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Formation and dynamic alterations of horizontal microdomains in sperm membranes during progesterone-induced acrosome reaction
BBRC Biochemical and Biophysical Research Communications 315 (2004) 763–770 www.elsevier.com/locate/ybbrc
Formation and dynamic alterations of horizontal microdomains in sperm membranes during progesterone-induced acrosome reactionq Mohammed Shoeb, Malini Laloraya, and Pradeep G. Kumar* Molecular Reproduction Unit, School of Life Sciences, Devi Ahilya University, Vigyan Bhawan, Khandwa Road, Indore 452 017, MP, India Received 9 January 2004
Abstract Capacitated mammalian spermatozoa undergo a fusion response of their head plasma membrane and the outer acrosomal membrane leading to vesiculation classically known as acrosome reaction. Acrosome reaction occurs in response to various acrosome reaction inducers including zona pellucida proteins, calcium ionophore, dibutyryl cAMP, progesterone, etc. All the acrosome reaction inducers cause a transient of calcium influx into the sperm through voltage-dependent cation channels. Efflux of chloride, stimulation of activity of phospholipases, and phosphorylation of proteins are other known changes introduced by acrosome reaction inducers. Macromolecular organization and dynamics of sperm membranes during the progression of this vesiculation are largely unexplored. In this study, we report that progesterone induced the formation of horizontal microdomains within the exofacial surfaces of sperm membranes, which showed progressive and independent alterations in molecular dynamics. In the light of this observation, we propose that sperm membrane rafts may contain both horizontal and vertical microdomains. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Acrosome reaction; Spermatozoa; Progesterone; Spin labeling; Raft
The acrosome reaction is an exocytotic process leading to the fusion of the plasma membrane of sperm overlying the acrosome and the outer acrosomal membrane at multiple points. Acrosome reaction is an essential step for fertilization. In mammalian sperm, the acrosome reaction is thought to be initiated in vivo by binding to the zona pellucida (ZP), the extracellular matrix of the egg, and only sperm that have completed the acrosome reaction can penetrate the ZP and fuse with the egg plasma membrane [1]. In general, it is thought that sperm binding to ZP3, one of the glycoprotein components of the ZP, induces a transient Ca2þ influx into the sperm through voltage-dependent nonselective cation channels, which in turn leads to activation of a pertussis toxin-sensitive trimeric Gi/o protein-coupled PLC [2]. A tyrosine kinase-regulated q
Abbreviations: BSA, bovine serum albumin; PLC, phospholipase C; ConA, Concanavalin A; GABA, c-aminobutyric acid; DMSO, dimethyl sulfoxide. * Corresponding author. Fax: +91-731-276-2984. E-mail address: [email protected] (P.G. Kumar). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.01.119
PLCc may also be activated during ZP3-binding [2]. Activation of PLCs generates IP3, thereby mobilizing [Ca2þ ]i from the sperm’s intracellular Ca2þ store, the acrosome, although the Ca2þ storing capacity of this organelle seems very limited [3]. Nonetheless, these early responses appear to promote a subsequent sustained Ca2þ influx signal via store-operated channels (SOCs) that results in the acrosome reaction [4–6]. Recent studies have provided evidence for the expression in sperm of transient receptor potential protein channels 1, 3, and 6 (Trp1, Trp3, and Trp6), all putative Ca2þ -permeant SOCs [4,7], and Trp2 has been described to play a role in the ZP3-induced acrosome reaction in mouse sperm [4,8]. However, the precise molecular mechanism by which the acrosome reaction occurs has remained unclear. In addition to ZP, thapsigargin, a specific blocker of the sarco/endoplasmic reticulum Ca2þ -ATPase, which causes Ca2þ depletion from internal stores and leads to capacitative Ca2þ entry [9], is also able to induce the acrosome reaction [10]. Furthermore, progesterone released from the cumulus cells, and thus one of the
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major components of follicular fluid, has also been shown to induce the acrosome reaction in a presumed physiological manner [11,12]. Although the mechanism of action of progesterone on sperm is not yet fully understood, it is thought to induce Ca2þ influx by activating a GABAA-like progesterone receptor/Cl channel [13]. A recent study shows that P promotes the AR in capacitated cauda sperm but is unable to do so in noncapacitated or immature sperm because the availability of PR increases during epididymal transit and after capacitation. The PR responsible for this behavior is different from a classical nuclear receptor on the basis of the immunostaining results and is probably a protein close to 44 kDa on the basis of the ligand assay results [14]. Progesterone, an agonist of acrosome reaction, induces a biphasic Ca2þ i -signal in human sperm comprising an initial transient Ca2þ elevation and a subsequent i ramp or plateau. Nifedipine, an inhibitor of voltageoperated Ca2þ channels, inhibits progesterone-induced acrosome reaction in human sperm. Treatment with nifedipine reduced the duration but not the amplitude of the progesterone-induced Ca2þ i -transient but fluorimetric studies have detected no effect of this compound on the progesterone-induced Ca2þ signal. Using Ca2þ i imaging of single sperm, it was shown that the spermatozoa displayed Ca2þ waves in response to ZP, progesterone, and thapsigargin, although the site of initation of the rises appears to differ with the agonist [15], supporting the view that various acrosome reaction inducers could act as effectors of grossly similar physicochemical alterations in spermatozoa [16]. End point assays have registered that capacitation was characterized by enhanced rotational freedom of molecules within the spermatozoa, with no significant changes in membrane packaging or the lateral diffusion of molecules. The AR inducers restricted the rotational freedom of molecules, while enhancing the lateral diffusion of highly ordered subdomains within the sperm membrane [16]. It is known that microdomain formation and dynamics within living membranes could be modulated by interplay of Ca2þ by modifying the local charge aggregation. However, it is not clear whether sperm membrane shows linear or oscillatory structural alterations during the progress of acrosome reaction. In this study, we have undertaken a time-course study of sperm membrane dynamics in response to progesterone-induced acrosome reaction. Materials and methods Reagents. Progesterone, 16 doxyl stearate (16DS), and 5 doxyl stearate (5DS) were from Sigma chemical, MO, USA. 4-Maleimido 2,2,6,6-tetramethyl piperidinooxyl (MalNet) was from Syva chemical, St .Louis, USA. All other chemicals used were of reagent grade.
