ABSTRACT
During spermatogenesis, phospholipids and fatty acids (FAs) play an important role both as structural components of spermatogenic cell plasma membranes and as molecular messengers that trigger the differentiation of the male germ cell line. However, spontaneous oxidation of plasma membrane phospholipids and FAs causes a decrease in mammalian fertility. In the present report, we examine the effects of non-enzymatically oxidized arachidonic acid (AAox) on mouse spermatogenic T-type Ca2+ currents (ICaT) due to their physiological relevance during spermatogenesis. AAox effects on the biophysical parameters of ICaT were significantly different from those previously reported for AA. AAox left shifted the I-V curve peak and both activation and steady-state inactivation curves. ICaT deactivation kinetics were slower in presence of AAox and the time for its recovery from inactivation increased significantly. Therefore, the fraction of inactivated Ca2+ channels of spermatogenic cells is increased at voltages where they are usually active. The inhibition of ICaT by AAox could contribute to the infertility phenotype and to the observed apoptotic state of spermatogenic cells induced by oxidized FAs.
INTRODUCTION
During mammalian spermatogenesis, the male germ cell line requires an increase in the intracellular Ca2+ concentration ([Ca2+]i), in order to continue with the cellular differentiation that produces mature sperm. The only voltage dependent Ca2+ channels thus far documented in spermatogenic cells (SCs) are T-type Ca2+ channels recorded as macroscopic currents (Santi et al, 1996; Arnoult et al, 1996). They are mainly encoded by the low voltage activated Ca2+ channel genes CaV3.2 (α1H) and CaV3.1 (α1G) in a lower extent (Treviño et al, 2004). Previous reports have suggested that T-type Ca2+ channels could be important to generate synchronized Ca2+ oscillations relevant for [Ca2+]i elevation and activation of transcriptional factors involved in spermatogenesis (Sánchez-Cárdenas et al, 2012).
In addition to [Ca2+]i increase, during spermatogenesis, phospholipids and fatty acids (FAs) also play important roles both as structural components of the SCs plasma membrane and as molecular messengers that trigger the first meiotic division within the male germ cell line, like the testicular meiotic-activating sterol (Keber et al, 2013). Sertoli cells provide the majority of phospholipids, sterols and FAs during spermatogenesis. The most representative ones, at least in human male gametes are: phosphatidylcholine and phosphatidylethanolamine, cholesterol and desmosterol, palmitic, arachidonic, and docosahexaenoic acids (Alvarez & Storey, 1995; Keber et al, 2013). In particular, AA is released from Sertoli cells in response to follicle-stimulating hormone (Jannini et al, 1994). These results strongly suggest that FAs originating in Sertoli cells could be part of the signaling mechanisms that modulate spermatogenic cell metabolism, proliferation, differentiation or death in the seminiferous tubule (Paillamanque et al, 2016). De novo metabolic synthesis of phospholipids and FAs in Sertoli cells is not enough to cover the spermatogenic cell requirements for these products. Thus, besides de novo synthesis, carrier mechanisms of different fatty acids by albumin have been proposed; free transport of different FAs salts is another option (Brash, 2001). Both hypothesis could be right because a deficient diet in essential FAs changes the lipid composition of Sertoli and spermatogenic cell plasma membranes in a period of 9-14 days; however, plasma membrane composition of the germinal cell line is completely recovered when essential FAs and lipids are reincorporated to the diet (Marzouki & Coniglio, 1982).
Arachidonic acid (AA) is a 20-carbon omega-6 polyunsaturated fatty acid and one of the main components of cellular membranes. In addition, AA is an important second messenger involved in signaling cascades being the substrate to produce many different metabolites like prostaglandins, leukotrienes etc. (Meves, 2008). This molecule possesses four cis-double bonds, which are the source of its flexibility and keep this pure fatty acid in a liquid state, even at subzero temperatures. AA participates in different cellular processes like cellular proliferation, where it generates a Ca2+ signaling cascade and nitric oxide release (Zuccolo et al, 2016). In rat pachytene spermatocytes and round spermatids, AA by itself, not its enzymatically-produced metabolites, induces [Ca2+]i increases caused by Ca2+ release from intracellular stores. This latter effect could be important for male germ line differentiation since this effect is stronger in spermatids than in spermatocytes (Paillamanque et al, 2016).
