Abstract
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) like superoxide and nitric oxide are produced by testis and spermatogenic cells in response to heat stress. However, the magnitude and mechanisms of this production in spermatogenic cells have not been described. In this work, we evaluated ROS/RNS production, its pharmacology, mitochondrial oxidative metabolism, membrane potential and antioxidant capacity at different temperatures in isolated rat pachytene spermatocytes and round spermatids. Our results showed an increment in ROS/RNS production by pachytene spermatocytes when increasing the temperature to 40 °C. Instead, ROS/RNS production by round spermatids did not change at temperatures higher than 33 °C. ROS/RNS production was sensitive to NADPH oxidase inhibitor diphenylene iodonium or the mitochondrial complex I inhibitor rotenone. No additive effects were observed for these two compounds. Our results suggest an important mitochondrial ROS/RNS production in spermatogenic cells. Oligomycin-insensitive oxygen consumption (uncoupled oxygen consumption) increased with temperature and was significantly larger in round spermatids than pachytene spermatocytes, indicating a likely round spermatid mitochondrial uncoupling at high temperatures. A similar conclusion can be reached by measuring the mitochondrial membrane potential using rhodamine 123 fluorescence in permeabilized cells or JC-1 fluorescence in intact cells. The antioxidant capacity was higher in round spermatids than pachytene spermatocytes at 40 °C. Our results strongly suggest that at high temperatures (40 °C) pachytene spermatocytes are more susceptible to oxidative stress, but round spermatids are more protected because of a temperature-induced mitochondrial uncoupling together with a larger antioxidant capacity.
Introduction
The increase in testicular temperature observed in cryptorchidia and varicocele has been associated with an increase in testicular oxidative stress (Peltola et al. 1995). Reactive oxygen species/reactive nitrogen species (ROS/RNS) seem to be involved in the oxidative damage in these pathologies (Peltola et al. 1995, Lue et al. 2003, Romeo et al. 2003). In experimental models, heat stress of testis and testicular cells has been associated with spermatocyte apoptosis (Yin et al. 1997, Ikeda et al. 1999, Lue et al. 1999, Rockett et al. 2001, Somwaru et al. 2004) and ROS formation (Ikeda et al. 1999, Ishii et al. 2005), with a likely activation of the intrinsic pathway of apoptosis (Hikim et al. 2003; see Moreno et al. (2012) for a review). The mechanisms associated with this special temperature sensitivity of spermatocytes have not been fully studied, although a role of testis-specific heat shock proteins has been suggested (Nakai et al. 2000, Izu et al. 2004). The intracellular source and associated mechanisms of ROS formation leading to the high heat sensitivity of pachytene spermatocytes or, conversely, to the relative heat resistance of other germ cells have not been studied. In this context, not only ROS formation is important in understanding the differential cellular effects of heat on germ cells, but also the antioxidant capacity that would determine the final outcome of an ROS challenge in these cells (Bauche et al. 1994).
In this work, we studied the extent and sources of ROS/RNS production in response to different temperatures by isolated pachytene spermatocytes and round spermatids, complemented by a determination of mitochondrial metabolism and membrane potential. Our results show that an important ROS/RNS production in these cells was from the mitochondria, both at 33 and 40 °C. Pachytene spermatocytes at high temperature (40 °C) had a higher ROS/RNS production compared with round spermatids. The lower ROS/RNS production induced by high temperature in round spermatids compared with pachytene spermatocytes was associated with a higher mitochondrial uncoupling, together with a larger antioxidant capacity in the latter cells. These properties of round spermatids can represent a protective mechanism from heat-induced oxidative stress in male reproductive cells. Furthermore, pachytene spermatocytes at high temperature (40 °C) were clearly subjected to a higher and differential oxidative stress compared with round spermatids, establishing a mechanistic basis for the high temperature sensitivity of the former cells.
