Regulation of Spermatogenic Cell T-Type Ca²⁺ Currents by Zn²⁺: Implications in Male Reproductive Physiology

Reagents

Disodium ethylenediaminetetraacetate (EDTA) was purchased from J.T. Baker (J.T. Baker, Mexico). Bovine serum albumin (BSA) and N,N,N,N-Tetrakis(2-pyridylmethyl)-1,2-ethylenediamine (TPEN) were acquired from Sigma–Aldrich (St. Louis, MO). TPEN was dissolved in ethanol (10%) and stored frozen until use. Other reagents and salts were from the highest quality commercially available.

Electrophysiology

CD1 mouse spermatogenic cells were prepared as described previously (Castillo et al., 2007). Disaggregated spermatogenic cells were placed on the stage of an inverted microscope (Diaphot 300, Nikon), and membrane currents were recorded with an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA) and filtered at 5 kHz (four-pole Bessel filter). Ion currents were digitized using a Digidata 1440A interface (Molecular Devices) and analyzed with the pCLAMP 10.5 and SigmaPlot 12.3 software suites. Linear capacitative currents were minimized analogically using the capacitative transient cancellation feature of the amplifier. All experiments were carried out at room temperature (∼22°C) and the holding potential (HP) was −120 mV. The recording extracellular solution contained (in mM): 125 TEACl, 5 CaCl₂, 10 n-2-hydroxyethylpiperazine-w-2- ethanesulfonic acid (HEPES) and 10 d-glucose; pH was 7.3 adjusted with TEAOH. Intracellular solution contained (in mM): 120 CsMeSO₄, 10 EGTA, 5 MgCl₂, 10 HEPES, 10 d-glucose and 60 glutamic acid. pH was adjusted to 7.4 with CsOH. The osmolality of the external and internal solutions were 290 and 265 mOsmol/kg, respectively. Patch pipettes were made from borosilicate glass and were pulled with a laser micropipette puller P-2000 (Sutter Instruments Co., Novato, CA). The typical micropipette electrical resistance was 3–8 MΩ when filled with internal solutions.

T-type Ca²⁺ current recordings

Macroscopic Ca²⁺ currents were acquired in response to 200 ms test pulses of variable amplitude at a sampling frequency of 20 kHz (50 μs). Current to voltage relationships (I–V curves) were obtained by plotting the peak amplitude of ion currents as a function of their respective membrane potential during the test pulse. Dose-response curves were fitted using the Hill equation:

(1)

where B is the normalized blocked current, IC₅₀ is the Zn²⁺ concentration giving half-maximal inhibition, and n is the Hill coefficient. Voltage dependence curves were obtained from the ratios of peak current amplitudes that were normalized to the maximal current amplitude by fitting to the following Boltzmann relation:

(2)

(3)

where G_max is the maximal conductance, I_max is the maximal activatable Ca²⁺ current, V₅₀ is the voltage at which half of the current is activated or inactivated, V_m is the membrane potential, k is the slope factor, and N is the power factor. Time constants for activation and inactivation were measured by fitting individual traces to the following kinetic equation:

(4)

where t is time, τ is the activation (τ_act) or inactivation (τ_inact) time constant, and I_∝ is an amplitude scaling factor. For the deactivation analysis, data were obtained by fitting a curve to the tail-currents following settling of 95% of the membrane capacitance transient. In all cases, tail-currents were well fitted by a single exponential equation (equation 4) to determine their fast component. Ensemble fluctuation analyses were performed on the tail phase to obtain the full open Ca²⁺ currents that were filtered at 5–KHz and sampled at 10 μs intervals. Ensembles of Ca²⁺ currents were elicited from 4 (control) to 6 (Zn²⁺ and BSA) cells with successive application of 15 identical stimulatory pulses (−45 mV, iteration rate 1 Hz), yielding 60 and 90 recorded pulses for experimental condition, respectively. The baseline noise measured prior to test pulse was used as the background, according to Clampfit 10.5 non-stationary fluctuation analysis. Maximal accepted baseline noise was 5 pA. The background variance was subtracted from the variance obtained by sequential trace subtraction (Alvarez et al., 2002). Single channel current amplitude and total number of available Ca²⁺ channels were calculated with the following equation:

(5)

where σ² is the sum of squared deviations from the mean, I is the macroscopic current amplitude, i is the single channel current amplitude and N is the total number of ion channels.