Animals. Mature Swiss male mice (Mus musculus) of 3–5 month age group were used for the experiments. Mice were killed by cervical dislocation and the spermatozoa from cauda epididymidis and vas deferens were obtained after mincing these tissues and dispersion in Hanks’ balanced salt solution (HBSS). The suspension was allowed to stand for 10 min at 37 °C. The cloudy upper layer of the suspension was recovered and passed through 80 lm Nitex screen to remove the cell debris. The spermatozoa were pelleted by centrifugation at 500g. The sperm pellets thus obtained were resuspended in KRB for induction of capacitation. Induction of capacitation and acrosome reaction. For the induction of capacitiation, the spermatozoa (1 million cells/ml) were incubated for 2 h in Krebs–Ringer bicarbonate buffer (KRB) containing sodium pyruvate and sodium lactate and in an atmosphere of 95% air and 5% CO2 . Sperm were confirmed to have undergone capacitation by observing their hyperactivation. Acrosome reaction was initiated with the introduction of 3.18 lM progesterone prepared in 0.05% DMSO (final concentration) into the suspension of capacitated spermatozoa in KRB. Concanavalin A-binding assay. The population of spermatozoa that underwent acrosome reaction was evaluated by analyzing the ConA-binding pattern of sperm head. For this purpose, the spermatozoa were withdrawn at intervals from the acrosome reaction setup and immobilized on poly-L -lysine coated coverslip for 2 min. Then they were drained and dipped in HBSS containing 0.05% BSA. The coverslips were placed on blotting paper with sperm-adhering side up. Twenty microliters of ConA + 40 ll HBSS containing BSA was added and coverslip was incubated in dark for 5 min. These coverslips were washed by gently dipping in HBSS containing BSA and deionized water, placed on a glass slide with sperm containing side on glass slide, and observed under NIKON Optiphot Microscope with epifluorescence attachment using Nikon B-2A filter for FITC conjugates. Slides were prepared for capacitated sperm sample and for each hour of incubation with progesterone. Sperm showing bright fluorescence over head were scored as capacitated while those showing patchy or diminished fluorescence were scored as acrosome reacted sperm. Spin labeling protocol. Forty microliter sperm aliquots were labeled with 5 ll spin label (200 lM final concentrations) for 50 min. Unincorporated spin labels were quenched by incubation of spermatozoa for 10 min with 5 ll NiCl2 (50 mM final concentration). The spin-labeled spermatozoa were transferred into 15 ll glass capillaries and one end was sealed with epoxy resin. EPR spectra were recorded on VARIAN E-104 electron spin resonance spectrometer equipped with TM110 cavity. Spectrometer settings and scanning were done according to standard methods described [16]. Instrument settings employed were: scan range, 100G; field set, 3237G; temperature, 27 °C; time constant, 1 s; modulation amplitude, 0.5 10; microwave power, 5 mW; microwave frequency, 9.01 GHz; recording time, 8 min; and receiver gain, mentioned as legend with respective panels of spectra. Sperm membrane fluidity profiling was done immediately prior to the addition of progesterone into the suspension of capacitated sperm, immediately after the addition of progesterone, and at successive 1-h intervals for a span of 5 h. Rotational freedom of the inserted labels was computed as 1/Tc , where Tc ¼ 6:51 1010 w0 ðh0 =hþ1 Þ1=2 . In this equation, w0 represents the width of the mid-field peak, h0 the height of the mid-field peak, and hþ1 is the height of the low field peak. Spin– spin exchange was empirically related to the broadening of the mid-field line (w0 ) and was taken as a measure of the lateral diffusion (DL ). Molecular ordering was measured by computing the total anisotropy in the system by calculating S3 using the relation S3 ¼ ð2ak 2a ? =2ak þ 2a ?Þ. Statistical analysis. All the experiments were repeated seven times unless otherwise indicated. Statistical analysis was done using Microsoft Excel and Sigma Plot. p values was computed using student’s t test.
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Results Induction of acrosome reaction in capacitated spermatozoa Induction of acrosome reaction was verified by monitoring the intactness of the sperm head membranes using ConA-binding. Fig. 1A shows an acrosome-intact spermatozoon stained with ConA, showing heavy fluorescence on the acrosomal domains, indicative of intact acrosome. Fig. 1B is the corresponding differential interference contrast image. Fig. 1C represents ConAbinding onto spermatozoa 1 h after the addition of progesterone. The fluorescence on the head region is diminished drastically signifying that acrosome reaction has occurred in these spermatozoa. In our assays, more than 90% of the spermatozoa underwent acrosome reaction during the first hour after the addition of progesterone. A corresponding DIC image is shown in Fig. 1D. Measurement of surface fluidity of sperm membranes Capacitated spermatozoa probed with 5DS presented a heavy level of immobilization that resulted in the generation of a ‘powder spectrum’ (Fig. 2A) as against signals from freely moving spin labels (Fig. 2G). Transient changes occurring on the membranes of the spermatozoa were monitored immediately after progesterone addition (Fig. 2B) and at subsequent 1 h intervals (Figs. 2B–F). Since we introduced progesterone as a solution in DMSO, we monitored the effect of DMSO, if any, on the overall membrane organization. As expected, DMSO brought about considerable modifications in the sperm membranes (Fig. 2H). In order to compensate for the possible artifacts introduced by DMSO in our experimental set-up, we performed a normalization of the observed values obtained in the progesterone-treated
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samples (which would contain progesterone in DMSO. This group is designated as +P) against that obtained in the DMSO-treated controls (designated as )P). Fig. 2H is a line graph summarizing the rotational freedom in sperm membranes in the +P and )P incubations. In the )P group, there was no detectable change in the rotational freedom of 5DS under our experimental conditions. On the contrary, the +P group presented dynamic increase in the mobility profiles (p < 0:01 and p < 0:008 when 0 h sample was compared with 1 h, and 1 h with 2 h, respectively) of the spin label during the first 120 min of incubation with progesterone (Fig. 2H). Thereafter, we noticed a progressive decline (p < 0:01) in fluidity during the following 80 min (Fig. 2I). Fig. 2J is a plot of the lateral diffusion on the membrane surface. Normalization of the data set of progesteronetreated spermatozoa against their respective DMSOcontrols revealed a significant restriction (p < 0:01) in the lateral diffusion in the membrane microdomains towards the outside, reaching minimal values towards 120 min. Thereafter, the lateral diffusion index reverted back to its original values in the next 2 h (Fig. 2K). Measurement of the internal fluidity of sperm membranes Using 16DS, we evaluated the fluidity of the hydrophobic interior of the sperm membranes. Fig. 3 summarizes our observations, with the representative EPR spectra shown in tracings A–G. 1/Tc values computed at various time points are presented as a line graph (Fig. 3H) and the normalized 1=Tc values are shown in Fig. 3I. We observed a highly significant (p < 0:05) restriction in the rotational freedom (Fig. 3I) and lateral diffusion (Figs. 3J and K) of molecules in this membrane subdomain during the earlier half of the incubation period, lasting for approximately 80 min. After this initial compaction, this zone exhibited a massive increase (p < 0:03) in rotational freedom (Figs. 3H and I) and lateral motion (Figs. 3J and K).
