O-6: Progesterone-Induced Reorganization of NOX-2 Components in Membrane Rafts is Criticafor Sperm Functioning in Capra Hircus

Background: Reactive oxygen species (ROS) is generated by spermatozoa from human (Aitken and Clarkson, 1987; Alvarez et al., 1987), rat (Vernet et al., 2001), mouse (Fisher and Aitken, 1997), rabbit (Holland et al., 1982) and buffalo (Roy and Atreja, 2008). ROS initiates signaling pathways regulating key processes including capacitation (Aitken et al., 1998;Aitken et al., 1996;deLamirande et al., 1997;deLamirande et al., 1998a;deLamirande and Gagnon, 1993b;deLamirande and Gagnon, 1993a;Griveau et al., 1994;Leclerc et al., 1997), acrosome reaction (deLamirande et al., 1998b;Furuya et al., 1992;Leyton and Saling, 1989;Liguori et al., 2005;Rivlin et al., 2004) and sperm-zona pellucida interaction (Aitken et al., 1989;Aitken and Clarkson, 1987). Progesterone induces acrosmal exocytosis in capacitated spermatozoa through rapid nongenomic pathways which include calcium influx, phosphorylation of proteins and activation of several signaling cascades, which are attenuated by a countering set of nongenomic signaling triggered by estrogen (Baldi et al., 2009). Estrogens and aromatase have a direct relation with semen quality and sperm motility (Carreau et al., 2009). Nitric oxide (NO) has been shown to activate cAMP-protein kinase A pathway during capacitation and protein kinase G pathway during acrosome reaction (Herrero et al., 2003). While attempts to identify a progesterone receptor on spermatozoa are still on, human spermatozoa have been shown to possess estrogen receptors (ER) ERα and ERβ (Aquila et al., 2004). The key molecules involved in superoxide anion production in spermatozoa have always been elusive. While mitochondrial origin of superoxide might appear highly plausible, localization of a phagocyte oxidase (PHOX) homolog known as NADPH oxidase 2 (NOX2) components on the anterior region of the sperm head in mouse spermatozoa suggested a non-mitochondrial origin of superoxide in spermatozoa (Shukla et al., 2005). NOX2 is a multicomponent enzyme consisting of two membrane located subunits, viz., gp91phox and p22phox (Vignais, 2002) and its regulatory modules including p67phox, p47phox and p40phox (Babior, 1999;El-Benna et al., 2009;Park et al., 1994). NOX subunits are present in the lipid raft (LR) compartment of neutrophils (Shao et al., 2003) and tobacco leaf (Mongrand et al., 2004). Clustering of LRs on the membrane of coronary endothelial cells produced aggregation and activation of NOX, thereby forming a redox signaling platform (Yang and Rizzo, 2007;Zhang et al., 2006). Spermatozoa possess caveolin- 1 enriched raft microdomains localized to regions appropriate for involvement with acrosomal biogenesis and exocytosis, as well as signaling pathways regulating such processes as capacitation and flagellar motility (Travis et al., 2001). This has led us to hypothesize that NOX components may be associated with membrane rafts in spermatozoa and sperm membrane rafts may be involved in the ROS signaling and thereby have a functional role in sperm capacitation and acrosome reaction. In this study, we evaluated the organization of NOX2 in membrane rafts of spermatozoa during capacitation and progesterone-indcued acrosome reaction in vitro. The effect of 17β-estradiol and methyl-β-cyclodextrin (MBCD) on raft architecture, NOX2 organization and superoxide production capabilities of spermatozoa were also assessed, which demonstrated a functional link between raft structure and NOX2 activity in spermatozoa. Materials and Methods: Reagents N-2-hydroxethyl piperazine-N-ethanesulfonic acid (HEPES), progesterone, estradiol, dimethyl sulfoxide (DMSO), Trizma hydrochloride, Trizma base, phenyl methyl sulphonyl fluoride (PMSF), ethylene glycol tetra acetic acid (EGTA), poly-L-lysine, sodium orthovanadate, Tween 20 and methyl beta cyclodextrin (MBCD) were from Sigma Chemical Company, Milwaukee, WI. Analytical grade sucrose for density gradients was from HiMedia Laboratories Mumbai, India. Triton X-100 was from Amersham Life Sciences, Cleveland, OH. LumiMax® Superoxide Anion Detection Kit was from Stratagene, La Jolla, CA. DC Protein Assay Kit was purchased from BioRad Laboratories, CA. Rabbit polyclonal antibodies gp91-phox (H-60), p22-phox (FL-195), p40-phox (H-300), p67-phox (H-300), p47-phox (H-195) and caveolin- 1 (N-20) were purchased from Santa Cruz Biotechnology Inc., CA. Goat anti-rabbit IgG-HRP conjugate and goat anti-rabbit IgG-FITC conjugate were from Bangalore Genei, Bangalore. Sperm preparation Testes from adult goat (Capra hircus), obtained