Statistical data analysis

Statistical analysis was performed using the Sigmaplot 12.3 software (Systat Software, Inc). Data were expressed as the mean ± standard error of the mean (SEM). Statistical significance was determined using Student paired t test or Analysis of variance (ANOVA) and Tukey's test for multiple comparisons. A P < 0.05 was considered significant. All the experiments were repeated at least three times.

Serum albumin (BSA) completely recovered the fraction of spermatogenic cells I_CaT inhibited by Zn²⁺

As previously demonstrated, I_CaT from spermatogenic cells can be evoked using a voltage protocol of 200 ms pulses from −80 up to +20 mV with 5 mV steps, from a holding potential (V_h) of −120 mV. Representative Ca²⁺ current traces show the transitory Ca²⁺ current with the classical criss–cross pattern for I_CaT (Fig. 1A, control upper traces). Consistently, the I_CaT activation threshold was −70 mV and the maximum current amplitude was reached around −40 mV (Fig. 1B, closed circles). Addition of Zn²⁺ (50 μM) almost completely inhibited the I_CaT of spermatogenic cells evoked by the same voltage protocol (Fig. 1A, lower traces; Fig. 1B closed triangles); whereas Zn²⁺ (5 μM) decreased I_CaT amplitude around 60% (Fig. 1B, open circles). Zn²⁺ inhibition of I_CaT was clearly concentration dependent with an IC₅₀ = 2.0 ± 0.8 μM and a Hill constant (n_Hill) = 0.44 ± 0.07 (Fig. 1C). The IC₅₀ value for the I_CaT inhibition by Zn²⁺ in spermatogenic cells is statistically similar to the IC₅₀ (3 μM) reported for the Ca_V3.2 (α1H) encoded-Ca²⁺ currents, confirming that I_CaT of these cells are mainly encoded by Ca_V3.2 subunits (Stamboulian et al., 2004; Treviño et al., 2004; Escoffier et al., 2007), with a n_Hill value indicating two potential binding sites for Zn²⁺ on T-type channels. The inhibitory effect of Zn²⁺ on I_CaT was partially reversible after washing (∼50%; Fig. 2A, open circles), suggesting the presence of a Zn²⁺ high affinity binding site on spermatogenic cells T-type Ca²⁺ channels as described for Ca_V3.2 (α_1H) pore subunits (Nelson et al., 2007; Kang et al., 2010). In neurons, such a Zn²⁺ high affinity binding to T-type Ca²⁺ channels is responsible for long lasting inhibition of an important fraction of the total Ca²⁺ current expressed in these cells, a phenomenon known as tonic inhibition (Nelson et al., 2007). A previous report showed that BSA (0.5%) increases I_CaT amplitude of spermatogenic cells (Espinosa et al., 2000). Consistently, we observed that perfusing spermatogenic cells with a BSA (0.5%) solution increased the I_CaT amplitude by 20% and abolished the rundown observed in control conditions (Fig. 2B, open diamonds vs. closed circles). Considering that BSA is an excellent Zn²⁺ carrier and chelator (Yang and Xie, 2006; Lu et al., 2008), it is possible that this protein increases I_CaT amplitude by removing Zn²⁺ ions previously bound to T-type Ca²⁺ channels in spermatogenic cells, as described for C-type nociceptors (Nelson et al., 2007). As a first approach to explore this possibility, we used BSA (0.5%) as a Zn²⁺ chelator, to further remove all the Zn²⁺ bound to spermatogenic cells Ca²⁺ channels and we were able to completely recover I_CaT confirming the high affinity binding of Zn²⁺ on T-type channels (Fig. 2C, gray circles). Furthermore, adding Zn²⁺ (50 μM) to the BSA (0.5%) solution eliminated the I_CaT amplitude increase and produced a partial I_CaT inhibition (around 40%) probably due to the presence of free Zn²⁺ in the solution (Fig. 2D, dark gray circles). To determine whether BSA (0.5%) increases I_CaT amplitude by removing Zn²⁺ ions previously bound to T-type Ca²⁺ channels releasing them from a putative tonic inhibition caused by Zn²⁺, we used two different high affinity Zn²⁺ chelators, namely TPEN and EDTA. Addition of TPEN at different concentrations (0.01–500 μM) did not induce the observed I_CaT amplitude increase (Fig. 2E, open circles; and F). Consistent with the previous results, TPEN (500 μM) also abolished the I_CaT inhibition by Zn²⁺ (50 μM) showing it was properly chelating this trace metal (Fig. 2F). EDTA (100 μM) addition suggests that the BSA (0.5%)-induced I_CaT increase is not due to a removal of tonic inhibition by Zn²⁺ from Ca_V3.2, as it happens in neurons (Fig. 2F).

