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Published as doi: 10.1096/fj.06-7716com.
 (The FASEB Journal. 2007;21:4121-4130.)
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Membrane-permeant Rab3A triggers acrosomal exocytosis in living human sperm

Cecilia I. Lopez, Silvia A. Belmonte, Gerardo A. De Blas and Luis S. Mayorga1

Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina

1Correspondence: Casilla de Correo 56, 5500 Mendoza, Argentina. E-mail: lmayorga{at}fcm.uncu.edu.ar


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The acrosome reaction is a regulated Ca2+-dependent secretion event required for sperm-egg interaction. Previous studies indicate that the process requires Rab3-dependent tethering of membranes, SNARE complex assembly, and Ca2+-mediated activation of synaptotagmin. Sperm are transcriptionally and translationally inactive; hence, most studies of the exocytosis mechanism are limited to membrane-permeant reagents. The effect of proteins involved in exocytosis has been assessed only in permeabilized cells. Polyarginine peptides are a powerful tool for delivering macromolecules to cells. Most reports indicate that membrane translocation of arginine-containing proteins requires endocytosis; therefore, this strategy might not be useful in sperm. However, our results indicate that GST and Rab3A, when fused with an arginine-rich peptide, were able to translocate into sperm. Moreover, membrane-permeant Rab3A initiated exocytosis when prenylated and activated with GTP. We show here that a key event after the cytoplasmic Ca2+ increase caused by progesterone is the activation of Rab3A. When active Rab3A is introduced into sperm, Ca2+ in the extracellular medium and in the cytoplasm is dispensable. However, a Ca2+ efflux from inside the acrosome is still required to achieve exocytosis. In conclusion, arginine-containing proteins can penetrate the sperm plasma membrane and thus are valuable tools to study sperm physiology in intact cells.—Lopez, C. I., Belmonte, S. A., De Blas, G. A., Mayorga, L. S. Membrane-permeant Rab3A triggers acrosomal exocytosis in living human sperm.


Key Words: membrane-permeant proteins • arginine-rich peptides • Rab3A • acrosome reaction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IN THE PROXIMITY OF THE EGG, MAMMALIAN sperm are stimulated by progesterone and glycoproteins present in the zona pellucida to release the content of the acrosomal granule, a key event in fertilization. This tightly regulated and complex secretion process is known as the sperm acrosome reaction. Upon activation, through a still not well-defined transduction mechanism, Ca2+ channels (likely voltage-dependent Ca2+ channels) in the plasma membrane are opened and a transient cytosolic Ca2+ increase occurs (1) . Later, store-operated Ca2+ (SOC) channels in the plasma membrane are opened, causing a second and sustained Ca2+ increase that initiates acrosomal exocytosis (2 3 4) . The molecular events involved in the release of the acrosomal content have been investigated intensively in our laboratories and in others (5) . Typically, the assays have been limited by the fact that sperm are transcriptionally and translationally inactive; hence the powerful technique of overexpressing wild-type and mutated proteins is inapplicable. To overcome this limitation, we have set up an exocytosis assay using streptolysin O (SLO) -permeabilized sperm. Experiments with this assay and several auxiliary techniques have generated a large body of evidence supporting the following working model for acrosomal exocytosis. In resting sperm, SNAREs are locked in inactive cis complexes on plasma and outer acrosomal membranes (6) . Ca2+ entry into the cytoplasm through SOC channels causes the activation of Rab3A, a key event that initiates secretion (7 , 8) . Rab3A effectors are directly or indirectly involved in tethering the acrosome to the plasma membrane and in the disassembly of cis SNARE complexes on both membranes mediated by N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein ({alpha}-SNAP) (8 , 9) . Monomeric SNAREs are then free to reassemble in loose trans complexes, causing irreversible docking of the acrosome to the plasma membrane (6) . At this point, Ca2+ is released from inside the acrosome through IP3-sensitive Ca2+ channels to trigger the final steps of membrane fusion, which require SNAREs and synaptotagmin (6 , 10 , 11) .

Permeabilized sperm are useful only to study events occurring after the opening of SOC channels because SLO pores will dissipate any ionic gradient across the plasma membrane. Thus, the model cannot be used to investigate earlier events or processes involving the regulation of gradients across the plasma membrane. Therefore, whenever possible, we have performed experiments in intact cells to confirm observations in the permeabilized sperm. However, we have been limited to the use of membrane-permeant inhibitors or activators.

To investigate the effect of exogenous proteins in intact cells, we have been increasingly interested in the possibility of using carrier peptides to introduce proteins into sperm. Several membrane-permeant peptides have been used to deliver proteins into living cells (12 13 14 15 16 17) . In particular, it has been shown that polyarginine peptides are efficient at conferring membrane permeability to proteins (13) . The mechanism for protein translocation is not well understood; however, results from several laboratories indicate that active endocytosis is required, suggesting that translocation occurs through the endocytic pathway (18 , 19) . Our initial goal was to assess whether arginine-rich peptides can be used to deliver proteins into nonendocytic cells, such as sperm.