However, the chemical properties of AA make it subject to multiple possible transformations. Their double bonds are quite propense to react with molecular oxygen. This process can happen non-enzymatically, as consequence of oxidative stress, or through the action of any of three different types of oxygenases: cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (Brash, 2001; Meves, 2008). While the biological effects of AA and its enzymatic oxidation products are widely discussed in literature, those produced by its non-enzymatic oxidation products are mostly unknown (Brash 2001). In the case of mammalian sperm, the spontaneous oxidation of their plasma membrane phospholipids and FAs has been reported to cause a decrease in fertility (Alvarez & Storey, 1995). For instance, loss of human sperm motility due to O2 was reported since 1943 by McLeod (rev. in Alvarez & Storey, 1995). In general, oxidized phospholipids within mammalian sperm produce plasma membrane damage (Jones & Mann, 1973, 1976, 1977, Jones et al, 1978, 1979), irrespective of the oxidation mechanism either spontaneous or enzymatic (Alvarez & Storey, 1995).
To contend with oxidized phospholipids and FAs, human sperm possess two principal protecting enzymes: superoxide dismutase (Mennella & Jones, 1980; Alvarez et al, 1987) and the group of glutathione peroxidase (GPX), glutathione reductase (GSH) and their substrate glutathione (Li, 1975; Alvarez et al, 1987). In addition, human sperm also have catalases (Jeulin et al, 1989; Zini et al, 1993) and phospholipases A2, which contribute by degrading hydroperoxylated phospholipids and FAs (Alvarez & Storey, 1995). As a consequence of their biological relevance, mutations on the above mentioned enzymes or inhibition of GPX by either depletion of reductive substrate GSH by reaction with H2O2 or direct inhibition of the enzyme with mercaptosuccinate, increases the rate of spontaneous peroxidation in human sperm by 20-fold and increase infertility in humans (Alvarez & Storey, 1989; Zini et al, 1993). In addition, chemical agents like nandrolone decanoate, a synthetic anabolic steroid analog of testosterone used in pig cattle; or cyclophosphamide, a chemotherapeutic agent; both increase phospholipid peroxylation, reduce antioxidant activity, produce spermatic abnormality, apoptosis and DNA fragmentation in mammalian testicles (Chabra et al, 2014; Mohamed & Mohamed, 2015). On the contrary, natural antioxidants (melatonin or Rosemary oil) protect against testicular damage produced by phospholipid peroxylation (Chabra et al, 2014; Türk et al, 2016).
Ion channels such as K+, Na+ and TRP channels are regulated by AA and some of its enzymatically oxidized forms in different cell types (Chen et al, 2001; Basora et al, 2003; Meves 2008; Wen et al, 2012). It has also been shown that AA inhibits N-, L-and T-type Ca2+ channels (Xiao et al, 1997; Talavera et al, 2004a; Roberts-Crowley & Rittenhouse, 2007; Roberts-Crowley & Rittenhouse, 2009). AA can inhibit α1G-encoded T-type Ca2+ currents directly, by exerting two independent effects: (a) reducing ion channel availability and (b) shifting the voltage dependence of steady-state inactivation to more negative potentials. Additionally, AA can interact with CaV3.1 (α1G) channels in both resting and inactivated states and the structural determinants of inactivation which modulate the AA affinity for this Ca2+ channel were suggested (Zhang et al, 2000; Talavera et al, 2004a; Talavera et al, 2004b; Roberts-Crowley & Rittenhouse, 2007; Zhang et al, 2013; Baeza et al, 2015). Consistently, it has been shown that inhibition of AA metabolism does not affect the AA inhibitory effect on T-type Ca2+ channels (Talavera et al, 2004b). This result indicates that AA directly, but not its enzymatic products, is inhibiting T-type Ca2+ currents in cells. On the other hand, the effects of AA metabolites produced by COX, LOX and cytochrome P450 have been explored on different ion channels (Marnett et al, 1999; McGiff & Quilley, 1999; Roman, 2002; Basora et al, 2003; Meves, 2008; Zuccolo et al, 2016). However, a better characterization of oxidized AA effects on T-type Ca2+ channels is lacking.
Considering both that AA is an important regulator of mammalian spermatogenesis and that there is incomplete information regarding how itself and its oxidized metabolites regulate ion channels, the goal of the present project was to investigate the effect of non-enzymatically oxidized AA metabolites (AAox) on T-type Ca2+ channels in spermatogenic cells.
RESULTS
Synthesis of non-enzymatically oxidized arachidonic acid (AAox
Due to the physiological relevance the early stages of FAs oxidation have on spermatogenesis, we obtained and evaluated how oxidized AA products impact spermatogenic cell T-type Ca2+ currents.