from local slaughterhouse were washed thoroughly in Hank’s Balanced Salt Solution (HBSS) maintained at 37°C. Cauda epididymidis was excised, and was washed 3 times in fresh HBSS, slit longitudinally and was placed in a watch glass containing 5 ml HBSS for 10 minutes at 37°C in a CO2 incubator to allow spermatozoa to swim out of the epididymis. The sperm suspension was collected and was filtered through a Nitex membrane (80 μM) to remove any cell debris. The suspension was centrifuged at 800 x g for 10 minutes, and the sperm pellet was resuspended in fresh HBSS adjusting the Abstracts of the 12th Royan International Congress on Reproductive Biomedicine International Journal of Fertility & Sterility (IJFS), Vol 5, Suppl 1, Summer 2011 28 sperm count to 2 million cells/ml. Induction of capacitation and acrosome reaction four aliquots of 1 ml each from the sperm suspension prepared as detailed above were transferred into sterile 5 ml screw cap tubes. One aliquot was pelleted by centrifugation at 1500 x g and was stored at -20°C for protein extraction (hereafter, this sample will be referred to as the cauda sperm sample). The remaining three aliquots were centrifuged to pellet the sperm as stated above, and were resuspended in 1 ml KRB buffer each and incubated for 2 hours at 37°C in a CO2 incubator to induce capacitation. At the end of incubation, capacitation was assessed by observing hyperactivated motility and the presence of a crescentshaped fluorescence resulting from Con-A-FITC binding on the principal acrosomal region of sperm head. One of the aliquots, referred to as capacitated sperm sample hereafter, was pelleted at 1500 x g and was processed for further experiments. The third aliquot received 3.18 μM of progesterone (Baumber et al., 2003) and the incidence of acrosome reaction was assessed microscopically by evaluating the presence or absence of the crescent shaped fluorescence resulting from Con-A-FITC binding. After 1 hour of progesterone treatment the rate of acrosome reaction was assessed by counting the number of spermatozoa that have lost the acrosomal cap as interpreted from Con-A-FITC binding. This sample was designated as acrosome-reacted spermatozoa. The fourth aliquot was treated with 3.18 μM of 17β- estradiol for 1 hour and the cells were evaluated in a fashion similar to that of the progesterone-treated spermatozoa. This sample was designated as estrogentreated spermatozoa. Both acrosome-reacted and estrogen- treated spermatozoa were pelleted at 1500 x g, and were processed for further experiments. Raft isolation Spermatozoa were resuspended in cold TNE buffer (25 mM Tris HCl, 150 mM NaCl, 5 mM EDTA, pH=7.5) containing 1% Triton X-100, 0.01% PMSF and Complete Mini Protease inhibitor tablet (Roche Diagnostics) for 30 minutes at 4°C. The sperm suspension was homogenized (5 strokes of 30 seconds duration each at 5000 rpm) using a teflon-glass mechanical homogenizer (Remi Motors, Mumbai). 3 ml of sperm homogenate was mixed with equal volume of 90% sucrose solution and was overlaid with 3 ml 35% sucrose and 3 ml 5% sucrose in 12 ml ultracentrifuge tube. The samples were centrifuged at 240000 x g in a Beckman L8-80 M ultracentrifuge using SW41 rotor for 15 hours at 4°C. After centrifugation, a thin white band which was visible at 5%-35% sucrose density interface was retrieved which contained the detergent resistant light membrane fraction. This fraction was used for detection of sperm membrane raft associated proteins. Protein quantitation was done using DC protein assay kit (Bio-Rad, Hercules, CA). SDS-PAGE and western blotting Protein extracts were diluted with an equal volume of Laemmli buffer (0.5 M Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.5% bromophenol blue and 2% β-mercaptoethanol) and were heated at 94°C for 4 minutes to denature. Electrophoresis was performed according to standard protocols (Laemmli, 1970). The samples (10 μg protein/lane) were separated on a discontinuous gel with 4% stacking gel and 12% resolving gel and were subjected to electrophoresis at 100 V and the gels were silverstained. For western blotting, the separated proteins on SDS PAGE were transferred onto PVDF membrane (Bio-Rad, Hercules, CA) in the presence of 20% v/v methanol, 25 mM Tris and 190 mM glycine (pH=8.2) at 30 mA constant current for 12 hours using Mini Trans- Blot cell (Bio-Rad, Hercules, CA). The blots were washed in PBS to remove blotting buffer and was incubated in 5% non-fat skimmed milk in PBS for 1 hour. The membranes were washed in three changes of PBS containing 0.1 % Tween-20 (PBST). The blots were incubated for 2 hours in 1:500 dilutions