Results
ROS or RNS production in round spermatids and pachytene spermatocytes incubated at different temperatures
When spermatogenic cells were incubated with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; 2.5 μM) at 33 °C in KH-lactate media, an increase in the fluorescence signal was observed due to dichlorofluorescein (DCF) formation (Supplementary Figure 2, see section on supplementary data given at the end of this article). A possible photooxidation of the probe by the light source was discarded from experiments performed with the probe (2.5 μM) in buffer (Supplementary Figure 2). Round spermatids and pachytene spermatocytes showed different kinetics of ROS/RNS formation (kROS/RNS) at different temperatures (Fig. 1). In pachytene spermatocytes, when the incubation temperature was increased from 25 to 40 °C, a progressive increase in ROS/RNS formation was observed, reaching 61% increase when the cells were taken from physiological temperature (33 °C) to a heat stress condition (40 °C) (Fig. 1). Instead, ROS/RNS production in round spermatids was not significantly different at the temperatures studied, showing a tendency to decrease at 40 °C (Fig. 1).
(A) ROS/RNS production rate in round spermatids and pachytene spermatocytes incubated at different temperatures. Each value represents the mean±s.e.m. of at least four different cell preparations. Round spermatids: 25 °C (n=8), 33 °C (n=6), 37 °C (n=8), 40 °C (n=8). Pachytene spermatocytes: 25 °C (n=5), 33 °C (n=6), 37 °C (n=4), 40 °C (n=5). *P<0.05, indicates significant differences in pachytene spermatocytes between 25 and 40 °C, and between 33 and 40 °C. The fluorescence values were standardized for each cell type using the average cell volume and the cell density used in the assays. The statistical analysis was performed using ANOVA and post-hoc tests (Tukey). (B) Arrhenius plot of kROS/RNS formation in pachytene spermatocytes at different temperatures. Each value represents the mean±s.e.m. of at least four different cell preparations. The number of measurements at each temperature is the same as in Figure 1A.
Citation: REPRODUCTION 145, 2; 10.1530/REP-12-0330
In order to compare the temperature dependence of the ROS/RNS formation process in spermatogenic cells, we determined the activation energy of ROS formation using an Arrhenius plot (Fig. 1B). We obtained a value of similar magnitude (30±8 kJ/mol) to those obtained for ROS formation described in animal mitochondria (e.g. Abele et al. 2002) and also not very different from the Ea of oxygen consumption in these same cells (see below), suggesting a likely mitochondria-associated ROS/RNS production in pachytene spermatocytes. As expected from the data, an Arrhenius plot of kROS formation in round spermatids at different temperatures gave a positive slope (not shown), strongly suggesting that temperature was also activating an opposing process to ROS/RNS formation in these cells.
In order to identify the different sources of ROS/RNS formation in pachytene spermatocytes and round spermatids, both at basal temperature and under heat stress, a pharmacological approach was used with different inhibitors of systems that generate ROS/RNS in cells. Thus, we tested the NADPH oxidase (flavoprotein) inhibitor diphenylene iodonium (DPI); the nitric oxide synthase inhibitor NG-methyl-l-arginine (LNMA); the mitochondrial complex I inhibitor rotenone (ROT); and the xanthine oxidase inhibitor allopurinol. Among these inhibitors, only DPI and ROT significantly inhibited the ROS/RNS production (P<0.05), both at basal temperature (33 °C) and at 40 °C (Fig. 2) in both cell types studied. In pachytene spermatocytes, DPI decreased the kROS formation by 23 and 14% at 33 and 40 °C respectively. Additionally, in pachytene spermatocytes, ROT decreased the kROS formation by 35 and 37% at 33 and 40 °C respectively (Fig. 2A and B). In round spermatids, DPI decreased the kROS formation by 27 and 30% at 33 and 44 °C respectively (Fig. 2C and D). In these same cells, ROT decreased the kROS formation by 60 and 25% at 33 and 40 °C respectively (Fig. 2C and D). No additive effects were observed when DPI and ROT or, when DPI and LNMA were added to the cells, suggesting that DPI and ROT could share the same or related targets as inhibitors of ROS/RNS production.