We generated non-enzymatically oxidized AA products (AAox) by dissolving AA in ethanol and injecting air into the flask, as described in the materials and methods section. After sample preparation, the AAox products were separated and identified chromatographically using HPLC. Our results show that AAox products started to appear after 1 h of air exposure and increased their concentration up to 20 h of treatment (Fig. 1). We found that the oxidation procedure lead to the appearance of 3 major peaks with retention times (RT) at 38 (a relatively small peak), 42 and 45 min, which are not present in the fresh AA sample (Fig. 1A, red line). The generation of AAox products with this strategy was highly reproducible. All separated peaks were re-purified to detect if the drying and dissolving procedures affected the separated compounds. Interestingly, each of the 3 re-purified peaks lead to obtaining p38, in insignificant amounts, and the p42 and p44 fractions in a higher extent (Fig. 1B). These results allowed us to suggest that the two main AAox peaks (p42 and p44) observed after the oxidation procedure could correspond to two different conformational stages, which could be in equilibrium and continuously produced by non-enzymatic oxidation. This continued oxidation and equilibration process precluded the purification of the individual fractions, and thus the inhibitory effect described was due to the mixture of both AAox products. In the text that follows we refer to both products as AAox to simplify naming them.
1H NMR experiments with AA clearly show that the CH from the double bonds at 5.35 ppm (Fig. 1C, upper spectrum). This signal completely disappeared in the 1H NMR spectra of the oxidized products and a new signal appears at 3.60 ppm (Fig. 1C, lower spectrum). This result confirms the complete oxidation of AA and the loss of all its double bonds with the formation of base oxygen carbons once the NMR spectrum was acquired.
This analyses plus GC-Ms of p42 and p45 spectra indicated that AA was hyper hydroxylated and completely loss its unsaturated bonds in presence of air, due to AA non-enzymatic oxidation (Fig. 1C). AAox contained six hydroxyl groups and one epoxide group due to the oxidation of its double bonds. Tautomerism of a hydroxyl and epoxide groups explains the formation of two compounds which interconvert one in each other. The most feasible positions for the epoxide group are double bonds 8 or 11, due to the capacity of interchange this position between them (Fig. 1D). However, the specific structure of AAox requires additional tests and is currently under study (Fig. EV1).
AAox addition shifts the peak of I-V curve to negative potentials
As reported, spermatogenic cells display the classical criss–cross pattern of transitory ICaT (Fig. 2A, control upper traces) (Santi et al, 1996; Arnoult et al, 1996). Consistently, the ICaT activation threshold was −70 mV and the maximum current amplitude was reached around −37 mV (Fig. 2B, closed circles). Previous reports showed that non-oxidized AA decreases the current amplitude encoded by heterologously expressed CaV3.1 channels (α1G) and does not significantly shift their I-V curve shape (Talavera et al, 2004). To examine how non-oxidized AA affects T-type Ca2+ currents in mouse spermatogenic cells, peak current amplitudes were measured applying 200 ms voltage pulses from a holding potential (Vh) of −120 mV up to −40 mV in 5 mV steps. Our results confirmed that AA (5 µM) decreased the ICaT current amplitude of mouse spermatogenic cells around 58% without altering their I-V peak current (Fig. EV2). Surprisingly, addition of a low AAox concentration (250 nM) inhibited ICaT of spermatogenic cells (Fig. 2A, lower traces; and B opened circles) around 54 ± 6%, and the inhibition extent was stable up to 15 min after AAox addition (Fig. 2B closed and opened triangles). In addition, the presence of AAox (250 nM) shifted the peak of the I-V curve around 10 mV to more negative potentials; from −37 ± 1.6 mV, for control, up to −45 ± 0.5 mV, after 10 min of AAox incubation (Fig. 2B and C).
AAox is a potent spermatogenic cell T-type Ca2+ current inhibitor
In order to confirm our above-mentioned observations, we compared the AAox inhibitory potency respect to AA-induced inhibition. Currents were elicited with a test pulse at −40 mV from a Vh of −120 mV. Representative Ca2+ current traces at −40 mV show that 5 and 8 µM AA (Fig 3A, upper panel) and 250 and 1000 nM AAox (Fig 3A, lower panel) significantly decrease their amplitude. For both AA and AAox, ICaT inhibition was dose dependent; however, the IC50 of AA (4.7 ± 0.4 µM) was significantly higher than the one of AAox (186 ± 12 nM) (Fig. 3B). Hill coefficients were 1.6 ± 0.3 and 1.4 ± 0.1 for AA and AAox, respectively.
The spontaneous oxidation of plasma membrane phospholipids and FAs has been involved in mammalian infertility (Alvarez & Storey, 1995). Because there is a lack of information regarding the effects of AAox, and they display a higher inhibitory potency (above shown) and differential effects on T-type Ca2+ channels, we decided to characterize the AAox effects on spermatogenic cell T-type Ca2+ currents.