Fig. 1. Photomicrographs of capacitated (A,B) and acrosome-reacted (C,D) visualized under epifluorescence optics showing strong PNA-binding pattern on the entire head (typical of capacitated spermatozoa) in (A), which was diminished remarkably after acrosome reaction as represented in (C). (B,D) Corresponding DIC images (800).
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Fig. 2. Analysis of the sperm membrane structure using electron spin resonance spectroscopy following spin labeling with 5-doxyl stearic acid. (A–F) Representative spectra originating from spermatozoa incubated with acrosome reaction inducer for 0, 1, 2, 3, 4, and 5 h respectively. (G) Free label spectrum. (H) Plot of the rotational freedom of 5DS in progesterone-treated and control groups. A normalization of the values of the progesteronetreated set against the control set is shown in (I). Lateral mobility profiles around the polar heads of the exofacial leaflet of capacitated spermatozoa before and after the addition of progesterone are plotted in (J). Normalized values of DL of spermatozoa treated with progesterone against control groups are represented in (K). Values represented are means SEM of seven replicates analyzed.
Measurement of the protein mobility in sperm membranes EPR spectra from spermatozoa labeled with a thiolreactive spin label MalNet and incubated with progesterone for 0 through 5 h with 1 h interval between
successive recordings are shown in Figs. 4A–G. The computed 1=Tc values from +P and )P groups are plotted as line graph (Fig. 4H) and the normalized 1=Tc values are shown in Fig. 4I. Protein rotation was significantly high (p < 0:002) during the first 200 min after
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Fig. 3. Analysis of the sperm membrane structure using electron spin resonance spectroscopy following spin labeling with 16-doxyl stearic acid. (A–F) Representative spectra originating from spermatozoa incubated with acrosome reaction inducer for 0, 1, 2, 3, 4, and 5 h, respectively. (G) Free label spectrum. (H) Plot of the rotational freedom of 16DS in progesterone-treated and control groups. A normalization of the values of the progesteronetreated set against the control set is shown in (I). Lateral mobility profiles around the hydrophobic tail regions of the exofacial leaflet of capacitated spermatozoa before and after the addition of progesterone are plotted in (J). Normalized values of DL of spermatozoa treated with progesterone against control groups are represented in (K). Values represented are means SEM of seven replicates analyzed.
the addition of progesterone, which was followed by a decline (p < 0:001) in the rotational freedom during the following 50 min (Figs. 4H and I). We observed a second instance of freely rotating proteins (p < 0:0002) on the spermatozoa towards the fag end of our incubations
(Figs. 4H and I). During the initial phase of the incubation with progesterone, thiol-containing proteins experienced reasonably free lateral motion, which was followed by a phase of gradual increase in freedom for lateral motion of proteins (Figs. 4J and K). Since MalNet
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Fig. 4. Analysis of the mobility of sperm membrane proteins using electron spin resonance spectroscopy following spin labeling with a thiol-reacting spin label MalNet. (A–F) Representative spectra originating from spermatozoa incubated with acrosome reaction inducer for 0, 1, 2, 3, 4, and 5 h, respectively. (G) Free label spectrum. (H) Plot of the rotational freedom of MalNet in progesterone-treated and control groups. A normalization of the values of the progesterone-treated set against the control set is shown in (I). Lateral mobility profiles around the hydrophobic tail regions of the exofacial leaflet of capacitated spermatozoa before and after the addition of progesterone are plotted in (J). Normalized values of DL of spermatozoa treated with progesterone against control groups are represented in (K). Order parameter was calculated as explained in methods and is presented in (L). Normalized values of order parameter are shown in (M). Values represented are means SEM of seven replicates analyzed.
produced clear high-field resonance line, we computed the molecular ordering of the protein. A plot of S3 from )P and +P groups is presented in Fig. 4L and the normalized values are presented in Fig. 4M. During the first hour of incubation of the spermatozoa with progesterone, protein ordering declined significantly (p < 0:05). During the subsequent period of incubation, there was a moderate increase in ordering,
yet the values were lower than those of the capacitated spermatozoa (Fig. 4M).