Extracellular Zn²⁺ modifies the voltage dependence of inactivation and the inactivation and deactivation kinetics of spermatogenic cell I_CaT

As previously mentioned, several reports indicate that Zn²⁺ differentially affects the biophysical parameters of T-type Ca²⁺ channels, expressed either endogenous or heterologously. However, the results are inconsistent and even contradictory (reviewed in Noh et al., 2010). In order to characterize the molecular mechanisms involved in the Zn²⁺ inhibitory effect on I_CaT from spermatogenic cells, we examined whether Zn²⁺ exposure could modify the steady-state activation and/or inactivation parameters of such Ca²⁺ currents. Addition of Zn²⁺ (5 μM) did not affect neither the activation curve (Fig. 3A left panel) nor the activation time constant (τ_act) values of I_CaT (Fig. 3B left panel). Half activation voltages (V₅₀ act) values were −52 ± 0.9 and −53 ± 0.6 mV in the absence (control, closed circles Fig. 3A) or presence of Zn²⁺ (open circles, Fig. 3A), respectively. However, this Zn²⁺ concentration shifted the steady-state inactivation curve to more positive voltages from a half inactivation voltage (V₅₀ inact) of −88 ± 0.9 to −73 ± 0.8 mV in presence of Zn²⁺ (Fig. 3A, right panel). In addition, Zn²⁺ significantly increased the inactivation time constant (τ_inact) compared to control conditions (Fig. 3B right panel, closed circles). Considering that inactivation but not activation parameters of I_CaT were altered by Zn²⁺, we wondered if recovery from inactivation of I_CaT could be also sensitive to Zn²⁺. Indeed, extracellular Zn²⁺ (5 μM) slowed the recovery from inactivation of I_CaT from a half time of recovery (T₅₀ rec) value of 48 for control (Fig. 4A, closed circles) to 87 ms in the presence of Zn²⁺ (Fig. 4A, open circles). A previous report indicates the increased deactivation rate of I_CaT as a novel mechanism for Zn²⁺ inhibition at least in thalamic neurons (Noh et al., 2010). To elucidate whether this mechanism also operates in spermatogenic cells, we applied a deactivation protocol in the absence or presence of Zn²⁺ (5 μM). Deactivation time constant (τ_Deact) values of I_CaT were larger in the presence of Zn²⁺ (Fig. 5 left panel, open circles) with respect to control condition (Fig. 5 left panel, closed circles) indicating that in spermatogenic cells a different mechanism is responsible for Zn²⁺ inhibition of I_CaT. Altogether, the Zn²⁺-induced modifications on the I_CaT steady-state and kinetics parameters described above are not enough to explain the reduction of I_CaT amplitude here reported.