AAox does not affect neither time-to-peak nor inactivation kinetics of spermatogenic cell ICaT
The shift observed in the peak of the I-V curve could imply changes in ICaT activation kinetics. In order to evaluate the AAox effect on ICaT kinetics, we analyzed both the time-to-peak (tp) and the time constant of inactivation (τinac) in the control condition and in the presence of AAox. AAox products have no effect on inactivation or time-to-peak of spermatogenic cell T-type Ca2+ channels (Fig 4A and B). A previous report showed that fresh AA does not affect these kinetic parameters in heterologously expressed T-type Ca2+ channels (Talavera et al, 2004b). We corroborated that fresh AA (3 µM) did not change the time-to-peak or inactivation kinetics of spermatogenic cell ICaT (Fig. EV3).
AAox left shifts both the steady state inactivation and activation curves of ICaT
Since AA shifts the inactivation curve to more negative potentials, but does not affect the activation curve of heterologous express CaV3.1 Ca2+ channels (Talavera et al, 2004), we tested how AAox affected steady-state inactivation (Fig. 5A) and activation (Fig. 5B) curves. In both cases, addition of AAox led to left shifts of the curves to more negative potentials, with changes of half inactivation voltage from V50= − 65 ± 1 mV in control to V50= − 74 ± 2 mV after AAox addition (Fig. 5A) and with changes of half activation voltage from V50= − 32 ± 0.5 mV in control to V50= − 43 ± 0.6 mV in the presence of AAox (Fig. 5B). As a consequence of both curve shifts, the ICaT current window was also left shifted 10 mV and reached its maximum around −57 mV in presence of AAox, and −47 mV in control conditions.
AAox slows spermatogenic cell ICaT deactivation kinetics
A previous report indicates that fresh AA does not modify the deactivation of α1G channels expressed in HEK cells (Talavera et al, 2004). To evaluate whether AAox regulates the ICaT deactivation in spermatogenic cells, we applied a deactivation protocol in the absence or presence of AAox. Deactivation of ICaT was recorded and tail currents at different deactivating potentials from −110 to −60 mV, after a 5 ms activation pulse, were fitted by a single exponential function to calculated τDeact. Deactivation time constant (τDeact) values of ICaT were larger in the presence of AAox (Fig. 6 left panel, open circles) with respect to the control condition (Fig. 6 left panel, closed circles), indicating that AAox differentially affects spermatogenic cell ICaT.
ICaT recovery from inactivation is delayed by AAox
Taking into account that AA does not affect the recovery from inactivation of α1G Ca2+ currents when applying a Vh more negative than −100 mV (Talavera et al, 2004), we evaluated the effect of AAox on this parameter of spermatogenic cell ICaT. Representative traces of recovery from inactivation show that AAox significantly increases the required time for channel recovery from inactivation state from τRec(control) = 125 ±7.0 ms in control (Fig. 7A and B, closed circles) to τRec(AAox) = 172 ±3.0 ms (Fig. 7B, open circles).
Albumin reverts AAox-induced ICaT inhibition, but reversion was both AAox concentration and time incubation dependent
It is know that AA-induced inhibition of α1G Ca2+ currents is rapidly reversed upon washing with BSA (Talavera et al, 2004). As previously reported, external BSA increases the ICaT amplitude of spermatogenic cells (Espinosa et al, 2000; López-González et al, 2016). Consistently, we observed that perfusing spermatogenic cells with a BSA (1%) solution increased the ICaT amplitude around 60% with respect to the control in the first 2 minutes of perfusion. Thereafter, the current amplitude decreased, and a plateau level was established being 40% higher than the control condition (Fig. 8A, gray vs closed circles). Considering that BSA is an excellent AA carrier (Brash, 2001) and also acts as a very potent protector against oxidized lipids in sperm due to its high affinity for them (Alvarez & Storey, 1995), we evaluated if this protein could protect the ICaT in spermatogenic cells from oxidized AA. As a first approach to explore this possibility, we used AAox preincubated with BSA (1%), in an equimolar stoichiometry. Adding an AAox/BSA (1%) containing solution eliminated the ICaT amplitude increase and produced a partial inhibition (∼50%) probably due to the transference of AAox into the spermatogenic cell plasma membrane (Fig. 8B, dark gray circles). This inhibition percentage was statistically similar to the observed inhibition in presence of a lower AAox concetration (250 nM; Fig. 8B, open circles). Interestingly, the AAox/BSA-induced ICaT inhibition was fast and spontaneously reverted in spermatogenic cells (Fig. 8B, dark gray circles).