Discussion Progesterone has been described to affect several sperm functions including motility, capacitation, and
M. Shoeb et al. / Biochemical and Biophysical Research Communications 315 (2004) 763–770
acrosome reaction. Progesterone can initiate the mammalian sperm acrosome reaction in vitro, and studies on the effect of progesterone and several other steroids have demonstrated an increased calcium influx following binding to the sperm membrane [17]. Progesterone may achieve this effect through the intervention of extragenomic receptors [18–20]. Progesterone is one of the physiological stimuli of human sperm acrosome reaction and is present in high levels at the site of fertilization (cumulus oophorus). Apart from the increase of intracellular calcium concentrations in spermatozoa shown by many laboratories, the effects of this steroid also extend to mediate efflux of chloride, stimulation of activity of phospholipases, and phosphorylation of proteins [21]. It has been indicated that PKC plays a role in progesterone-induced acrosome reaction and that progesterone-stimulated PKC activation is downstream to stimulation of calcium influx by the steroid [22]. Progesterone-dependent calcium influx appears to be a rapid process [23–25], followed by a sustained rise lasting for several minutes (plateau phase). Both these calcium transients are dependent upon entry of extracellular calcium. The nature of the calcium channel that mediates the effects of progesterone is, currently, unknown. It has been postulated that it may be: (i) part of the progesterone receptor; (ii) voltage-dependent; or (iii) operated by second messengers following activation of the progesterone receptor [26]. However, it should be noted that calcium flux is induced specifically by progesterone in capacitated and uncapacitated sperm cells, whereas only capacitated spermatozoa are able to subsequently complete the acrosome reaction [27], clearly showing that capacitated spermatozoa respond differently to progesterone by manifesting membrane vesiculation. Many investigators find that the introduction of progesterone into the capacitation medium has a positive effect on capacitation [28,29]. It is also shown that a minimum capacitation time of 6 h was required for induction of the acrosome reaction in both progesteronefree and progesterone-supplemented capacitation media [30]. Thus, as against the rapid dynamics of calcium influx in response to progesterone, vesiculation events appear to be slow and progressive. In this study, we attempted to evaluate the progressive changes in the molecular mobility and ordering of sperm membranes of capacitated spermatozoa exposed to progesterone for various time intervals. Three microdomains of the spermatozoa were probed using two lipophilic and one protein-binding spin labels. One of the stearic acids used in this study had the doxyl group covalently attached to the 5th carbon (5DS), while the other one had the doxyl group on the 16th carbon (16DS). MalNet was used as a thiol-reacting spin label that could be anchored onto thiol-containing proteins. During the first hour of incubation of the spermatozoa with progesterone, the rotational freedom
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of the spin label 16DS decreased significantly, while that of 5DS and MalNet showed a considerable enhancement in rotational freedom when compared with the respective values observed in capacitated spermatozoa. Interestingly, both 5DS and MalNet reported a significant decline in the lateral diffusion. This indicates the formation of microdomains within the exofacial surfaces of the sperm membrane with lipids and proteins experiencing rotational freedom and restrictions in lateral mobility at the same time. An earlier report demonstrated an increase in molecular ordering in sperm membranes after incubation of capacitated sperm with progesterone for 1 h [31]. Thus, the initial alteration in sperm membranes after the introduction of progesterone is the formation of lipid-disordered and protein-disordered submicrodomains within an ordered macromolecular cluster within which lateral motion of components is highly restricted. During the second hour of incubation of capacitated spermatozoa with progesterone, the phospholipid tail microdomains regained freedom of free rotation, and this state prevailed for another hour, after which the rotational freedom of this domain got restricted a second time (Fig. 3I). Interestingly, towards the end of the second hour, the polar head microdomains showed highest freedom of rotational motion, after which the rotational motion was restricted a second time (Fig. 2I). Computation of lateral diffusion patterns also revealed a similar phase alteration profile, where the freedom of molecular dynamics towards the outer and inner halves of the exofacial surfaces of sperm membranes showed mutually opposing alterations (Figs. 2I and K and 3I and K). However, the protein mobility patterns were grossly similar to the lipid mobility patterns on the outer half of the exofacial surfaces (Figs. 4I and K). A potentially important event during sperm capacitation leading to acrosome reaction is the loss of sperm membrane cholesterol. Although the exact mechanisms mediating this loss are not known, albumin and highdensity lipoprotein have been proposed as lipid acceptors [32]. Cholesterol efflux has been linked to the activation of membrane and transmembrane signaling events leading to the activation of a unique signaling pathway involving the cross-talk between cAMP and tyrosine kinase second messenger systems [33]. It is also proposed that the human sperm acrosome reaction has many similarities to the somatic cell exocytotic events which occur during the regulated pathway of secretion. One or more oocyte stimuli result in the activation of protein kinases, likely (but not necessarily) via activation of G-protein-coupled receptors on the sperm plasma membrane and the formation of second messengers. The kinases phosphorylate and activate proteins, continuing the biochemical cascade that ultimately results in the acrosome reaction [34].
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Since membranes consist of an exofacial leaflet and a cytofacial leaflet that differ in fluidity and cholesterol distribution [35], removal of cholesterol actively from the exofacial surfaces could lead to increase in fluidity in that region. Effective efflux of cholesterol from the polar zones of the exofacial side and/or the enrichment of the hydrophobic interior with cholesterol through transmembrane cholesterol transport could introduce opposing phase alterations in two horizontal segments of the exofacial membrane. Mammalian sperm cells are activated prior to fertilization by high-bicarbonate levels, which facilitate lipoprotein-mediated cholesterol efflux, which suggested the formation of apical membrane rafts in the sperm head surface that enables albuminmediated efflux of cholesterol [36]. Our observations revealing the differences in motional freedom at two depths in the exofacial leaflet of the sperm membrane during acrosome reaction suggest the formation of horizontal subdomains in addition to the previously proposed vertical subdomains [37] during the progression of exocytotic events. Our results support the concept that the dynamic changes in membrane organization during acrosome reaction are slow and oscillatory as against rapid transients in ion fluxes. This novel finding might render a new perspective to lipid and protein trafficking during membrane vesiculation events.