Rate of ROS/RNS formation in round spermatids and pachytene spermatocytes in the presence of inhibitors: nitric oxide synthase (LNMA, 10 μM), NADPH (flavin) oxidases (DPI, 20 μM), mitochondrial complex I (rotenone (ROT), 2 μM) and xanthine oxidase (allopurinol, 100 μM). The inhibitors were present 5 min before ROS/RNS measurements. Asterisks indicate significant differences with respect to controls without inhibitors (*P<0.05, **P<0.01, ***P<0.001). The statistical analysis was performed with ANOVA and post hoc tests (Tukey). (A) Round spermatids at 33 °C: control (n=23), DPI+LNMA (n=6), DPI (n=6), LNMA (n=6), ROT (n=6), ROT+DPI (n=4), allopurinol (n=4). (B) Round spermatids at 40 °C: control (n=15), DPI+LNMA (n=4), DPI (n=5), LNMA (n=6), ROT (n=6), ROT+DPI (n=4), allopurinol (n=4). (C) Pachytene spermatocytes at 33 °C: control (n=20), DPI+LNMA (n=6), DPI (n=6), LNMA (n=6), ROT (n=6), ROT+DPI (n=4), allopurinol (n=4). (D) Pachytene spermatocytes at 40 °C: control (n=15), DPI+LNMA (n=4), DPI (n=5), LNMA (n=6), ROT (n=6), ROT+DPI (n=4), allopurinol (n=4).
Citation: REPRODUCTION 145, 2; 10.1530/REP-12-0330
Control experiments performed in order to check if some of the inhibitors used could affect mitochondrial oxygen consumption showed that DPI, LNMA or allopurinol did not affect oxygen consumption at 33 °C (Supplementary Figure 3, see section on supplementary data given at the end of this article). At this temperature, as expected, ROT and sodium cyanide (NaCN) inhibited the oxygen consumption of round spermatids by 80 and 95% respectively. At 40 °C, DPI showed a significant decrease in round spermatid oxygen consumption (24%), indicating some cross inhibition of DPI in the mitochondrial electron chain (see also Hutchinson et al. (2007)). At 40 °C, ROT and NaCN inhibited the round spermatid oxygen consumption by 92 and 97% respectively. Therefore, our results suggest that an important source of ROS/RNS production in spermatogenic cells at basal temperature and under a heat stress condition was their mitochondria.
In order to determine specifically the effect of temperature on mitochondrial ROS/RNS formation, we estimated the ROT-sensitive ROS/RNS formation as kROS/RNSbasal−kROS/RNSrotenone(Fig. 3). The data of mitochondrial ROS/RNS formation at different temperatures gave a very similar relation to the ROS/RNS formation for the overall process (cf. Figs 1 and 3) both in pachytene spermatocytes and round spermatids, strongly suggesting a differential effect of temperature on pachytene spermatocyte and round spermatid mitochondrial redox processes. These differences could be accounted for, either by a possible uncoupling of round spermatid mitochondrial oxidative phosphorylation at 40 °C (e.g. Mailloux & Harper 2011) or a higher total antioxidant capacity of round spermatids at 40 °C.
Mitochondrial (rotenone-dependent) ROS/RNS formation rate in round spermatids and pachytene spermatocytes incubated at different temperatures. Each value represents the mean±s.e.m. of at least four different experiments. *P<0.05 indicates significant differences in round spermatids between 33 and 40 °C. ***P<0.001 indicates significant differences in pachytene spermatocytes between 40 °C and all the other temperatures. The statistical analysis was performed using ANOVA and post hoc tests (Tukey). Round spermatids: 25 °C (n=8), 33 °C (n=6), 37 °C (n=8), 40 °C (n=8). Pachytene spermatocytes: 25 °C (n=5), 33 °C (n=6), 37 °C (n=4), 40 °C (n=5).
