1. Systems Biology in Reproductive Medicine, 56:37–43, 2010 Copyright & Informa Healthcare USA, Inc. ISSN: 1939-6368 print/1939-6376 online DOI: 10.3109/19396360903497217 Research Communication + Tetraethylammonium-Sensitive K Current in the Bovine Spermatozoa and its Blocking by the Venom of the ChileanSyst Biol Reprod Med Downloaded from informahealthcare.com by University Autonoma Barcelona Latrodectus mactans Jorge Parodi, Patricia Navarrete, The morphology and size of spermatozoa hinder the study of the functional Marcelo Marconi, ´ ´ ´ Raul Sanchez Gutierrez, properties of the spermatozoa plasma membrane. However, some studies have ´ ´ Ataulfo Martınez-Torres, and revealed the presence of a number of ion channels in this cell. We set out to Fernando Romero Mejıas ´ measure the endogenous currents and to study the effect of the venom of the Centro de neurociencia y Chilean black widow spider (Latrodectus mactans). By patch-clamping bovine For personal use only. ´ Biologia de peptidos-BIOREN, spermatozoa our results indicate the presence of an outwardly rectifying Departamento de Ciencias current, sensitive to changes in K + concentration (30–140 mM) and to tetra- ´ Preclınicas, Facultad de Medicina, Universidad de la ethylammonium (TEA, 10–100 mM). The application of the venom (7.5 g/ml) Frontera, Chile blocks these K+ currents and then alters the passive properties of the plasma membrane. This leads to the entry of Ca ++ , reﬂected by a change in basal ´ ´ Ataulfo Martınez-Torres ﬂuorescent units (572 at 35710 FAU). The Ca ++ inﬂux follows a reduction in Laboratorio Neurobiologıa ´ Molecular y Celular II, the membrane conductance (control 2272; venom 1071 pS), as calcium Departamento de Neurobiologıa ´ channels open in accord with voltage dependence. Molecular y Celular, Instituto de ´ Neurobiologıa, Campus KEYWORDS ionic currents, Latrodectus venom, membrane potential, spermatozoa ´ Juriquilla-Queretaro, UNAM, ´ Mexico INTRODUCTION Abbreviations: TEA: tetraethyl- ammonium; FAU: ﬂuorescent arbitrary There is strong experimental evidence that mature mammalian sperma- units; pS: membrane conductance; tozoa have several ionic-conductors, including those driven by voltage CCVD: calcium channel voltage dependent; PBS: phosphate saline dependent K + channels [Darszon et al. 1996; Labarca et al. 1995; Nuccitelli buffer solution. and Ferguson 1994]. Recent reports using whole cell patch-clamping to Received 13 August 2009; study ion-conductance, have described the functional properties of Ca ++ accepted 19 September 2009. channels (CATsper), which are key components of capacitation [Darszon Address correspondence to et al. 2005; Wennemuth et al. 2000]. Jorge Parodi, Centro de Neuro- ´ ´ ciencias y Biologıa de Peptidos, The mammalian spermatozoa acquire the functional capacity to fertilize ´ ´ Nucleo cientıﬁco BIOREN, an egg during their trajectory along the female genital tract [Boni et al. 2007]. Universidad de la Frontera, Montevideo 0870 Temuco, Chile. In this process the plasma membrane potential is hyperpolarized by the E-mail: firstname.lastname@example.org activation of pH-sensitive K + -channels. This leads to an increase in Ca ++ 37
2. permeability [Kumar et al. 2000; Linares-Hernandez with sensicam, for time lapse assessment with fast et al. 1998; Shi and Ma 1998]. However, this increase perfusion of either venom or a high concentration of of intracellular Ca ++ is not the sole cause of K + (30 mM). Notice the rise in ﬂuorescence and the capacitation, it has also been suggested that the immediate decrease of the emission after a few s as intracellular pH and transient plasma membrane measured by its change in the midpiece. The depolarization play a role [Fraire-Zamora and ﬂuorometric data is summarized in Figure 1D Gonzalez-Martinez 2004; Neri-Vidaurri Pdel et al. showing that both venom and K + increased the 2006] similar to the series of events that occur in free Ca ++ up to 35 ﬂuorescent arbitrary units (FAU) somatic cells [Baker et al. 1973]. The detailed as compared to the uninduced control. Together this mechanism that leads to spermatozoa capacitation data suggests that spermatozoa calcium increases remains to be detailed. after depolarization or perfusion with venom. In a previous report we described that the venom of the Chilean black widow spider Latrodectus mactans increased the concentration of