of respective primary antibodies diluted in PBST, washed in three changes of fresh PBST and were incubated for 1 hour in anti-rabbit IgG-HRP conjugate at a dilution of 1:2000 at room temperature. The membranes were washed in PBST and the immunoblots were developed by incubation in PBS containing 0.05% diaminobenzidine (DAB), 0.1% H2O2 and 0.04% nickel chloride until the desired contrast was obtained. The blots were imaged on a BioRad gel documentation system. Intensities of bands of interest were calculated using Advanced Phoretix 1.0 (Nonlinear dynamics, CA). Immunofluorescence assay The spermatozoa were smeared onto clean 0.05% poly- L-lysine coated glass coverslips and were allowed to dry at room temperature. The spermatozoa were fixed by immersing the coverslips in 4 % formalin for 10 minutes neutralized in 0.5 M ammonium chloride, and permeabilized in 0.25% TritonX-100 for 10 minutes. These coverslips were incubated for 2 hours in PBS containing 2 mg/ml BSA and 100 mM glycine to block the nonspecific binding of cellular proteins to the primary antibodies. The coverslips were washed in 3 changes of PBS and were subsequently incubated with respective primary antibody at a dilution of 1:200 in PBS for 2 hours at room temperature. The cover slips were again washed 3 times in PBS and were incubated with goat anti rabbit-FITC conjugate for 1 hour in dark. The coverslips were washed with PBS and stored in dark till imaging. For imaging, the coverslips were mounted on a clean slide and immunofluorescence images were taken on a confocal laser scan microscope (Leica TCS SP-II AOBS system, Germany). Raft disruption by MBCD treatment Cauda epididymidal spermatozoa were treated with 10 mM MBCD in HBSS for 30 minutes at 37°C, and a parallel mock treatment was done by suspending another aliquot of spermatozoa in HBSS alone as control. At the end of incubation the cells were washed twice by pelleting at 800 g and immunofluorescence slides were prepared as described above. Measurement of ROS Superoxide production by the spermatozoa was measured using LumiMax superoxide anion detection kit (Stratagene, La Jolla, CA) following the instructions provided by the manufacturer. The spermatozoa suspensions were prepared as detailed earlier and the density was adjusted to 1 million cells/ml. 100 μl sperm suspension was mixed with an equal volume of the reagent mixture having luminol (100 μM final concentration) and enhancer (125 μM final concentration) in the superoxide anion (SOA) assay medium. To study the effect of progesterone and estrogen treatment, these hormones were added to the capacitated sperm sample aliquots just before taking the reading. To study the effect of raft disruption on superoxide generation, the capacitated sperm sample was treated with raft disruptor MBCD for 30 minutes at 37°C. Chemiluminescence was measured using Wallac Tri-Lux luminescence counter (Microbeta 1450, Perkin Elmer) using Abstracts of the 12th Royan International Congress on Reproductive Biomedicine 2 9 International Journal of Fertility & Sterility (IJFS), Vol 5, Suppl 1, Summer 2011 1450-105 RIGID 96 Cassette and a 96 well plate. Counting time was 2 seconds and each sample was counted 10 times at 1 minute interval. Results were expressed as luminescence count per second (LCPS). A blank sample was also read which had all the reagents except for the spermatozoa, which was used to subtract the background signal from the sample readings. The experiment was done in 5 replicates and the mean and standard deviations were computed. Statistical analysis All the experiments were replicated more than three times and the mean ± SEM was computed. Analysis of variance and t-test were conducted using Microsoft Excel and the graphs were prepared using Sigma Plot 11 (Systat Software, Inc. San Jose, CA, USA). Results: Caveolin-1 positive Membrane lipid Rafts in the sperm: The light buoyant band at 5-35% interface of the density gradient was recovered in fractions 3 and 4, and was caveolin-positive. These fractions constituted the detergent resistant light membrane (DRM) fraction enriched in membrane rafts and raft associated proteins. Fractions 5 and 6 also were moderately caveolin-positive, though higher molecular weight aggregates of caveolin were also present. However, we used fractions 3 and 4 for all subsequent experiments. The levels of caveolin decreased significantly when the spermatozoa recovered from cauda epididymidis were subjected to capacitation in vitro and progesterone-induced acrosome reaction thereafter. Treatment of capacitated spermatozoa with 17β-estradiol also resulted in reduced levels of