Acknowledgments This work was supported by Grants BT/PR1316/Med/09/212/98 from Department of Biotechnology and F.3-118/2001(SR-II) from University Grants Commission, New Delhi. Mohammad Shoeb received a Junior Research Fellowship from Council of Scientific and Industrial Research, New Delhi.
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Formation and dynamic alterations of horizontal microdomains in sperm membranes during progesterone-induced acrosome reactionq Mohammed Shoeb, Malini Laloraya, and Pradeep G. Kumar* Molecular Reproduction Unit, School of Life Sciences, Devi Ahilya University, Vigyan Bhawan, Khandwa Road, Indore 452 017, MP, India Received 9 January 2004
Abstract Capacitated mammalian spermatozoa undergo a fusion response of their head plasma membrane and the outer acrosomal membrane leading to vesiculation classically known as acrosome reaction. Acrosome reaction occurs in response to various acrosome reaction inducers including zona pellucida proteins, calcium ionophore, dibutyryl cAMP, progesterone, etc. All the acrosome reaction inducers cause a transient of calcium influx into the sperm through voltage-dependent cation channels. Efflux of chloride, stimulation of activity of phospholipases, and phosphorylation of proteins are other known changes introduced by acrosome reaction inducers. Macromolecular organization and dynamics of sperm membranes during the progression of this vesiculation are largely unexplored. In this study, we report that progesterone induced the formation of horizontal microdomains within the exofacial surfaces of sperm membranes, which showed progressive and independent alterations in molecular dynamics. In the light of this observation, we propose that sperm membrane rafts may contain both horizontal and vertical microdomains. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Acrosome reaction; Spermatozoa; Progesterone; Spin labeling; Raft
The acrosome reaction is an exocytotic process leading to the fusion of the plasma membrane of sperm overlying the acrosome and the outer acrosomal membrane at multiple points. Acrosome reaction is an essential step for fertilization. In mammalian sperm, the acrosome reaction is thought to be initiated in vivo by binding to the zona pellucida (ZP), the extracellular matrix of the egg, and only sperm that have completed the acrosome reaction can penetrate the ZP and fuse with the egg plasma membrane [1]. In general, it is thought that sperm binding to ZP3, one of the glycoprotein components of the ZP, induces a transient Ca2þ influx into the sperm through voltage-dependent nonselective cation channels, which in turn leads to activation of a pertussis toxin-sensitive trimeric Gi/o protein-coupled PLC [2]. A tyrosine kinase-regulated q
Abbreviations: BSA, bovine serum albumin; PLC, phospholipase C; ConA, Concanavalin A; GABA, c-aminobutyric acid; DMSO, dimethyl sulfoxide. * Corresponding author. Fax: +91-731-276-2984. E-mail address: [email protected] (P.G. Kumar). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.01.119
PLCc may also be activated during ZP3-binding [2]. Activation of PLCs generates IP3, thereby mobilizing [Ca2þ ]i from the sperm’s intracellular Ca2þ store, the acrosome, although the Ca2þ storing capacity of this organelle seems very limited [3]. Nonetheless, these early responses appear to promote a subsequent sustained Ca2þ influx signal via store-operated channels (SOCs) that results in the acrosome reaction [4–6]. Recent studies have provided evidence for the expression in sperm of transient receptor potential protein channels 1, 3, and 6 (Trp1, Trp3, and Trp6), all putative Ca2þ -permeant SOCs [4,7], and Trp2 has been described to play a role in the ZP3-induced acrosome reaction in mouse sperm [4,8]. However, the precise molecular mechanism by which the acrosome reaction occurs has remained unclear. In addition to ZP, thapsigargin, a specific blocker of the sarco/endoplasmic reticulum Ca2þ -ATPase, which causes Ca2þ depletion from internal stores and leads to capacitative Ca2þ entry [9], is also able to induce the acrosome reaction [10]. Furthermore, progesterone released from the cumulus cells, and thus one of the
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major components of follicular fluid, has also been shown to induce the acrosome reaction in a presumed physiological manner [11,12]. Although the mechanism of action of progesterone on sperm is not yet fully understood, it is thought to induce Ca2þ influx by activating a GABAA-like progesterone receptor/Cl channel [13]. A recent study shows that P promotes the AR in capacitated cauda sperm but is unable to do so in noncapacitated or immature sperm because the availability of PR increases during epididymal transit and after capacitation. The PR responsible for this behavior is different from a classical nuclear receptor on the basis of the immunostaining results and is probably a protein close to 44 kDa on the basis of the ligand assay results [14]. Progesterone, an agonist of acrosome reaction, induces a biphasic Ca2þ i -signal in human sperm comprising an initial transient Ca2þ elevation and a subsequent i ramp or plateau. Nifedipine, an inhibitor of voltageoperated Ca2þ channels, inhibits progesterone-induced acrosome reaction in human sperm. Treatment with nifedipine reduced the duration but not the amplitude of the progesterone-induced Ca2þ i -transient but fluorimetric studies have detected no effect of this compound on the progesterone-induced Ca2þ signal. Using Ca2þ i imaging of single sperm, it was shown that the spermatozoa displayed Ca2þ waves in response to ZP, progesterone, and thapsigargin, although the site of initation of the rises appears to differ with the agonist [15], supporting the view that various acrosome reaction inducers could act as effectors of grossly similar physicochemical alterations in spermatozoa [16]. End point assays have registered that capacitation was characterized by enhanced rotational freedom of molecules within the spermatozoa, with no significant changes in membrane packaging or the lateral diffusion of molecules. The AR inducers restricted the rotational freedom of molecules, while enhancing the lateral diffusion of highly ordered subdomains within the sperm membrane [16]. It is known that microdomain formation and dynamics within living membranes could be modulated by interplay of Ca2þ by modifying the local charge aggregation. However, it is not clear whether sperm membrane shows linear or oscillatory structural alterations during the progress of acrosome reaction. In this study, we have undertaken a time-course study of sperm membrane dynamics in response to progesterone-induced acrosome reaction. Materials and methods Reagents. Progesterone, 16 doxyl stearate (16DS), and 5 doxyl stearate (5DS) were from Sigma chemical, MO, USA. 4-Maleimido 2,2,6,6-tetramethyl piperidinooxyl (MalNet) was from Syva chemical, St .Louis, USA. All other chemicals used were of reagent grade.