intra- Patch-Clamping Reveals a TEA- cellular Ca ++ in spermatozoa [Romero et al. 2007]. Sensitive K+ CurrentSyst Biol Reprod Med Downloaded from informahealthcare.com by University Autonoma Barcelona Furthermore, this venom is known to block the TEA- Spermatozoa exhibits varied permeability to ions sensitive K + currents in neurons [Parodi and Romero reﬂecting several channels, including the voltage- 2008] as well as endogenous K + currents of Xenopus sensitive K + and ÀCa ++ [Darszon et al. 2006]. As laevis oocytes [Parodi et al. 2008]. To assess the effect illustrated in Figure 2A, whole-cell patch-clamping of this venom on the plasma membrane potential of spermatozoa showed inward and outward cur- of bovine spermatozoa we used the nystatine- rents. P pulses that ranged between À100 mV to perforated patch-clamp technique. The results that + 100 mV were more sensitive to 50 mM TEA. These begin to dissect the molecular components of currents are plotted as a current/ voltage curve (I/V) capacitation are reported below. For personal use only. (Fig. 2B). The TEA-sensitivity of the outward current is consistent with the presence of a voltage depen- dent potassium channel (Kv channel; IUPHAR classiﬁcation). These channels are crucial towards RESULTS maintaining the plasma membrane potential Exposure to Venom Increases Free [Gutman et al. 2003]. Increasing the concentration Intracellular Calcium of extracellular K + shifted the equilibrium potential to more negative values (Fig. 2C). This is in accord It has been shown by spectrophotometry that with that predicted by the Nerst’s equation (Fig. 2D exposing spermatozoa to the venom of the Chilean and Table 1) indicating that the current was due to L. mactans leads to an increase in pH and free K + TEA-sensitive channels. intracellular Ca ++ [Romero et al. 2007]. To extend these results we assessed the changes in the intracellular Ca ++ by ﬂuorometry and patch-clamp. Venom Alters the TEA-Sensitive The spermatozoa loaded with Fluo-3 is shown in Figure 1A. As expected, the basal level of free K+ Current intracellular Ca ++ is low, with most of the cells Outwardly-rectifying currents were efﬁciently and emitting some faint ﬂuorescence. In contrast, reversibly blocked when the spermatozoa were 30 min after the addition of the venom, the number exposed to the venom (Fig. 3A). The changes in of cells exhibiting ﬂuorescence increased consider- membrane conductance are summarized in Figure 3B. ably (Fig. 1B). This was consistently observed Venom reduces the membrane conductance from even after 3 repetitions suggesting that sudden 2271 pS to approximately 1072 pS (Fig. 3B). In depolarization of the plasma membrane may induce contrast upon exposure to venom, the membrane similar effects. resistance changed from 200 mO712 to 800 mO723, Figure 1C shows typical ﬂuorescence traces of the suggesting an important change in the passive mem- spermatozoa midpiece loaded with Fluo-3 acquired brane properties. 38 J. Parodi et al.
3. Syst Biol Reprod Med Downloaded from informahealthcare.com by University Autonoma Barcelona For personal use only. FIGURE 1 Venom-induced rise of intracellular Ca + + . Sample images of Fluo-3 AM loaded spermatozoa in the absence (A), or presence (B) of total venom (7.5 mg/ml). (C), ﬂuorometric records. (D), ﬂuorescent arbitrary units (FAU) of spermatozoa in high K + (30 mM) or venom (7.5 mg/ml); the bars represent means7 SD of three different experiments. Calibration bar ¼ 5 mm. We explored whether venom also modulates expected the pre-pulse currents were approximately spermatozoa K + currents by increasing the free 300% higher than the control. Interestingly, 10 mM intracellular Ca ++ as known to occur in somatic cells TEA did not affect the current. [Romero et al. 2007]. Brieﬂy hyperpolarizing the spermatozoa plasma membrane from À60 mV to À80 mV activates a calcium-dependent K + current DISCUSSION (Fig. 3C, insert). The tail-current derived from this A previous study has indicated that the venom response in the presence of either venom, high K + , induced cellular effect is mediated by low molecular or TEA is plotted in Figure 3C. In most cases at weight heat sensitive peptides present in the venom, 7.5 mg/ml venom with 30 mM potassium or 50 to [Parodi and Romero 2008]. As described above, 100 mM TEA the K + currents were blocked. As the venom of the Chilean L. mactans is effective Effect of Venom on Mature Spermatozoa Currents 39