Zn²⁺ acts as a pore blocker without affecting single channel current amplitude of spermatogenic cells I_CaT

Reduction in I_CaT of spermatogenic cells could be explained if the single channel unitary current (i) and/or the number of available channels (N) of spermatogenic cells were affected by Zn²⁺ addition. To evaluate both possibilities, we performed a non-stationary fluctuation analysis of I_CaT tails. Our results showed that single channel current amplitude (i) was statistically similar in the three different experimental conditions: control, Zn²⁺ (5 μM) or BSA (0.5%), with values of −1.2 ± 0.2, −1.3 ± 0.2, and −1.0 ± 0.2 pA, respectively (Fig. 6B, left panel). The calculated single channel conductance for these single current amplitudes in our experimental conditions ranged between 6 and 10 pS, similar to the single channel conductance reported for T-type channels (5–7 pS) (Cribbs et al., 1998; Perez-Reyes et al., 1998). However, the number of available Ca²⁺ channels dramatically changed in each experimental condition. The total number of available ion channels (N) in control condition was 326 ± 36 per spermatogenic cell (Fig. 6B, right panel closed bar), whereas in the presence of Zn²⁺ (5 μM), the N mean value was 200 ± 20 channels per cell (around 39% less; Fig. 6B right panel, gray bar). Consistently, BSA (0.5%) treatment of spermatogenic cells, an experimental control condition that induced larger amplitude of Ca²⁺ currents by itself and without any previous exposition of these cells to Zn²⁺, produced an increased N value of 546 ± 48 channels per cell (Fig. 6 right panel, dark gray bar).

Extracellular Zn²⁺ in male reproductive organs could regulate I_CaT in spermatogenic cells

The regulation of I_CaT from different neuronal cell types by Zn²⁺ has been widely reported (Noh and Chung, 2003; Nelson et al., 2007; Kang et al., 2010; Noh et al., 2010; Mor et al., 2012). However, despite the Zn²⁺ relevance for spermatogenesis progression and male fertility, how this trace metal regulates spermatogenic cell I_CaT has been poorly explored (Bedwal and Bahuguna, 1994). As previously mentioned, the male reproductive organs contains a high Zn²⁺ concentration (Bedwal and Bahuguna, 1994) and spermatogenic cells accumulate increasing Zn²⁺ concentrations through spermatogenesis (Vallee and Falchuk, 1993; Celino et al., 2011). Considering that the Zn²⁺ IC₅₀ for spermatogenic cell T-type Ca²⁺ channels that we found (2.0 ± 0.8 μM; Fig. 1C) is close to the one reported in heterologous expressed Ca_V3.2 α subunit (3.0 ± 0.2 μM) but different from the obtained IC₅₀ value for Ca_V3.1 α subunit (82.2 ± 6.9 µM) (Kang et al., 2010), this observation is consistent with the proposal that the main component of I_CaT in spermatogenic cells is encoded by Ca_V3.2, as previously reported (Stamboulian et al., 2004; Treviño et al., 2004; Escoffier et al., 2007). Our findings indicate that testis physiological Zn²⁺ concentrations (∼20–200 μg/g dry weight) could influence Ca²⁺ influx via Ca_V3.2 in these cells. Partial reversibility of I_CaT inhibition by Zn²⁺ after washing can be explained considering that Ca_V3.2 Ca²⁺ channels possess a high affinity binding site for Zn²⁺ (Fig. 2A). The fast I_CaT inhibition by Zn²⁺ and the partial rapid recovery of I_CaT after washing suggest a direct Zn²⁺ interaction rather than an indirect regulation of this Ca²⁺ current. However, these observations do not exclude a potential intracellular regulation of I_CaT by Zn²⁺. On the other hand, the need to use a BSA (0.5%) solution to recover the remaining current indicates that Zn²⁺ is strongly bound to spermatogenic cell T-type channels, and is removed by this Zn²⁺ chelator (Fig. 2C). BSA has only one high-affinity binding site for Zn²⁺ named site A, and its dissociation constant is 1 μM (Lu et al., 2008). The I_CaT partial inhibition observed in a BSA (0.5%) solution preincubated with Zn²⁺ (50 μM) is probably caused by the remaining free Zn²⁺ in this experimental condition (Fig. 2D). To explore a potential tonic inhibition by Zn²⁺ of spermatogenic cell I_CaT as the molecular mechanism behind the BSA-induced I_CaT amplitude increase, we used two additional Zn²⁺ chelators named TPEN and EDTA. Our results indicated that contrary to previous observations on neuronal I_CaT (Nelson et al., 2007), spermatogenic cell I_CaT do not present a tonic inhibition by Zn²⁺ in our experimental conditions (Fig. 2F). However, they do not disprove that tonic I_CaT inhibition by Zn²⁺ could occur after sperm transit through the epididymis and/or in the presence of seminal fluid. Alternatively, the BSA-induced I_CaT amplitude increase could be due to this protein's interaction with β-estradiol (Espinosa et al., 2000) and/or with membrane long-chain fatty acids (Lu et al., 2008). Interestingly, the long-chain fatty acid binding site (FA2) and the Zn²⁺ binding site (site A) on BSA are mutually exclusive (Lu et al., 2008).