The reversibility of AAox inhibitory effect on T-type Ca2+ currents of spermatogenic cells was studied under various conditions. Cells were incubated in the absence (control) or presence of 325 (Fig. 8C, left panel) or 500 nM (Fig. 8C, right panel) of AAox, for 5 or 20 min-lasting incubations for both concentrations. After incubation with AAox, cells were subsequently perfused with external media or with BSA-containing media. Our results showed that the reversibility of AAox inhibitory effect on T-type current depends on both incubation time and concentration. For instance, the recovery of T-type Ca2+ current amplitude (∼80%) after 2 min of AAox-induced inhibition in presence of 325 or 500 nM showed no statistical difference when it was washed with either BSA-free or BSA-containing extracellular media (Fig. 8C, gray bars). On the contrary, the reversibility of the T-type Ca2+ current inhibition after 20 minutes of incubation with 325 nM AAox was partial (60%) but it was irreversible in presence of 500 nM AAox, even when exposed to BSA-containing media (Fig. 8C, dark gray bars).
DISCUSSION
Various groups have reported deleterious effects of spontaneous oxidation of plasma membrane phospholipids and FAs in mammalian sperm. In general, phospholipids and FAs oxidation of mammalian sperm produces plasma membrane damage (Jones & Mann, 1973, 1976, 1977, Jones et al, 1978, 1979; Alvarez & Storey, 1995), a reduction in antioxidant activity, spermatic abnormality, apoptosis, and DNA fragmentation (Chabra et al, 2014; Mohamed & Mohamed, 2015). This multifactorial phenotype is a consequence of different oxidized plasma membrane compounds which regulate a diversity of intracellular molecular targets.
Here we focused on the specific effect of non-enzymatically oxidized AA products on T-type Ca2+ channels of spermatogenic cells, considering the physiological relevance of both ICaT and AA in the male germ cell line (Sánchez-Cárdenas et al, 2012; Paillamanque et al, 2016, 2017). Furthermore, there are few reports that show the functional modification of spermatogenic cells, by secreted molecules from Sertoli cells, especially at the meiotic and post-meiotic stages (reviewed in Paillamanque et al, 2016). Even less is known about the early effects of specific oxidized FAs on ion channels in spermatogenesis.
As a first approach to characterize the early effects of oxidized FAs on spermatogenic cells physiology, we induced the non-enzymatic oxidization of AA and obtained two main oxidized products which seem to be in equilibrium (Fig. 1). Unlike FAs which are oxidized by enzymes with a high degree of positional and conformational specificity, unsaturated FAs are also vulnerable to many types of oxidation by non-enzymatic mechanisms that result in the formation of many different oxidized forms. Our findings indicate that non-enzymatic oxidation of AA continuously produced AAox products, two of which seem to be in constant equilibrium, precluding their independent evaluation. Therefore, the inhibition of spermatogenic cell ICaT, here reported, must be caused by the mixture of both AAox products. According to MS and NMR spectra, the AAox products lose their four double bonds and all of them were completely hydroxylated, whereas they could contain at least one epoxide group at position 8 or 11 (Fig. 1).
AA and AAox inhibit ICaT by different mechanisms
In the present study we investigated the mechanism of inhibition of spermatogenic cell T-type current by AAox as an essential step in understanding the influence of oxidized FAs on the physiology of male germ cell line. We show that AAox inhibits ICaT more potently than non-oxidized AA (Fig. 2). The majority of earlier reports indicate that AA regulates both α1G-and α1H-encoded Ca2+ currents in the micromolar range (Zhang et al, 2000; Chemin et al, 2002; Talavera et al, 2004). Regardless of the Ca2+ current type (N, L, or T) and cell type (rev in Meves, 2008), the estimated IC50 was between 3 and 10 µM. Our results confirmed that AA inhibits spermatogenic cell ICaT in the same concentration range (IC50 = 4.7 µM); however, AAox showed a significantly higher inhibitory potency (IC50 = 186 nM).
Even though the inhibition of T-type Ca2+ channels by AA has been described in several studies, the contribution of its metabolites is not fully understood. Some reports suggest that Ca2+ current inhibition is caused by AA itself. For instance, Zhang et al (2000) showed that application of lipoxygenase (nordihydroguaiaretic acid) and cyclooxygenase (indomethacin) inhibitors does not prevent the AA-induced inhibition of α1H-encoded Ca2+ current, indicating a direct inhibition by AA. Results of Talavera et al (2004) and Schmitt & Meves (1995) support this hypothesis since addition of 17-ODYA, an AA-derivate metabolite, shows no effect on α1G currents. Furthermore, these authors state that ETYA, a non-metabolizable analogue and inhibitor of the AA metabolism, did not influence the inhibition by AA, suggesting that AA and not its metabolites, is directly involved in T-type Ca2+ current regulation. However, other reports suggest the AA metabolites do participate in Ca2+ current inhibition. Addition of 8,9-EET, a specific cytochrome P-450 produced metabolite, together with AA led to 31% reduction of Gmax, suggesting that the epoxygenase metabolite of AA (8,9-EET) can be partially involved in the inhibition of α1H-encoded channel activity. Interestingly, ETYA by itself led to partial inhibition of α1G current even though its potency was significantly lower than that of AA (Talavera et al, 2004). Contrary to the above mentioned studies, our findings indicate non-enzymatically produced AAox compounds inhibit spermatogenic T-type Ca2+ by acting themselves. They can be easily removed from spermatogenic cell plasma membranes by washing which is mildly enhanced by including BSA (Fig. 8).