Animals. Mature Swiss male mice (Mus musculus) of 3–5 month age group were used for the experiments. Mice were killed by cervical dislocation and the spermatozoa from cauda epididymidis and vas deferens were obtained after mincing these tissues and dispersion in Hanks’ balanced salt solution (HBSS). The suspension was allowed to stand for 10 min at 37 °C. The cloudy upper layer of the suspension was recovered and passed through 80 lm Nitex screen to remove the cell debris. The spermatozoa were pelleted by centrifugation at 500g. The sperm pellets thus obtained were resuspended in KRB for induction of capacitation. Induction of capacitation and acrosome reaction. For the induction of capacitiation, the spermatozoa (1 million cells/ml) were incubated for 2 h in Krebs–Ringer bicarbonate buffer (KRB) containing sodium pyruvate and sodium lactate and in an atmosphere of 95% air and 5% CO2 . Sperm were confirmed to have undergone capacitation by observing their hyperactivation. Acrosome reaction was initiated with the introduction of 3.18 lM progesterone prepared in 0.05% DMSO (final concentration) into the suspension of capacitated spermatozoa in KRB. Concanavalin A-binding assay. The population of spermatozoa that underwent acrosome reaction was evaluated by analyzing the ConA-binding pattern of sperm head. For this purpose, the spermatozoa were withdrawn at intervals from the acrosome reaction setup and immobilized on poly-L -lysine coated coverslip for 2 min. Then they were drained and dipped in HBSS containing 0.05% BSA. The coverslips were placed on blotting paper with sperm-adhering side up. Twenty microliters of ConA + 40 ll HBSS containing BSA was added and coverslip was incubated in dark for 5 min. These coverslips were washed by gently dipping in HBSS containing BSA and deionized water, placed on a glass slide with sperm containing side on glass slide, and observed under NIKON Optiphot Microscope with epifluorescence attachment using Nikon B-2A filter for FITC conjugates. Slides were prepared for capacitated sperm sample and for each hour of incubation with progesterone. Sperm showing bright fluorescence over head were scored as capacitated while those showing patchy or diminished fluorescence were scored as acrosome reacted sperm. Spin labeling protocol. Forty microliter sperm aliquots were labeled with 5 ll spin label (200 lM final concentrations) for 50 min. Unincorporated spin labels were quenched by incubation of spermatozoa for 10 min with 5 ll NiCl2 (50 mM final concentration). The spin-labeled spermatozoa were transferred into 15 ll glass capillaries and one end was sealed with epoxy resin. EPR spectra were recorded on VARIAN E-104 electron spin resonance spectrometer equipped with TM110 cavity. Spectrometer settings and scanning were done according to standard methods described [16]. Instrument settings employed were: scan range, 100G; field set, 3237G; temperature, 27 °C; time constant, 1 s; modulation amplitude, 0.5 10; microwave power, 5 mW; microwave frequency, 9.01 GHz; recording time, 8 min; and receiver gain, mentioned as legend with respective panels of spectra. Sperm membrane fluidity profiling was done immediately prior to the addition of progesterone into the suspension of capacitated sperm, immediately after the addition of progesterone, and at successive 1-h intervals for a span of 5 h. Rotational freedom of the inserted labels was computed as 1/Tc , where Tc ¼ 6:51 1010 w0 ðh0 =hþ1 Þ1=2 . In this equation, w0 represents the width of the mid-field peak, h0 the height of the mid-field peak, and hþ1 is the height of the low field peak. Spin– spin exchange was empirically related to the broadening of the mid-field line (w0 ) and was taken as a measure of the lateral diffusion (DL ). Molecular ordering was measured by computing the total anisotropy in the system by calculating S3 using the relation S3 ¼ ð2ak 2a ? =2ak þ 2a ?Þ. Statistical analysis. All the experiments were repeated seven times unless otherwise indicated. Statistical analysis was done using Microsoft Excel and Sigma Plot. p values was computed using student’s t test.
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Results Induction of acrosome reaction in capacitated spermatozoa Induction of acrosome reaction was verified by monitoring the intactness of the sperm head membranes using ConA-binding. Fig. 1A shows an acrosome-intact spermatozoon stained with ConA, showing heavy fluorescence on the acrosomal domains, indicative of intact acrosome. Fig. 1B is the corresponding differential interference contrast image. Fig. 1C represents ConAbinding onto spermatozoa 1 h after the addition of progesterone. The fluorescence on the head region is diminished drastically signifying that acrosome reaction has occurred in these spermatozoa. In our assays, more than 90% of the spermatozoa underwent acrosome reaction during the first hour after the addition of progesterone. A corresponding DIC image is shown in Fig. 1D. Measurement of surface fluidity of sperm membranes Capacitated spermatozoa probed with 5DS presented a heavy level of immobilization that resulted in the generation of a ‘powder spectrum’ (Fig. 2A) as against signals from freely moving spin labels (Fig. 2G). Transient changes occurring on the membranes of the spermatozoa were monitored immediately after progesterone addition (Fig. 2B) and at subsequent 1 h intervals (Figs. 2B–F). Since we introduced progesterone as a solution in DMSO, we monitored the effect of DMSO, if any, on the overall membrane organization. As expected, DMSO brought about considerable modifications in the sperm membranes (Fig. 2H). In order to compensate for the possible artifacts introduced by DMSO in our experimental set-up, we performed a normalization of the observed values obtained in the progesterone-treated
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samples (which would contain progesterone in DMSO. This group is designated as +P) against that obtained in the DMSO-treated controls (designated as )P). Fig. 2H is a line graph summarizing the rotational freedom in sperm membranes in the +P and )P incubations. In the )P group, there was no detectable change in the rotational freedom of 5DS under our experimental conditions. On the contrary, the +P group presented dynamic increase in the mobility profiles (p < 0:01 and p < 0:008 when 0 h sample was compared with 1 h, and 1 h with 2 h, respectively) of the spin label during the first 120 min of incubation with progesterone (Fig. 2H). Thereafter, we noticed a progressive decline (p < 0:01) in fluidity during the following 80 min (Fig. 2I). Fig. 2J is a plot of the lateral diffusion on the membrane surface. Normalization of the data set of progesteronetreated spermatozoa against their respective DMSOcontrols revealed a significant restriction (p < 0:01) in the lateral diffusion in the membrane microdomains towards the outside, reaching minimal values towards 120 min. Thereafter, the lateral diffusion index reverted back to its original values in the next 2 h (Fig. 2K). Measurement of the internal fluidity of sperm membranes Using 16DS, we evaluated the fluidity of the hydrophobic interior of the sperm membranes. Fig. 3 summarizes our observations, with the representative EPR spectra shown in tracings A–G. 1/Tc values computed at various time points are presented as a line graph (Fig. 3H) and the normalized 1=Tc values are shown in Fig. 3I. We observed a highly significant (p < 0:05) restriction in the rotational freedom (Fig. 3I) and lateral diffusion (Figs. 3J and K) of molecules in this membrane subdomain during the earlier half of the incubation period, lasting for approximately 80 min. After this initial compaction, this zone exhibited a massive increase (p < 0:03) in rotational freedom (Figs. 3H and I) and lateral motion (Figs. 3J and K).