Citation: REPRODUCTION 145, 2; 10.1530/REP-12-0330
Heat stress induces mitochondrial uncoupling in round spermatids but not in pachytene spermatocytes
Our results of ROS/RNS production in pachytene spermatocytes and round spermatids showed a differential effect of high temperature on this process in these two cell types. In order to elucidate the role of mitochondrial function in the differential effect of heat stress in these cells, we performed direct oxygen consumption measurements and also estimated the mitochondrial membrane potential in round spermatids and pachytene spermatocytes at 33 °C (physiological temperature) and 40 °C.
At the physiological temperature of rat testis, the basal oxygen consumption (QO2basal) of round spermatids in KH-lactate media was 69% of the uncoupled oxygen consumption (maximal QO2, QO2max), showing that round spermatid mitochondria at this temperature still had an unused oxidative capacity of 30% of the QO2max (Fig. 4A). Instead, pachytene spermatocytes showed a basal QO2 equivalent to 40% QO2 max, indicating that pachytene spermatocyte mitochondria still had an unused oxidative capacity of 60% of the QO2max (Fig. 4B). At 40 °C, the basal QO2 in round spermatids reached almost the maximum oxygen consumption capacity (94% of the QO2max), whereas in pachytene spermatocytes it was only 52% of the QO2max (Fig. 4A and B). At both temperatures, the QO2 was almost completely blocked by antimycin A or KCN (inhibitors of mitochondrial electron transport), confirming that most cellular oxygen consumption in pachytene spermatocytes and round spermatids was a result of mitochondrial respiration. Pachytene spermatocyte and round spermatid basal O2 consumption gave Arrhenius activation energies of 53±18 and 64±15 kJ/mol respectively (Supplementary Figure 4, see section on supplementary data given at the end of this article).
Rate of oxygen consumption (QO2) in (A) round spermatids and (B) pachytene spermatocytes incubated at different temperatures and in the absence (basal) and presence of different effectors of oxidative phosphorylation. Each value represents the mean±s.e.m. of at least four different experiments. Round spermatids: 25 °C (n=6), 33 °C (n=5), 40 °C (n=7). Pachytene spermatocytes: 25 °C (n=3), 33 °C (n=3), 40 °C (n=4).
Citation: REPRODUCTION 145, 2; 10.1530/REP-12-0330
As an estimation of uncoupled oxygen consumption (Fig. 5), we analyzed the oligomycin-insensitive oxygen consumption in spermatogenic cells. At the physiological temperature (33 °C), the uncoupled QO2 in round spermatids was 80% higher than in pachytene spermatocytes. Under heat stress conditions (40 °C), the uncoupled QO2 in round spermatids was 92% higher than in pachytene spermatocytes. These differences were statistically significant (P<0.0001), confirming that round spermatids presented a partial uncoupling of oxidative phosphorylation with increasing temperatures.
Oligomycin-insensitive (uncoupled) QO2 (expressed as percent QO2max) in round spermatids and pachytene spermatocytes incubated at different temperatures. QO2max: QO2 of cells incubated with CCCP (2 μM). ***P<0.0001 indicates significant differences between round spermatids and pachytene spermatocytes (paired t-test). Round spermatids: 25 °C (n=6), 33 °C (n=5), 40 °C (n=7). Pachytene spermatocytes: 25 °C (n=3), 33 °C (n=3), 40 °C (n=4).
Citation: REPRODUCTION 145, 2; 10.1530/REP-12-0330
Figure 6 shows the changes in the rhodamine 123 (R123) fluorescence ratio (an estimation of mitochondrial membrane potential) induced by heat stress (40 °C) in round spermatids and pachytene spermatocyte mitochondria. All the values were expressed using the R123 fluorescence ratios in the presence of carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 0 mV) as a reference. In round spermatids, at 40 °C, the R123 fluorescence ratios decreased significantly (28% reduction; P<0.05), indicating a reduction in mitochondrial membrane potential (Fig. 6A), corroborating the notion that high temperature induced mitochondrial uncoupling in these cells. Instead, in pachytene spermatocytes, the R123 fluorescence ratios did not change significantly when the temperature was increased to 40 °C (Fig. 6B). Similar to the R123 measurements, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) measurements showed a significant decrease in round spermatid mitochondrial membrane potential exposed to heat stress conditions (Fig. 6C, 53% reduction; P<0.05) but not in the case of pachytene spermatocytes (Fig. 6D).