The effects of AAox on some biophysical parameters of ICaT were clearly different from those previously reported for AA. For instances, non-oxidized AA induces a decrease of the current amplitude but does not significantly modify the I-V curve shape (Talavera et al, 2004); whereas AAox shifted the peak of I-V curve around 10 mV to more negative potentials (Fig. 3). AA shifts the steady-state inactivation curve to more negative potentials, but it does not affect the activation curve (Talavera et al, 2004). AA-induced Ca2+ current inhibition has been explained as a consequence of a decrease in channel open probability (Liu & Rittenhouse, 2000), which causes an increase in the number of channels in a non-conducting conformation, that is in either closed or inactivated states (Meves, 2008). In contrast, AAox left shifted both the activation and steady-state inactivation curves (Fig. 5). Whereas AA does not modify the deactivation of T-type Ca2+ channels (Talavera et al, 2004), ICaT deactivation kinetics was slower in presence of AAox (Fig. 6). Lastly, AA does not affect recovery from inactivation with holding potentials more negative than −100 mV (Talavera et al, 2004) while AAox significantly increased the time for recovery from inactivation of ICaT at a holding potential of −120 mV (Fig. 7).
Altogether, these data indicate that the molecular mechanism of the AAox-induced inhibition of ICaT is different from that proposed for AA. In the case of AAox, the activation threshold of ICaT I-V curve and the activation curve were both left shifted; this effect could be explained by an increase in the transitions between the closed states of the activation pathway. Our results indicate AAox slowed the deactivation kinetics of ICaT, suggesting that it does affect the transition from the open to the nearest closed state. Thus, in presence of AAox, spermatogenic cell T-type Ca2+ channels could transit from the open to inactivated states and spend more time inactivated rather than going back to close states. Indeed, AAox profoundly affected the inactivation properties of spermatogenic cell ICaT. AAox shifted the voltage dependence of steady-state inactivation by 10 mV to more negative potentials and slowed the kinetics of the recovery from inactivation. Therefore, one of the most important conclusions in this report is that in presence of AAox, the fraction of inactivated Ca2+ channels of spermatogenic cells is increased at voltages where they are usually not inactivated and their recovery from inactivation is slowed. The change in both processes related to Ca2+ channel inactivation reduce both the fraction of Ca2+ channels able to open and their contribution to the macroscopic current amplitude, therefore reducing the Ca2+ current.
AAox-induced ICaT inhibition may occur in a membrane-delimited manner
As previously mentioned, it has been argued that AA inhibits α1G channels partitioning into the plasma membrane (Talavera et al, 2004). When added to cell-free inside-out patches, Ca2+ current inhibition by AA was associated to the compound itself, as its metabolites could not be produced by enzymes given the lack of intermediary reagents in the bath solutions (Arreaza et al, 1997). Furthermore, AA-induced inhibition of Ca2+ current was removed within 0.5 min with a BSA-containing external solution suggesting AA was inhibiting Ca2+ channels in a membrane-delimitated manner (Talavera et al, 2004), though the comparison washing with only media was not performed.
Our results showed that preincubation of AAox with BSA, in an equimolar manner, reduced the AAox-induced ICaT inhibition which then reverted (Fig. 8). As previously mentioned, albumin is a very potent protector against lipid peroxidation in sperm due to its high affinity for oxidized FAs, and also acts as an excellent AA carrier (Alvarez & Storey, 1995; Brash, 2001). Our preincubation assays could suggest that AAox is incorporated to the plasma membrane of spermatogenic cells as a first step, then the oxidized FA reaches a partition equilibrium between the plasma membrane and extracellular free-BSA, recovering the ICaT amplitude. To better understand these effects, we evaluated the effect of washing with media ±BSA at different AAox concentrations and incubation times. Our findings showed that the reversibility of AAox inhibitory effect on ICaT depends on both AAox incubation time and concentration. For instance, Ca2+ current amplitude recovery after 2 minutes of 325 or 500 nM AAox-induced inhibition was statistically similar washing with extracellular media ± BSA. In contrast, ICaT reversibility after 20 minutes of incubation with 325 nM AAox was partial, but it was completely irreversible at 500 nM AAox, even after washing with a BSA-containing media. It is likely that when incubating 20 min with 500 nM AAox, a significant percentage of the oxidized FAs flip to the internal lipid layer precluding their removal from plasma membrane by the BSA containing solution and preventing Ca2+ current amplitude recovery during washing. This result is consistent with the proposal that at high AAox concentration and incubation times inhibition of ICaT is at least partially membrane delimitated. Indeed, washing spermatogenic cells with BSA-containing media slightly improves the recovering of ICaT amplitude from a AAox short-time inhibition.