4. Syst Biol Reprod Med Downloaded from informahealthcare.com by University Autonoma Barcelona For personal use only. FIGURE 2 A TEA-sensitive outwardly rectifying K + . (A), sample records of membrane currents obtained in the absence or the presence of 5 mM TEA. (B), I/V relation from the sample currents (n ¼ 6). (C), I/V plot in increasing concentrations of K + . TABLE 1 Calculated [I/pA ¼ f (Vm/mV)] and Experimental venom of L. mactans increases the free intracellular Reversal Membrane Potentials. Ca ++ negatively impacting the TEA-sensitive K + [K + ] Predicted I/V (mV) Experimental I/V (mV) spermatozoa current. All conductances are crucial to spermatozoa capacitation. 30 mM À40 mV À43 7 7 mV 80 mM À14 mV À16 7 4 mV After several min of exposure of the spermatozoa to 140 mM 0 mV 57 2 mV the venom, free intracellular Ca ++ rises. As shown in Figures 3A and B, the change in the membrane con- ductance after exposure to venom suggests depolari- even in the absence of a-latrotoxin, which is the zation and calcium channel voltage dependent primary component described for this venom in (CCVD) activation in the spermatozoa cells. This another species of spider [Ushkaryov et al. 2008]. may reﬂect the blockage of voltage-dependent K + The results reported in this study suggest that the channels leading to the entrance of Ca ++ from the 40 J. Parodi et al.
5. Syst Biol Reprod Med Downloaded from informahealthcare.com by University Autonoma Barcelona FIGURE 3 Venom blocks the TEA-sensitive current. (A), I/V relation in the absence or presence of 7.5 mg/ml venom. (B), the membrane conductance is reduced in spermatozoa exposed to the venom. (C), comparative effect of venom, high K + , and increasing concentra- tions of TEA (10 to 50 mM) on the prepulse current. The insert shows sample recordings of the prepulse in control cells and cells exposed to venom. The bars represent means7SD of six different experiments.n extracellular medium in accord with previous studies observed. Nevertheless the functional data reported that suggested that the venom inhibits several K + here and elsewhere [Darszon et al. 2006; Marconi conductances [Parodi and Romero 2008; Parodi et al. et al. 2008; Navarro et al. 2008] is consistent with 2008]. Like the oocyte Kv1.1 s [Parodi et al. 2008], this the view that Kv channels are integral to the For personal use only. channel is also present in the spermatozoa midpiece spermatozoa. [Darszon et al. 1999]. Since Kv1.1 is integral to Other natural sources of spermicidal action have membrane potential regulation [Gutman et al. 2003] been reported, including scorpion toxins and plant we compared the effect with high K + added in the extracts [Harat et al. 2008; Lopez-Gonzalez et al. medium. In both cases the concentration of free Ca ++ 2003]. Together with the previous studies of the exhibited a transient rise and sharp fall as detected spermicidal properties of the venom of the Chilean by spermatozoal midpiece ﬂuorescence. However, L. mactans we were enabled, with a new set of a rise in Ca ++ was observed throughout the cell pharmacological tools, to identify the active after long periods of time. This is consistent with the compound(s) that mediate the modulation of ionic- view that a CCVD was present [Darszon et al. 2005]. conductances. Perhaps they could provide a A direct pharmacological study is required to specify resource for a new generation of contraceptives. the Ca ++ and K + currents involved. The molecular identity of the K + channel modulated by the venom was indirectly assessed by the activation kinetics and TEA-sensitivity of the MATERIALS AND METHODS currents. At 5 mM TEA-blocks the outward-rectifying K + current and likely represents a Kv1-like current Spider Retrieval [Marconi et al. 2008], although mouse brain large- Female adult L. mactans were captured in Chile conductance (mSlo) has also been identiﬁed in during the summer months (December 2006 and spermatozoa [Navarro et al. 2008]. The currents January 2007) from the area of ‘‘Alto Bio Bio’’ in the blocked by the spider venom appear similar to those Bio-Bio state (721160 5100 W, 71450 2400 S) as previously found in frog oocytes and neurons in culture [Parodi described [Romero et al. 2003]. Care was taken not to and Romero 2008; Parodi et al. 2008] although damage the breeding zones. The specimens were differences in the expression proﬁles of Kv channels separately maintained in individual jars. To stimulate [Darszon et al. 2006] among species have been the production and concentration of venom in the Effect of Venom on Mature Spermatozoa Currents 41