Mitochondrial membrane potential measurements. Rhodamine 123 (R123) fluorescence ratio (520/490 nm excitation, emission 530 nm) in (A) digitonin-permeabilized spermatids and (B) pachytene spermatocytes at 33 and 40 °C. JC-1 fluorescence ratio (590/560 nm emission, excitation 490 nm) in (C) round spermatids and (D) pachytene spermatocytes at 33 and 40 °C. (n=3; mean±s.e.m.) paired t-test, *P<0.05 R123 spermatids. (n=4; mean±s.e.m.) paired t-test, *P<0.05 JC-1 spermatids.
Citation: REPRODUCTION 145, 2; 10.1530/REP-12-0330
Thus, both QO2 measurements and mitochondrial membrane potential estimates agree that round spermatid but not pachytene spermatocyte mitochondria undergo partial uncoupling when exposed to 40 °C.
Cellular antioxidant capacity in round spermatids and pachytene spermatocytes incubated at different temperatures
Relative round spermatid and pachytene spermatocyte antioxidant capacities were evaluated from the net ROS/RNS levels detected by H2DCFDA under basal and oxidative stress conditions. Figure 7 shows that the kROS ratios (kROS oxidative/kROS basal; see Materials and Methods) determined with H2DCFDA at 33 °C were not significantly different between round spermatids and pachytene spermatocytes. However, under heat stress (40 °C), round spermatids showed a significantly lower ROS production ratio (oxidative/basal) than pachytene spermatocytes (30% less, P<0.05) strongly suggesting an increased total antioxidant capacity (enzymatic and nonenzymatic) of round spermatids at the highest temperature tested (40 °C).
Cellular antioxidant capacity of round spermatids and pachytene spermatocytes estimated as the net ROS detection at 33 °C (A) and 40 °C (B) in the absence and presence of an oxidative challenge (kROS formation ratio). The oxidative challenge was induced with Fe+3 (197 μM)/ascorbate (19.7 mM) that generates O2− and OH radicals. Each value represents the mean±s.e.m. of at least three different experiments. ***P<0.05 indicates significant differences between round spermatids and pachytene spermatocytes (unpaired t-test).
Citation: REPRODUCTION 145, 2; 10.1530/REP-12-0330
Discussion
Heat stress of testis and testicular cells has been associated with ROS formation (Ikeda et al. 1999, Ishii et al. 2005), spermatocyte apoptosis (Yin et al. 1997, Ikeda et al. 1999, Lue et al. 1999, Rockett et al. 2001, Hikim et al. 2003, Somwaru et al. 2004, Vera et al. 2005) and activation of the intrinsic pathway of apoptosis in these cells (Vera et al. 2005). This sensitivity of spermatocytes to heat stress appears to be associated with the heat shock transcription factor 1 (Nakai et al. 2000, Izu et al. 2004). However, the mechanisms that activate the spermatocyte intrinsic pathway of apoptosis and make spermatocytes differentially sensitive to heat stress, or, conversely, the mechanisms that make other spermatogenic cells resistant to heat stress, are still unknown. In this respect, Herrera et al. (2001) showed that increasing the incubation temperature of rat pachytene spermatocytes and round spermatids to 37–40 °C increased intracellular [Ca2+] ([Ca2+]i) to levels that effectively can modify signaling in these cells (e.g. Reyes et al. 2010). Also, the intracellular pH in these cells was found to decrease with increasing temperature. These changes occurred within 1 min of the increase in temperature and can be classified as early events in the response of these cells to heat stress. Interestingly, the same combination of changes in these cellular parameters (increase in [Ca2+]i and decrease in intracellular pH) was associated with apoptotic cells in the testis (Lizama et al. 2007), strongly suggesting that high temperatures per se can set physiological conditions in spermatogenic cells that make them prone to other noxious or proapoptotic stimuli from Sertoli cells.