As previously reported, external BSA increases the ICaT amplitude of spermatogenic cells (Espinosa et al, 2000; López-González et al, 2016). Consistently, we observed that perfusing spermatogenic cells with a BSA solution increased the ICaT amplitude around 40% higher than the control condition (Fig. 8). This result suggests BSA could increase the ICaT amplitude by removing the tonic inhibition from a fraction of Ca2+ channels by endogenous AA/AAox present in the spermatogenic cell plasma membrane.
MATERIALS AND METHODS
Fresh (AA) and oxidized arachidonic acid (AAox) preparation
The sodium salt of Arachidonic acid from Mortierella alpina (AA) was ordered from SIGMA-ALDRICH (SML1395) and dissolved according to instruction with 100% Ethanol to a final concentration of 150 mM (50 mg/ml). Part of freshly prepared AA was used for analyzes and another part was saved for preparation of non-enzymatically oxidized sample. For oxidation, 5 mg of AA dissolved in 100 µL of ethanol were placed in a glass flask with light protection and stirred for 24 h at room temperature (∼24°C) aerating the flask for 30 s each 4-5 hours. Thereafter, the volume of the sample was checked and adjusted to the initial level (100 µL) to preserve the calculated concentration (150 mM). The samples obtained were characterized using HPLC and peaks were separated to isolate AAox forms.
Purification of AAox products by reverse phase HPLC
For HPLC, 10 µL of 150 mM AAox was dissolved in 400 µL 25% Acetonitrile + 0.1% TFA solution and vortexed for 30 s until sample become transparent. Shortly after vortexing, peaks that corresponds to oxidized AA forms were separated by rpHPLC using an analytical CN column (Varian Microsorb-MV 100 CN, 250 x 4.6 mm) thru a gradient from solvent A (0.1 % TFA in water) to solvent B (0.1 % TFA in acetonitrile) starting with a 30:70 ratio of solvent B to A, respectively with then further increasing the B solvent concentration 1%/min. The flow rate was 1mL/min. Effluent absorbance was monitored at 230 nm Components corresponding to fractions p42 and p45 were dried under vacuum to prevent further decomposition and dissolved in ethanol. These fractions were tested for their effect on T-type Ca2+ currents in spermatogenic cells and re-purification. For each peak, procedure of separation, drying and testing was performed at least 3 times.
Mass spectrometry of AAox products
The HPLC-MS experiments were performed on a HPLC Agilent 1260 Infinity coupled to an MS-Q-TOF 6530 with Jet Stream ESI using a C18 column PoroShell 120EC-C18 2.1X100 mm 2,7 µm (Flores-Solis et al, 2016). For each analysis, 10 µL of oily sample were dissolved in 20 mL of 60/40 methanol/water solution. Isocratic conditions were used as solution to dissolve the sample. HPLC chromatograph and mass spectra are shown in Fig. EV1.
NMR experiments
NMR samples were prepared with CDCl3 (99.99 % D). 1H NMR experiments were acquired in a 500 MHz Varian Inova and 300 MHz Bruker Advance at 297 K. NMR spectra of arachidonic acid was used directly from Aldrich. TMS was used as internal standard.
Electrophysiology
Spermatogenic cells were collected from CD1 mouse and prepared as described previously (López-González et al, 2016). After disaggregation procedure spermatogenic cells were placed on the stage of an inverted microscope (Diaphot 300, Nikon) and kept there for 5 min to adhere to the coverslip surface. Membrane currents were recorded with an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). Macroscopic Ca2+ currents were acquired at a sampling frequency of 20 kHz (50 ms) and filtered at 5 kHz (four-pole Bessel filter) during recording, and then digitized using a Digidata 1440A interface (Molecular Devices). Linear capacitive 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. Borosilicate glass was used for preparation of patch pipettes by pulling with a laser micropipette puller P-2000 (Sutter Instruments Co., Novato, CA). The typical micropipette electrical resistance was 6–8 MΩ when filled with internal solutions. The recording extracellular (EM) solution contained (in mM): 125 TEACl, 5 CaCl2, 10 N-2-hydroxyethylpiperazine-W-2-ethanesulfonic acid (HEPES) and 10 D-glucose, pH 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 7.4 was adjusted with CsOH. Osmolarity of external (290 mOsmol/kg) and internal (265 mOsmol/kg) media was monitored using a vapor pressure osmometer (Wescor). Obtained data were analyzed with the Clampfit 10.7 (Molecular Devices, Sunnyvale, CA).
