FIGURE 4 No venom-induced DNA fragmentation. The figure shows microphotographic images of spermatozoa in different conditions.
A shows sample images of bright field and fluorescence of DNA fragmentation spermatozoa in positive control (DNase I) (a and b) and
control (c and d), respectively, calibration bar ¼ 5 mm. The images are representative of 5 individual experiments. B shows the images in
A as plots of percent TUNEL positive spermatozoa in control, DNase I, high potassium (10 mM), and venom (10 mg/mL). The bar is mean7E.R.
of 5 different experiments. nSignificance po0.05.
that the venom alters genomic stability, although a observed (data not shown). This data suggests the
more detailed analysis is needed. participation of voltage-dependent calcium channels
For personal use only.
(VDCCs) [Brandelli et al. 1996]. The change in the
membrane potential is consistent with the view that at
DISCUSSION the cellular level this is one of the mechanisms used.
Previous reports showed that after several minutes Here we show that the venom induces other effects
of exposure of the sperm cells to the L. mactans on spermatozoa, which are secondary to the calcium
venom the free Ca++ rises [Romero et al. 2007]. This
i entrance and do not cause morphological or toxic
could be explained by the blockage of voltage- effects. Nevertheless, the same conditions altered intra-
dependent K + channels, leading to the entrance of cellular calcium along the spermatozoa (Fig. 1B).
Ca++ from the extracellular medium. This is supported This global increase in the intracellular calcium has
by the experiments in absence of calcium that showed been reported before [Romero et al. 2007], but the
a reduced effect [Romero et al. 2007], but does not precise location where calcium increases was not
discount the participation of intracellular calcium. described. Many others have proposed that calcium
Whatever, the mechanism modulating membrane entry is key to induce spermatozoa capacitation [Parrish
potential, the participation of extracellular calcium is et al. 1999] and the acrosomal reaction [Coronel and
involved. This report and previous experiments Lardy 1992]. In the present report, we observed that
suggested that the venom inhibits several K + con- calcium entry is dependent on venom concentration
ductances [Parodi and Romero 2008; Parodi et al. (Fig. 2B), and it has been proposed that this calcium is
2008; 2010], and thus we compared this effect with the a signal that triggers the acrosomal reaction after
addition of high K + in the medium. In both cases the spermatozoa capacitation [Coronel and Lardy 1992].
concentration of free Ca++ was observed, and a This evidence supports the idea of venom-induced
transient rise and sharp fall of the fluorescence was functional alteration, mediated by calcium entry, as
monitored in the midpiece of the spermatozoa. a secondary depolarization mechanism. We observed
Thus, the rise of Ca++ was observed throughout the a reduction in viability and suggest that this alteration
cell after long periods of time. In the absence of is a consequence of capacitation and the acrosomal
extracellular calcium the rise in the calcium was not reaction [Medeiros et al. 2002]. We explain the
Effect of Venom on Mature Spermatozoa Function 307

6. reduction in viability as methodological condition without food and given only water in order to
and cell death mediated by spermatozoa activation stimulate the production and concentration of venom
and the acrosomal reaction. This notion is supported in the glands.
by the observation of the DNA integrity measured by
DNA fragmentation, in the absence or presence of
venom. No DNA fragmentation was observed under
Venom Retrieval
any condition (Fig. 4). The controls with DNase I The spiders were immersed in liquid nitrogen and
showed typical fragmentation and support our after 1 min transferred to a phosphate saline buffer
suggestion that there is no toxic effect of the venom solution (PBS: 0.1 M NaH2PO4, 0.01 M Na2HPO4,
in our model. 1.35 M NaCl, pH 7.4) at 41C. The glands were removed
Previous studies have provided information about and the membrane that binds them to the base was
the spermicidal properties of the venom of the sectioned. Each gland was placed into a tube con-
Chilean spider L. mactans. Other natural sources of taining PBS (25 pairs of glands for 100 mL of PBS) and
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spermicidal action have been reported, such as homogenized. The homogenate was immediately
scorpion toxins and plant extracts [Harat et al. 2008; centrifuged at 1000 Â g for 15 min and the supernatant
Lopez-Gonzalez et al. 2003]. All these studies suggest was subsequently aliquoted, and frozen at À 201C.
new lines of research to identify the active The protein content of the venom was determined
compound(s) that mediate the modulation of ionic- by a modification of the Bradford method (BioRad
conductances, and that could provide a resource for Protein Assay, BioRad, Hercules, CA, USA).
a new generation of contraceptives.