Zn²⁺ effects on the biophysical parameters of I_CaT in spermatogenic cells are different than in neuronal cells

In contrast to previous reports in neurons (Cataldi et al., 2007; Traboulsie et al., 2007), Zn²⁺ did not affect the steady-state activation curve nor the activation kinetics of spermatogenic cell I_CaT. However, the presence of Zn²⁺ modified the steady-state inactivation curve and inactivation and deactivation kinetics of spermatogenic cell I_CaT. Altogether, these observations suggest that, at least in male germinal cells, the Zn²⁺ binding site(s) could be only accessible in the open configuration of Ca²⁺ channels, but this hypothesis deserves further investigation. Furthermore, the strong I_CaT inhibition by Zn²⁺ cannot be explained by the Zn²⁺ effects on the biophysical parameters on these Ca²⁺ currents: a positive shift in the steady-state inactivation curve and slower inactivation and deactivation kinetics. In addition, the recovery from inactivation was also slowed in the presence of Zn²⁺. Regarding deactivation, the effect of Zn²⁺on this process seems to depend on the Ca_V3 isoform. A previous study in thalamic neurons indicates an increased deactivation rate for Ca_V3.1-encoded I_CaT as a novel mechanism for Zn²⁺ inhibition (Noh et al., 2010); however, Zn²⁺ slows down the deactivation kinetics of I_CaT generated by Ca_V3.3 (Cataldi et al., 2007) or Ca_V3.2 subunits (this work, Fig. 5). Most of these Zn²⁺-induced alterations of I_CaT biophysical parameters would not contribute to the I_CaT amplitude reduction by this trace metal. Nevertheless, the Hill coefficient obtained for Zn²⁺ inhibition (0.44 ± 0.07) suggests, at least, two different Zn²⁺ binding sites with a negative cooperative binding. One of these metal binding sites is constituted by an Asp-Gly-His motif on the transmembrane segments IS3-IS4 plus one Asp residue in IS2 transmembrane segments of Ca_V3.2 Ca²⁺ channels, (Kang et al., 2010). Zn²⁺ occupancy of this metal binding site could be responsible for its effects as a gating modifier and it would be independent of the binding site inside the channel pore where Zn²⁺ directly blocks Ca_V3 Ca²⁺ channels (Kang et al., 2010). The ability of Zn²⁺ to decrease gating charge movement (Kang et al., 2010) could explain the slowing in T-type channel transitions among different channel states (from open to inactive or closed channel states, a less effective recovery from inactivation, etc.; Figs. 3-5). Certainly, this hypothesis deserves to be further tested. In addition, it is quite possible that the interaction of Zn²⁺ with the most external Zn²⁺ binding site (IS3-IS4 and IS2 transmembrane segments) precludes Zn²⁺ blocking of spermatogenic cell I_CaT in a negative cooperative binding mode. As reported by different groups, the main effect of Zn²⁺ on T-type Ca²⁺ channels is reducing Ca²⁺ flux due to its role as a pore blocker (Noh and Chung, 2003; Nelson et al., 2007; Kang et al., 2010; Noh et al., 2010). Consistently, our results indicated that Zn²⁺ reduces the total number of available Ca²⁺ channels of spermatogenic cells without affecting single channel current amplitude (Fig. 6). This effect is the main cause of I_CaT reduction in spermatogenic cells.