T-type Ca2+ currents analysis
Current to voltage relationships (I–V curves) were obtained by plotting the peak amplitude of Ca2+ currents as a function of their respective membrane potential during the test pulse. Dose-response curves were fitted using the Hill equation: where B is the normalized blocked current, IC50 is the AAox concentration giving half-maximal inhibition, and n is the Hill coefficient. Steady-state inactivation and activation curves were obtained from the ratios of peak current amplitudes normalized to the maximal current amplitude by fitting to the following Boltzmann relation:
where Gmax is the maximal conductance, Imax is the maximal Ca2+ current amplitude, V50 is the voltage at which half of the current is activated or inactivated, Vm 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: where t is time, τ is the activation (τact) or inactivation (τinact) time constant, and Iα 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.
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 (S.E.M.). 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.
AUTHORS’ CONTRIBUTIONS
ILG and AD conceived and coordinated the study, and contributed to the analyses of the experiments and to write the final version of the manuscript. OB and ILG acquired and analyzed electrophysiological data. HPLC separation of oxidized compounds was conducted by OB, FLS and GC. FDP acquired and analyzed the MS and NMR data. All authors reviewed the results and approved the final version of the manuscript.
COMPETING INTEREST
Authors declare that they have no financial and/or non-financial competing interests.
EXPANDED VIEW FIGURE LEGENDS
Figure EV1. Mass spectra of three AAox products separated by HPLC
A) Methylation was made before GC-MS analysis. Fraction 38 (p38, upper panel) differs from fractions 42 (p42, middle panel) and 44 s (p44, lower panel). B) Peak fragments of fractions 42 and 44 are the same (p42 vs p44, middle and lower panels) and differ from fragments of fraction 38 (p38, upper panel). These results confirm that p42 and p44 fractions are very similar compounds.
Figure EV2. Arachidonic acid reduces the ICaT amplitude but does not displace the I-V curve peak current
A) Representative current-voltage relationships obtained with 200 ms test pulses of 10 mV steps in the −80 to +40 mV range from a Vh of −120 mV. Incubation with AA (5 µM, open circles) inhibited the ICaT amplitude current around 60% with respect to the control (closed circles). B) Voltage of the I-V curve peak in absence (control: −40 ± 2 mV, closed bar) or presence of AA (5 mM: −40 ± 1 mV), respectively. In all cases, symbols represent the mean ± S.E.M. (n=4).
Figure EV3. AA does not alter time-to-peak or the inactivation kinetics of spermatogenic cells ICaT
A) Voltage-dependence of ICaT activation (time to peak). Addition of AA (3 µM, open circles) does not alter the activation kinetics values compared to the control condition (closed circles) (n=4). B) ICaT inactivation constant values (τinact) were statistically similar after incubation with AA (3 µM, open circles) compared to control conditions (closed circles). In all cases, symbols represent mean ± S.E.M. (n=4).
ACKNOWLEDGEMENTS
The authors thank Shirley Ainsworth, for technical assistance; Juan Manuel Hurtado, Roberto Rodríguez, Omar Arriaga and Arturo Ocádiz for computer services; and Dr. Otto Geiger for his expertise and assistance in chromatography. OB was depositary of a fellowship from Dirección General de Asuntos del Personal Académico/ Universidad Nacional Autónoma de México (DGAPA/ UNAM). This work was supported by CONACyT-México (71 and 128566 to AD and 84362 to ILG) and DGAPA/ UNAM (IN205516 to AD and IN205518 to ILG). In its final stage, this work was supported by the Ministry of Education, Youth and Sports of the Czech Republic - projects “CENAKVA” (No. CZ.1.05/2.1.00/01.0024), “CENAKVA II” (No. LO1205 under the NPU I program), CZ.02.1.01./0.0/0.0/16_025/0007370 Reproductive and genetic procedures for preserving fish biodiversity and aquaculture and by the Grant Agency of the University of South Bohemia in Ceske Budejovice (125/2016/Z) and by the Czech Science Foundation (No. 18-12465Y).