In this work, our results show that an increase in temperature increases ROS/RNS formation in pachytene spermatocytes but not in round spermatids. Pachytene spermatocyte and round spermatid basal QO2 gave Arrhenius activation energies (Ea) that did not differ from each other. Pachytene spermatocyte ROS/RNS production gave an Ea lower, but not significantly different, from QO2 Ea. These Ea values are comparable with those reported for oxidative processes in yeast mitochondria (Watson et al. 1975) or rat liver mitochondria (Smith 1978, Wander & Berdanier 1985, Samartsev et al. 2003). In fact, the unexpected result in our experiments was the tendency for ROS/RNS production to decrease with higher temperature in round spermatids. Thus, in this work we studied the source of ROS/RNS production in pachytene spermatocytes and round spermatids at 33 and 40 °C, and additionally, elucidated the mechanisms associated with the lack of increase in ROS/RNS production with increased temperatures in round spermatids.
As for the source of ROS/RNS production in pachytene spermatocytes and round spermatids, our results using a pharmacological approach suggested that mitochondria was an important source of ROS/RNS in these cells. Although we did not detect inhibition of QO2 by DPI at 33 °C, the fact that at 40 °C this compound partially inhibited QO2 in round spermatids (see also Hutchinson (2007)), together with the absence of additive effects of DPI plus ROT on ROS/RNS production, strongly suggests that DPI could be acting in the same target as ROT, and that an important source of ROS/RNS at 33 and 40 °C in pachytene spermatocytes and round spermatids was the mitochondria. As is evident in Fig. 2, there was an H2DCFDA oxidation that was not inhibited by any of the redox processes inhibitors used in this work. This unaccounted for-DCF production was more pronounced at higher temperatures and could come from any redox process that produced hydrogen peroxide (H2O2) in spermatogenic cells (e.g. cytochrome P450 and other oxidases). In fact, the results that ROT can inhibit 80–95% of oxygen consumption in the cells (most QO2 can be attributed to mitochondria) but it inhibited 25–30% of ROS/RNS formation at 40 °C strongly suggest that it is highly likely that H2DCFDA could undergo ROS-independent oxidation in these cells. Nevertheless, and being the focus of our work, the ROT-sensitive ROS/RNS production can be clearly attributed to mitochondria as judging from the effects of ROT on QO2 in these cells (Supplementary Figure 3). Our results and analysis of the QO2 of pachytene spermatocytes and round spermatids showed an increased oligomycin-insensitive oxygen consumption (uncoupled respiration) in round spermatids at 40 °C (92% higher than pachytene spermatocytes) (Fig. 5), in agreement with a decreased ROS/RNS production at that temperature in these latter cells. The conclusion of a temperature-dependent uncoupling in round spermatids but not spermatocytes was also supported by the measurements of mitochondrial membrane potential. Thus, high temperature (40 °C) clearly induced a decrease in mitochondrial membrane potential in round spermatids but not in pachytene spermatocytes, consistent with heat-induced uncoupling in round spermatid mitochondria. As has been suggested in other works, a likely consequence of this mitochondrial uncoupling would be a lesser production of ·O2− in the electron transport chain (Finkel & Holbrook 2000, Mailloux & Harper 2011). Thus, round spermatids appear ‘protected’ from heat stress in terms of mitochondrial ROS/RNS production compared with pachytene spermatocytes. Furthermore, our results strongly suggest that under heat stress conditions, round spermatids have a higher total antioxidant capacity than pachytene spermatocytes, contributing even further to the protection from ROS in these cells. Our results, showing an increased oxidative stress in pachytene spermatocytes at high temperature, provide a mechanism that, linked to other noxious stimuli in the heat-stressed testis (see also Herrera et al. (2001)) can help to explain the higher temperature sensitivity of pachytene spermatocytes. The heat stress-induced uncoupling of round spermatid mitochondria and its associated control of ROS/RNS production can certainly provide an explanation for their higher temperature resistance compared with spermatocytes in testis exposed to high temperature.