Zn²⁺and the I_CaT of male germinal cell line

In the sperm physiological context, increasing Zn²⁺ concentrations in the germ cell line could contribute to silence T-type Ca²⁺ channels and, as a consequence, reducing I_CaT in late spermatogenesis stages. Interestingly, in vivo chelator-induced or diabetes-caused Zn²⁺ deficiency, increases spermatogenic cell apoptosis in rat testis, suggesting a physiological relevant role of Zn²⁺ in spermatogenesis (Zhao et al., 2012, 2013). Recently, it has been reported that subpopulations of spermatogenic cells display spontaneous Ca²⁺ oscillations which involved Ca²⁺ influx through Ca_V3 channels (Sánchez-Cárdenas et al., 2012). However, the role of Ca²⁺ signaling in spermatogenesis is poorly understood. The increase of intracellular Ca²⁺ concentration in Sertoli cells in response to testosterone and follicle-stimulating hormone is associated to cell migration and proliferation during spermatogenesis (Loss et al., 2011). Considering the above mentioned information, in a pathological condition such as diabetes, spermatogenic cell I_CaT could be larger than in physiological conditions due to Zn²⁺ deficiency and it could contribute to spermatogenic cell apoptosis. Interestingly, testicular mitochondrial Ca²⁺ buffering capacity is diminished in diabetic organisms (Amaral et al., 2009), which could exacerbate phenotypes associated to dysregulation of Ca²⁺ homeostasis in male germinal cells. Lastly, maximal I_CaT inhibition could be reached at the stage of ejaculated sperm when mixed with seminal fluid, where Zn²⁺ concentration is 800 up to 3000 μg/g dry weight (Vallee and Falchuk, 1993; Bedwal and Bahuguna, 1994). The presence and functionality of Ca_V3.2 channels in mature and capacitated sperm was previously demonstrated using a Ca_V3.2 deficient mouse line, where the Ca²⁺ influx amplitude in Ca_V3.2^−/− sperm in response to a membrane depolarization was 30% smaller compared to wild-type sperm (Escoffier et al., 2007). In contrast, no macroscopic I_CaT currents but CatSper Ca²⁺ currents were found in epididymal sperm (Xia and Ren, 2009), highlighting the down regulation of I_CaT currents during sperm maturation. The fraction of other voltage activated Ca²⁺ channels which could be functional in mature sperm is not known, but at least it could include Ca_V3.2 and Ca_V2.3 Ca²⁺ channels (Escoffier et al., 2007; Cohen et al., 2014). In a speculative way, once in the female genital tract, ejaculated sperm would be in the presence of a physiologically relevant amount of albumin which could release the remaining fraction of T-type Ca²⁺ channels from their inhibition by the Zn²⁺ present in seminal fluid. While the physiological relevance of macroscopic CatSper Ca²⁺ currents is clear in regulating sperm hyperactivation, a particular form of motility which generates a more powerful swimming force required for successful fertilization; their potential contribution to the acrosome reaction requires further examination. Catsper knockout mice maintain intact spermatogenic cell I_CaT (Ren et al., 2001), and the ability of their spermatozoa to undergo the acrosome reaction is similar to that of those from wild-type mice (Xia and Ren, 2009; Singh and Rajender, 2015). Spermatozoa from CatSper1 null mice show a delayed ZP-induced Ca²⁺ rise that leads to acrosome reaction therefore, the potential contribution of Ca_V2.3 or Ca_V3.2 channels in this phenomenon should be further studied.