Fig. 1. Photomicrographs of capacitated (A,B) and acrosome-reacted (C,D) visualized under epifluorescence optics showing strong PNA-binding pattern on the entire head (typical of capacitated spermatozoa) in (A), which was diminished remarkably after acrosome reaction as represented in (C). (B,D) Corresponding DIC images (800).
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Fig. 2. Analysis of the sperm membrane structure using electron spin resonance spectroscopy following spin labeling with 5-doxyl stearic acid. (A–F) Representative spectra originating from spermatozoa incubated with acrosome reaction inducer for 0, 1, 2, 3, 4, and 5 h respectively. (G) Free label spectrum. (H) Plot of the rotational freedom of 5DS in progesterone-treated and control groups. A normalization of the values of the progesteronetreated set against the control set is shown in (I). Lateral mobility profiles around the polar heads of the exofacial leaflet of capacitated spermatozoa before and after the addition of progesterone are plotted in (J). Normalized values of DL of spermatozoa treated with progesterone against control groups are represented in (K). Values represented are means SEM of seven replicates analyzed.
Measurement of the protein mobility in sperm membranes EPR spectra from spermatozoa labeled with a thiolreactive spin label MalNet and incubated with progesterone for 0 through 5 h with 1 h interval between
successive recordings are shown in Figs. 4A–G. The computed 1=Tc values from +P and )P groups are plotted as line graph (Fig. 4H) and the normalized 1=Tc values are shown in Fig. 4I. Protein rotation was significantly high (p < 0:002) during the first 200 min after
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Fig. 3. Analysis of the sperm membrane structure using electron spin resonance spectroscopy following spin labeling with 16-doxyl stearic acid. (A–F) Representative spectra originating from spermatozoa incubated with acrosome reaction inducer for 0, 1, 2, 3, 4, and 5 h, respectively. (G) Free label spectrum. (H) Plot of the rotational freedom of 16DS in progesterone-treated and control groups. A normalization of the values of the progesteronetreated set against the control set is shown in (I). Lateral mobility profiles around the hydrophobic tail regions of the exofacial leaflet of capacitated spermatozoa before and after the addition of progesterone are plotted in (J). Normalized values of DL of spermatozoa treated with progesterone against control groups are represented in (K). Values represented are means SEM of seven replicates analyzed.
the addition of progesterone, which was followed by a decline (p < 0:001) in the rotational freedom during the following 50 min (Figs. 4H and I). We observed a second instance of freely rotating proteins (p < 0:0002) on the spermatozoa towards the fag end of our incubations
(Figs. 4H and I). During the initial phase of the incubation with progesterone, thiol-containing proteins experienced reasonably free lateral motion, which was followed by a phase of gradual increase in freedom for lateral motion of proteins (Figs. 4J and K). Since MalNet
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Fig. 4. Analysis of the mobility of sperm membrane proteins using electron spin resonance spectroscopy following spin labeling with a thiol-reacting spin label MalNet. (A–F) Representative spectra originating from spermatozoa incubated with acrosome reaction inducer for 0, 1, 2, 3, 4, and 5 h, respectively. (G) Free label spectrum. (H) Plot of the rotational freedom of MalNet in progesterone-treated and control groups. A normalization of the values of the progesterone-treated set against the control set is shown in (I). Lateral mobility profiles around the hydrophobic tail regions of the exofacial leaflet of capacitated spermatozoa before and after the addition of progesterone are plotted in (J). Normalized values of DL of spermatozoa treated with progesterone against control groups are represented in (K). Order parameter was calculated as explained in methods and is presented in (L). Normalized values of order parameter are shown in (M). Values represented are means SEM of seven replicates analyzed.
produced clear high-field resonance line, we computed the molecular ordering of the protein. A plot of S3 from )P and +P groups is presented in Fig. 4L and the normalized values are presented in Fig. 4M. During the first hour of incubation of the spermatozoa with progesterone, protein ordering declined significantly (p < 0:05). During the subsequent period of incubation, there was a moderate increase in ordering,
yet the values were lower than those of the capacitated spermatozoa (Fig. 4M).