Calcium influx in response to membrane depolar-
ization with potassium has been reported as one of
Spermatozoa Preparation
the key early events leading to the process of sperm Bovine spermatozoa were obtained from samples
capacitation. Therefore, it is possible that compo- cryopreserved in liquid nitrogen, which were thawed
For personal use only.
nents in the venom from the widow spider may at 301C for 30 s and suspended in SP-TALP medium.
influence membrane properties in sperm leading to Briefly, 1 mL SP-TALP medium supplemented with
capacitation. In conclusion, we find that bovine 6 mg/mL BSA was underlayered with bovine sperma-
spermatozoa increases the Ca++ when exposed to
i tozoa in a centrifuge tube. The tube was inclined
aracnotoxin from L. mactans and that this pheno- to 45 degrees and incubated for 40 min at 38.51C,
menon produces the subsequent acrosome reaction 5% CO2. After incubation the upper 800 mL of the
in the spermatozoa, most probably through the medium (containing motile spermatozoa) was removed
blocking of voltage-dependent potassium channels. with a sterile transfer pipette [Deppe et al. 2008].
Thus, molecules derived from venom could be The concentration of spermatozoa was measured
isolated for biotechnological applications, including in a Neubauer counting chamber and diluted in
the design of new contraceptives. New data is an appropriate volume of external solution to give
presently being gathered that demonstrates the a final concentration of 3 Â 106 spermatozoa ml À 1.
effects of the purified protein and peptides. The spermatozoa were exposed to total venom
(10 mg/mL) or high potassium (10 mM).
MATERIALS AND METHODS Ca++ Dynamics
i
Spider Retrieval Spermatozoa (2 Â 106 cells) were diluted in
Female adult L. mactans from Chile were captured external solution (NaCl 150 mM, KCl 5.4 mM, CaCl2
during the summer months (December 2008 and 2 mM, MgCl2 1 mM, glucose 10 mM, HEPES 10 mM,
January 2009) from the ‘Alto Bio Bio’ area in the sucrose 50 mM) and incubated with 1 mM Fluo-4 AM
Bio-Bio Region (72116’5100 W, 7145’2400 S) as pre- (Molecular Probes, Eugene, OR, USA) for 30 min at
viously described [Romero et al. 2007], taking care not 371C, 5% CO2. They were washed with external
to damage breeding zones. The specimens were solution and centrifuged at 200 Â g for 10 min.
maintained separately in individual jars for 30 days The pellet was suspended in external solution and
308 P. Navarrete et al.

7. transferred to a continuously stirred quartz cuvette. a negative control without the addition of TdT enzyme
Calcium fluorescence probes were measured at and a positive control with DNAse I (Promega, Madison,
559 nm in a fluorimeter 814 photomultiplier detec- WI, USA) treatment were included [Fatehi et al. 2006].
tion system, with a brytebox using FeliX32 software
(PTI system, USA) at room temperature with
Statistical Analysis
excitation at 488 nm, plotted as arbitrary fluorescent
unit (AFU). Autofluorescence of the spermatozoa was Representative results from the experiments are
not significant. The spermatozoa were exposed to shown in the figures. Basal levels are expressed as
total venom (1 to 25 mg/mL) or high potassium mean7SEM. Test data were analyzed using the
(10 mM) in the cuvette, and changes in the emission GraphPad Prism 5.0 software. Differences between
of fluorescence in the total mass of the cells were the different groups were analyzed using the paired
recorded immediately. The imaging analysis of test. A p value less than 0.05 (po0.05) was considered
calcium entry was evaluated in a Zeiss fluorescence as statistically significant.
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microscope (Axiolab, Carl Zeiss Inc., Thornwood, NY,
USA) and ImageJ software was used for quantification. ACKNOWLEDGMENTS
´l ´
Fernando Romero, Rau Sanchez, and Jorge Parodi
Acrosome Reaction were supported by the FONDEF-CONICYT Chile No.
After depolarization of the membrane or exposure DO5I10416. Atau´lfo Martinez-Torres was supported
to the venom for 15 min, the spermatozoa were then by CONACYT and UNAM-PAPIIT 204806. Jorge Parodi
incubated during 30 min at 381C, 5% CO2 with was postdoctoral fellow from CTIC-UNAM and from
protease inhibitors as previously described [Deppe MIDEPLAN-Chile. Patricia Navarrete is supported by
et al. 2008]. The spermatozoa were subsequently a CONICYT grant.
incubated with Tripan blue 2% for 15 min at 371C in a Declaration of Interest: The authors report no
For personal use only.
water bath and were centrifuged at 480 Â g for 3 min. conflicts of interest. The authors alone are respon-
The spermatozoa were immediately incubated with sible for the content and writing of the paper.
formaldehyde 4% for 10 min at 371C in a water bath
and were washed with external solution at 480 Â g for
3 min. The supernatant was removed and smears of
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310 P. Navarrete et al.