6. glands, the spiders were raised without food and and Marty 1988] at room temperature, using a patch- only given water for 30 days. clamp ampliﬁer (Warner PC-501A). For whole-cell recordings, the external solution contained 150 mM NaCl, 5.4 mM KCl, 2.0 mM CaCl2, 1.0 mM MgCl2, Venom Retrieval 10 mM glucose buffered with 10 mM HEPES, pH 7.4 The spiders were immersed in liquid nitrogen and buffer. The internal solution contained 120 mM KCl, after 1 min transferred to a phosphate saline buffer 2.0 mM MgCl2, 2 mM ATP-Na2-ATP, 10 mM BAPTA, solution (PBS: 0.1 M NaH2PO4, 0.01 M Na2HPO4, 0.5 mM GTP buffered with 10 mM HEPES, pH 7.4 1.35 M NaCl, pH 7.4) at 41C. The glands were buffer. For perforated patch-clamp 10 mg/ml nystatine removed and the membrane that binds them to the was added [Ermishkin et al. 1976]. For some experi- base was sectioned. Each gland, was placed into ments, the external solution included either 7.5 g/ml a tube containing PBS (25 pairs of glands for 100 ml of venom or 5–100 mM TEA. Spermatozoa were of PBS) and homogenized. The homogenate was clamped at a holding potential of À60 mV. The ionic immediately centrifuged at 1,000 Â g for 15 min. The currents were captured online and digitized at a supernatant was subsequently aliquoted, and frozen sampling rate of 1.2 kHz using pClamp 9 (MolecularSyst Biol Reprod Med Downloaded from informahealthcare.com by University Autonoma Barcelona at À 201C. The protein content of the venom was Devices, Axon) after being ﬁltered (internal 4 Bessel; determined by a modiﬁcation of the Bradford frequency cutoff ¼ 3 kHz). Pulse protocols, data method (BioRad Protein Assay). capture, and recording analysis were performed using pClampﬁt 9.0 software (Molecular Devices, Axon). Spermatozoa Bovine spermatozoa were obtained from samples Fluo-3 AM Loading cryopreserved in liquid nitrogen, which were Bovine spermatozoa were loaded with 5 mM Fluo-3 thawed at 351C for 1 min then suspended in human AM in pluronic acid/DMSO, 10% w/v (Molecular For personal use only. tubal ﬂuid (HTF) medium supplemented with 3% Probes, Eugene, OR, USA) and mounted in a fast- BSA. Spermatozoa were selected by the swim-up switching ﬂow perfusion chamber adapted on the technique (recommendation of [World Health stage of an inverted ﬂuorescent microscope (Eclipse Organization 1999]) and motile spermatozoa were TE, Nikon) equipped with a xenon lamp and a 40 Â placed in a recording chamber coated with poly-L- objective (22–241C). The cells were brieﬂy illumi- lysine (0.2 mg/mL). The recording chamber was then nated (200 ms) for fast record real time analysis, using washed with a standard external solution that a computer-controlled Lambda 10-2 ﬁlter wheel contained 150 mM NaCl, 5.4 mM KCl, 2.0 mM CaCl2, (Sutter Instruments). Regions of interest (ROI) were 1.0 mM MgCl2, 10 mM glucose buffered with 10 mM simultaneously selected on midpiece containing HEPES, pH 7.4 buffer. Fluo-3 ﬂuorescence (excitation 480 nm, emission 510 nm). Images were collected at 2–5 s intervals during a continuous 5-min period or at 0 or 30 min. Micropipette Preparation The imaging was carried out with a 12 bit cooled Borosilicate glass capillary tubes 1.5 mm of internal SensiCam camera (PCO, Germany), using the imag- diameter (Sutter Instrument Co., CA, USA) were ing software Axon Workbench 2.2 (Axon Instru- placed in a vertical puller (Narishige PP 830 m ments). The spermatozoa were exposed to 7.5 g/ml Barishige, Tokyo, Japon) and then treated with a of total venom () or 30 mM potassium () by bath micropolisher (Narishige MF 900) to obtain a micro- perfusion, and changes in the emission of midpiece pipette tip with electrical resistances ranging from ﬂuorescence, measured in FAU and scored. 10 to 12 MO. ACKNOWLEDGMENTS Electrophysiology Fernando Romero, Raul Sanchez, and Jorge Parodi Electrical currents were recorded by using the were supported by the Fondef-Conicyt Chile No perforated whole-cell patch-clamp technique [Horn ´ DO5I10416. Ataulfo Martinez-Torres was supported 42 J. Parodi et al.
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