Discussion Progesterone has been described to affect several sperm functions including motility, capacitation, and
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acrosome reaction. Progesterone can initiate the mammalian sperm acrosome reaction in vitro, and studies on the effect of progesterone and several other steroids have demonstrated an increased calcium influx following binding to the sperm membrane [17]. Progesterone may achieve this effect through the intervention of extragenomic receptors [18–20]. Progesterone is one of the physiological stimuli of human sperm acrosome reaction and is present in high levels at the site of fertilization (cumulus oophorus). Apart from the increase of intracellular calcium concentrations in spermatozoa shown by many laboratories, the effects of this steroid also extend to mediate efflux of chloride, stimulation of activity of phospholipases, and phosphorylation of proteins [21]. It has been indicated that PKC plays a role in progesterone-induced acrosome reaction and that progesterone-stimulated PKC activation is downstream to stimulation of calcium influx by the steroid [22]. Progesterone-dependent calcium influx appears to be a rapid process [23–25], followed by a sustained rise lasting for several minutes (plateau phase). Both these calcium transients are dependent upon entry of extracellular calcium. The nature of the calcium channel that mediates the effects of progesterone is, currently, unknown. It has been postulated that it may be: (i) part of the progesterone receptor; (ii) voltage-dependent; or (iii) operated by second messengers following activation of the progesterone receptor [26]. However, it should be noted that calcium flux is induced specifically by progesterone in capacitated and uncapacitated sperm cells, whereas only capacitated spermatozoa are able to subsequently complete the acrosome reaction [27], clearly showing that capacitated spermatozoa respond differently to progesterone by manifesting membrane vesiculation. Many investigators find that the introduction of progesterone into the capacitation medium has a positive effect on capacitation [28,29]. It is also shown that a minimum capacitation time of 6 h was required for induction of the acrosome reaction in both progesteronefree and progesterone-supplemented capacitation media [30]. Thus, as against the rapid dynamics of calcium influx in response to progesterone, vesiculation events appear to be slow and progressive. In this study, we attempted to evaluate the progressive changes in the molecular mobility and ordering of sperm membranes of capacitated spermatozoa exposed to progesterone for various time intervals. Three microdomains of the spermatozoa were probed using two lipophilic and one protein-binding spin labels. One of the stearic acids used in this study had the doxyl group covalently attached to the 5th carbon (5DS), while the other one had the doxyl group on the 16th carbon (16DS). MalNet was used as a thiol-reacting spin label that could be anchored onto thiol-containing proteins. During the first hour of incubation of the spermatozoa with progesterone, the rotational freedom
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of the spin label 16DS decreased significantly, while that of 5DS and MalNet showed a considerable enhancement in rotational freedom when compared with the respective values observed in capacitated spermatozoa. Interestingly, both 5DS and MalNet reported a significant decline in the lateral diffusion. This indicates the formation of microdomains within the exofacial surfaces of the sperm membrane with lipids and proteins experiencing rotational freedom and restrictions in lateral mobility at the same time. An earlier report demonstrated an increase in molecular ordering in sperm membranes after incubation of capacitated sperm with progesterone for 1 h [31]. Thus, the initial alteration in sperm membranes after the introduction of progesterone is the formation of lipid-disordered and protein-disordered submicrodomains within an ordered macromolecular cluster within which lateral motion of components is highly restricted. During the second hour of incubation of capacitated spermatozoa with progesterone, the phospholipid tail microdomains regained freedom of free rotation, and this state prevailed for another hour, after which the rotational freedom of this domain got restricted a second time (Fig. 3I). Interestingly, towards the end of the second hour, the polar head microdomains showed highest freedom of rotational motion, after which the rotational motion was restricted a second time (Fig. 2I). Computation of lateral diffusion patterns also revealed a similar phase alteration profile, where the freedom of molecular dynamics towards the outer and inner halves of the exofacial surfaces of sperm membranes showed mutually opposing alterations (Figs. 2I and K and 3I and K). However, the protein mobility patterns were grossly similar to the lipid mobility patterns on the outer half of the exofacial surfaces (Figs. 4I and K). A potentially important event during sperm capacitation leading to acrosome reaction is the loss of sperm membrane cholesterol. Although the exact mechanisms mediating this loss are not known, albumin and highdensity lipoprotein have been proposed as lipid acceptors [32]. Cholesterol efflux has been linked to the activation of membrane and transmembrane signaling events leading to the activation of a unique signaling pathway involving the cross-talk between cAMP and tyrosine kinase second messenger systems [33]. It is also proposed that the human sperm acrosome reaction has many similarities to the somatic cell exocytotic events which occur during the regulated pathway of secretion. One or more oocyte stimuli result in the activation of protein kinases, likely (but not necessarily) via activation of G-protein-coupled receptors on the sperm plasma membrane and the formation of second messengers. The kinases phosphorylate and activate proteins, continuing the biochemical cascade that ultimately results in the acrosome reaction [34].
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Since membranes consist of an exofacial leaflet and a cytofacial leaflet that differ in fluidity and cholesterol distribution [35], removal of cholesterol actively from the exofacial surfaces could lead to increase in fluidity in that region. Effective efflux of cholesterol from the polar zones of the exofacial side and/or the enrichment of the hydrophobic interior with cholesterol through transmembrane cholesterol transport could introduce opposing phase alterations in two horizontal segments of the exofacial membrane. Mammalian sperm cells are activated prior to fertilization by high-bicarbonate levels, which facilitate lipoprotein-mediated cholesterol efflux, which suggested the formation of apical membrane rafts in the sperm head surface that enables albuminmediated efflux of cholesterol [36]. Our observations revealing the differences in motional freedom at two depths in the exofacial leaflet of the sperm membrane during acrosome reaction suggest the formation of horizontal subdomains in addition to the previously proposed vertical subdomains [37] during the progression of exocytotic events. Our results support the concept that the dynamic changes in membrane organization during acrosome reaction are slow and oscillatory as against rapid transients in ion fluxes. This novel finding might render a new perspective to lipid and protein trafficking during membrane vesiculation events.
Acknowledgments This work was supported by Grants BT/PR1316/Med/09/212/98 from Department of Biotechnology and F.3-118/2001(SR-II) from University Grants Commission, New Delhi. Mohammad Shoeb received a Junior Research Fellowship from Council of Scientific and Industrial Research, New Delhi.
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