Materials and Methods
Materials
H2DCFDA was obtained from Molecular Probes (Invitrogen). Sodium l-lactate, DMSO, allopurinol, ROT, DPI, LNMA, oligomycin, CCCP, NaCN, enzymes, salts and buffers were obtained from Sigma–Aldrich Chemical Co. Peroxynitrite (ONOO−) was synthesized in a quenched flow reactor according to Beckman et al. (1990), with some modifications. Peroxynitrite concentration was determined spectrophotometrically in 1 M NaOH (ε302 nm=1670/M per cm).
Animals
Sprague–Dawley adult rats were housed with free access to food and water, and under a 12 h light:12 h darkness photoperiod. The animals were killed by cervical dislocation after being narcotized with CO2 for 45 s. Animal procedures were performed in agreement with the Principles of Laboratory Animal Care, advocated by the National Society of Medical Research, and the Guide for the Care and Use of Laboratory Animal (Institute of Animal Laboratory Resources, 1996). All animal protocols were endorsed by the Comision Nacional de Investigacion en Ciencia y Tecnologia, Chile.
Preparation of rat spermatogenic cells
Spermatogenic cell populations were prepared from adult rat testicles (50–70 days old) using velocity sedimentation separation in a 2–4% BSA gradient, as described by Romrell et al. (1976). Pachytene spermatocyte (85±5% purity) and round spermatid fractions (92±4% purity) were identified both by their size and by the typical aspect of their nucleus stained with Hoechst 33342 (Reyes et al. 1997).
ROS/RNS production measurements
Rat spermatogenic cells were suspended in KH buffer containing (in mM) 144 NaCl, 4.2 KCl, 1.6 MgCl2, 1.6 KH2PO4, 10 HEPES, pH 7.4, supplemented with 0.5 CaCl2 and 10 lactate to a final concentration of 3×105 cells/ml. Cells were preincubated for 5 min at different temperatures (25–40 °C), and then H2DCFDA (2.5 μM) was added to the cell suspension. Fluorescence measurements of DCF (the product of H2DCFDA oxidation) (excitation 495 nm, emission 529 nm) were made at 4-s intervals for 15 min in a Fluoromax-2 fluorometer (Jobin Yvon-Spex, Edison, NJ, USA).
As described by Crow (1997), in vitro addition of peroxynitrite (80 μM) oxidized the probe H2DCFDA (2.5 μM) at 33 °C and pH 7.4. Neither superoxide (1.3 μM ·O2−/min) nor H2O2 (100 μM) could oxidize the probe in the same conditions (Supplementary Figure 1A, see section on supplementary data given at the end of this article). In the presence of peroxidase activity or cells, H2O2 was able to oxidize H2DCFDA (Supplementary Figure 1B). Hence, H2DCFDA oxidation reflects intracellular RNS or ROS formation in cells.
Standardization of the probe oxidation was made with peroxynitrite at different concentrations in the same conditions as the samples. Background signals correspond to the cells (3×105 cells/ml) suspended in KH buffer without the probe. The results were expressed as DCF fluorescence minus the background and normalized per cell volume (μl) using the average diameter of round spermatids (11 μm) and pachytene spermatocytes (16 μm). When the results were expressed in terms of ONOO− formed, the fluorescence signal of DCF was converted to nanomoles of ONOO− equivalents (ONOO−eq) by performing a standard curve of DCF fluorescence vs ONOO− concentration.
ROS/RNS formation data analysis
The ROS/RNS formation rate constants correspond to the slope of the line obtained by linearization of the time course DCF fluorescence (expressed as nanomoles of ONOO− equivalents) and described by the following equation:
Where, Ct are the nanomoles of ONOO− equivalents at time t (s), Co are the nanomoles of ONOO− equivalents at time zero and kROS/RNS formation is the ROS/RNS formation rate constant (nmol of ONOO−eq×μl−1×s−1).
