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-sensitiveSyst Biol Reprod Med Downloaded from informahealthcare.com by University Autonoma Barcelona on 11/02/10 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: firstname.lastname@example.org nents in the capacitation process [Darszon et al. 2005; Wennemuth et al. 303
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, thisSyst Biol Reprod Med Downloaded from informahealthcare.com by University Autonoma Barcelona on 11/02/10 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.
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 theSyst Biol Reprod Med Downloaded from informahealthcare.com by University Autonoma Barcelona on 11/02/10 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
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.Syst Biol Reprod Med Downloaded from informahealthcare.com by University Autonoma Barcelona on 11/02/10 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.
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
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) andSyst Biol Reprod Med Downloaded from informahealthcare.com by University Autonoma Barcelona on 11/02/10 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.
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.Syst Biol Reprod Med Downloaded from informahealthcare.com by University Autonoma Barcelona on 11/02/10 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 REFERENCES Baker, P. F., Meves, H. and Ridgway, E. B. (1973) Calcium entry in the treated spermatozoa were spread on two slides, response to maintained depolarization of squid axons. J Physiol coated with giemsa 10%, and incubated for 40 min at 231:527–548. room temperature in darkness. Finally, at least 100 Boni, R., Gualtieri, R., Talevi, R. and Tosti, E. (2007) Calcium and other ion dynamics during gamete maturation and fertilization. Theriogenology spermatozoa per slide were counted and evaluated in 68(Suppl 1):S156–S164. a Zeiss optical microscope (Axiolab). Brandelli, A., Miranda, P. V. and Tezon, J. G. (1996) Voltage-dependent calcium channels and Gi regulatory protein mediate the human sperm acrosomal exocytosis induced by N-acetylglucosaminyl/mannosyl neoglycoproteins. J Androl 17:522–529. DNA Fragmentation Coronel, C. E. and Lardy, H. A. (1992) Functional properties of caltrin proteins from seminal vesicle of the guinea pig. Mol Reprod Dev DNA fragmentation was assessed using the In situ 33:74–80. Cell Death Detection kit (Roche, Basel, Switzerland). Darszon, A., Lievano, A. and Beltran, C. (1996) Ion channels: key Sperm samples incubated previously during 15 min elements in gamete signaling. Curr Top Dev Biol 34:117–167. Darszon, A., Nishigaki, T., Wood, C., Trevino, C. L., Felix, R. and Beltran, C. were ﬁxed in 4% formaldehyde for 15 min at 41C. (2005) Calcium channels and Ca2+ ﬂuctuations in sperm physiology. Cells were washed twice and permeabilized with 5% Int Rev Cytol 243:79–172. Deppe, M., Morales, P. and Sanchez, R. (2008) Effect of protease Triton X-100 in PBS for 30 min at room temperature. inhibitors on the acrosome reaction and sperm-zona pellucida binding Cells were washed, and incubated with 50 ml of TdT in bovine sperm. Reprod Domest Anim 43:713–719. enzyme and Fluorescein isothiocyanate (FITC) conju- Fatehi, A. N., Bevers, M. M., Schoevers, E., Roelen, B. A., Colenbrander, B. and Gadella, B. M. (2006) DNA damage in bovine sperm does not gated dUTP labeling mixture for 1 h at 371C in the block fertilization and early embryonic development but induces dark. Cells were washed twice in PBS and at least 200 apoptosis after the ﬁrst cleavages. J Androl 27:176–188. Fraire-Zamora, J. J. and Gonzalez-Martinez, M. T. (2004) Effect of spermatozoa were counted and evaluated in the Zeiss intracellular pH on depolarization-evoked calcium inﬂux in human ﬂuorescence microscope (Axiolab). In each batch, sperm. Am J Physiol Cell Physiol 287:C1688–C1696. Effect of Venom on Mature Spermatozoa Function 309
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