caveolin in the raft preparations. NOX associated with the sperm membrane Raft: The NOX subunits gp91phox, p22phox, p67phox, p47phox and p40phox were detected in the DRM fraction of spermatozoa. During capacitation in vitro, the DRM fraction of spermatozoa showed a significant decrease in the levels p40 phox, while the other subunits did not exhibit any change in their levels. The levels of gp91phox, p22phox and p40phox in the DRM were elevated during progesterone-induced acrosome reaction and in estrogen-treated cells as compared to the capacitated sperm. NADPH Oxidase subunits are localized in Caveolin rich domains of Spermatozoa. Caveolin showed strong localization over the principal acrosomal region with moderate distribution on the post-acrosomal region and mid piece. The equatorial region and the tail were negative for caveolin. Capacitated spermatozoa showed caveolin distribution which was similar to that of cauda spermatozoa. In acrosome-reacted spermatozoa, the distribution of caveolin was diffused and significantly reduced. However, estrogen treatment did not bring about any noticeable change in the distribution or levels of caveolin in spermatozoa. All the NOX components tested showed a strong localization over the principal acrosomal region and mid piece segment. Interestingly, p47phox and p40phox showed clear localization on the post-acrosomal region as well. Capacitation in vitro increased the intensity of fluorescence due to the localization of gp91phox, p22phox, p47phox and p40phox over the principal acrosomal domain, while p67phox exhibited a moderate reduction in its levels. This was associated with a concomitant reduction in the levels of gp91phox and p22phox in the mid piece segment and that of p47phox and p40phox from the post-acrosomal segment. Induction of acrosome reaction resulted in the loss of all the NOX subunits from the principal acrosomal domain. However, substantial quantities of gp91phox, p47phox and p40phox reappeared on the post-acrosomal region. Conversely, p22phox and p67phox experienced significant reduction in their levels on their head region. Methyl beta cyclodextrin treatment reduces caveolin as well NADPH oxidase subunits fluorescence on the sperm membrane Treatment of cauda epididymal spermatozoa with 10 mM MBCD for 30 minutes brought about a significant decrease in the distribution of caveolin on spermatozoa. The organization of all the NOX components was also sensitive to MBCD treatment, indicating their association with membrane rafts. Levels of superoxide generation changes during sperm maturation Cauda epididymal spermatozoa produced detectable levels of superoxide anion radical, which increased significantly (p<0.02) during capacitation in vitro. Progesterone-induced acrosome reaction resulted in further elevation in the levels of superoxide anion radical production by the spermatozoa. However, treatment of the spermatozoa with 17β-estradiol could not elevate the levels of superoxide anion produced, and the quantity obtained was comparable to that of the cauda spermatozoa. Treatment of the capacitated spermatozoa with progesterone after 10 mM MBCD treatment blocked the enhancement in the production of superoxide anion significantly (p<0.001) indicating that the integrity of membrane raft is a prerequisite for NOX activation in spermatozoa undergoing capacitation and acrosome reaction. A time course analysis indicated that superoxide anion production showed a transient peak within 4 minutes after the addition of progesterone to capacitated spermatozoa, which was again sensitive to MBCD treatment. However, treatment of capacitated spermatozoa with 17β-estradiol failed to induce this effect. Conclusion: Spermatozoa produce increasing levels of reactive oxygen species (ROS) during development. We examined the functional organization of NOX-2 subunits in developing spermatozoa and its involvement in ROS generation. We found NOX-2 components associated predominantly with caveolin-rich microdomains of sperm head and mid piece membranes. Capacitation in vitro enriched NOX-2 components within the caveolin-positive sperm membrane rafts. Progesterone, but not 17β- estradiol, brought about loss of caveolin and extensive reorganizations in NOX-2 distribution on sperm head microdomains and accompanying activation in the levels of superoxide. The sensitivity of superoxide production by spermatozoa to methyl-β-cyclodextrin confirms that NOX-2 function in spermatozoa is tightly dependent on raft-organization. We conclude that progesteroneinduced reorganization of NOX-2 components in sperm membrane rafts is critical for sperm functioning