Oxygen consumption measurements
Oxygen consumption (QO2) measurements were performed using a Clark-type O2 sensing electrode (YSI, Yellow Springs, OH, USA) in a sealed glass chamber (1.1 ml) surrounded by a water jacket kept at different temperatures (25–40 °C). Isolated spermatogenic cells were preincubated for 1 min in KH buffer at the specified temperatures before being transferred to the QO2 chamber. QO2 values were obtained from the slope of the O2 tension vs time curve between 0.5 and 2.0 min after each experimental addition.
R123 and JC-1 mitochondrial membrane potential measurements
The mitochondrial membrane potential changes were estimated using the lipophilic cationic probe R123. Spermatogenic cells in suspension (2 million cells/ml) were permeabilized with 20 and 25 μg/ml digitonin (spermatids and pachytene spermatocytes respectively) (see also Reyes & Benos (1984)) in a high-K+ medium (HK+) containing (mM) 140 KCl, 5 KH2PO4, 1.6 MgCl2, 10 HEPES, 1 EGTA, pH 7.4 and exposed to 300 nM R123 for 5 min. Subsequently, the excitation spectra (450–520 nm) at an emission wavelength of 530 nm were taken at the different temperatures tested. R123 undergoes quenching when accumulated by a negative mitochondrial inner membrane potential, with also a red shift in the excitation spectrum (Emaus et al. 1986). The excitation ratio 520/490 with an emission wavelength of 530 nm reflects the mitochondrial membrane potential and would decrease upon depolarization of the membrane potential (Emaus et al. 1986, Scaduto & Grotyohann 1999).
JC-1 can also be used to estimate the mitochondrial membrane potential changes. Spermatogenic cells in suspension with KH buffer supplemented with 0.5 mM CaCl2 and 10 mM lactate (1 million cells/ml) were incubated with JC-1 (200 nM) for 15 min. JC-1 dye exhibits potential-dependent accumulation in mitochondria and aggregates formation, indicated by a fluorescence emission shift maxima from green (∼529 nm) to red (∼590 nm). The emission ratio 590/560 with an excitation wavelength of 514 nm reflects the mitochondrial membrane potential and would decrease upon depolarization of the membrane potential (e.g. Smiley et al. 1991).
Cellular antioxidant capacity of spermatogenic cells
The antioxidant capacity of the spermatogenic cells was studied by exposing the cells (3×105 cells/ml in KH buffer supplemented with 0.5 mM CaCl2 and 10 mM lactate) to an ROS-generating system simultaneously with DCF measurements (see above) for 10 min at 33 and 40 °C. The ROS-generating system (oxidative condition) was composed of FeCl3 (197 μM) and ascorbate 19.7 mM, producing ·O2−, H2O2 and ·OH (e.g. Davies et al. 2007). With a constant ROS challenge, the net ROS concentration estimated by H2DCFDA would depend on the total antioxidant capacity of the cells. The results are expressed as the ratio between the kROS under oxidative condition over the kROS under basal condition (kROS oxidative/kROS basal). Thus, the kROS ratio would be closer to 1 for higher cell antioxidant capacities, i.e. cells that could inactivate the ROS generated under an oxidative condition.
Statistical analysis
The data were analyzed using ANOVA followed by Tukey's post hoc test. In some cases, paired t-tests were used as indicated (GraphPad Prism, GraphPad Software, Inc., La Jolla, CA, USA). Kinetic data analysis was performed with the ORIGIN Software package 6.0 (OriginLab, Northampton, MA, USA).
Supplementary data
This is linked to the online version of the paper at http://dx.doi.org/10.1530/REP-12-0330.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
Funding was provided by CONICYT doctoral fellowship to J A Pino (grant numbers 21080629 and 24100103), Fondo Nacional de Ciencia y Tecnología grant (grant number 1110267) and a PUCV-VRIEA grant to J G Reyes and Fondo Nacional de Ciencia y Tecnología grant (grant number 1110778) to R D Moreno.
Acknowledgements
Interesting and stimulating conversations with Drs Gloria Celedon and Gustavo Gonzalez on the subject of this article are gratefully acknowledged.
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