To understand whether the mechanism of entry is strictly dependent on endocytosis, we directly challenged living sperm cells with proteins containing a polyarginine peptide (R). In particular, we used GST with a C terminus R peptide (GST-R) and Rab3A with an N terminus R peptide (His6-R-Rab3A). Transduced recombinant proteins were also analyzed for their ability to exert biological activity. The use of membrane-permeant Rab3A allows us to elucidate the different requirement of Ca2+ for the acrosomal exocytosis before and after activation of Rab3A and to establish the relationship of this protein with the exocytosis mechanism initiated by the physiological agonist progesterone.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
HTF (human tubal fluid medium, 5.94 g/L NaCl, 0.35 g/L KCl, 0.05 g/L MgSO4.7H2O, 0.05 g/L KH2PO4, 0.3 g/L CaCl2.2H2O, 2.1 g/L NaHCO3, 0.51 g/L D-glucose, 0.036 g/L Na pyruvate, 2.39 g/L Na lactate, 0.06 g/L penicillin, 0.05 g/L streptomycin, 0.01 g/L phenol red) was purchased from Irvine Scientific (Santa Ana, CA, USA); methyl-β-cyclodextrin (CyD) and CyD loaded with cholesterol (1000 mg of the complex contains 53 mg of cholesterol, CyDC) were from Aquaplex, Cyclodextrins Technologies Development (High Springs, FL, USA); H89 was purchased from LC Laboratory (Woburn, MA, USA), nifedipine, thapsigargin, xetospongine C, 2-aminoethoxydiphenylborate (2-APB), anti-GST, and anti-His6 antibody were from Calbiochem (MERCK Química Argentina SAIC, Buenos Aires, Argentina); BAPTA, BAPTA-AM, tetra-methyl-rhodamine iodoacetamide, Oregon Green 488 iodoacetamide, and Alexa Fluor 488-labeled anti-rabbit antibody were from Molecular Probes (Invitrogen Argentina Ltda., Buenos Aires, Argentina), glutathione-Sepharose 4B was from Amersham (GE Healthcare Life Sciences, Buenos Aires, Argentina); verapamil, amphotericin, glutathione, and progesterone were from Sigma (Sigma-Aldrich Argentina SA, Buenos Aires, Argentina); Ni-NTA-agarose was from Qiagen (Tecnolab SA, Buenos Aires, Argentina); A23187 was from Alomone (Alomone Labs. Ltd., Jerusalem, Israel); BamHI was from New England Biolabs (Migliore Laclaustra SRL, Buenos Aires, Argentina); EcoRI was from Promega (Biodynamics S.R.L., Buenos Aires, Argentina); TOPO cloning kit, primers, and Taq polymerase were from Invitrogen (Invitrogen Argentina Ltda., Buenos Aires, Argentina); isopropyl-β-D-thiogalactopyranoside (IPTG), albumin, guanosine 5'-O-(3-thiotriphosphate), tetralithium salt (GTP{gamma}S), guanosine 5'-O-(2-thiodiphosphate), trilithium salt (GDPβS), and FITC-Pisum sativum lectin were from ICN (Eurolab SA, Buenos Aires, Argentina); horseradish peroxidase (HRP) -coupled anti-rabbit antibody was from Jackson Immunochemicals (Sero-Immuno Diagnostics, Inc., Tucker, GA, USA); and rhodamine-labeled goat anti-mouse was from Kirkegaard and Perry Laboratories (Gaithersburg, MD, USA).

Plasmids
Oligonucleotides (5' P-gat ccc gtc gcc gtc agc gtc gca aac gcc gtc gcc agc tag ctc tag 3' and 5' P-aat tct aga gct agc tgg cga cgg cgt ttg cga cgc tga cgg cga cgg 3') were annealed, then ligated with pGEX-2T pretreated with BamHI and EcoRI (pGEX-2T-R). Rab3AQ81L was subcloned from pGEX-2T into EcoRI site of pGEX-2T-R through a PCR protocol using forward primer 5' cgg tcg aat tca tgg cct cag cca ca 3' and reverse primer 5' ccc ccg aat tct cag cag gcg caa tcc tga tg 3' with an intermediate TOPO cloning kit construct. R-Rab3AQ81L was subcloned from pGEX-2T-R into the BamHI site of pQE80L through a PCR protocol using forward primer 5' gtg gtt ccg cgt gga tcc cgt 3' and the same reverse primer used before with an intermediate TOPO cloning kit construct. Rab3A was subcloned from pGEX-2T into BamHI site of pQE80L using forward primer 5' cca gca agt ata tag cat gg 3' and the same reverse primer used before with an intermediate TOPO cloning kit construct.

Acrosome reactions assays
Human semen samples were obtained from healthy donors. Highly motile sperm were recovered after a swim-up separation for 1 h in HTF medium supplemented with 5 mg/ml of bovine serum albumin at 37°C in an atmosphere of 5% CO2/95% air. Cell concentration was then adjusted to 5–10 x 106 sperm/ml with HTF and incubation was continued (capacitating conditions) for at least 2 h. This relatively short period of incubation was chosen to allow a direct comparison with our previous published results (11 , 20) in permeabilized sperm. Furthermore, we have previously shown that, after swim-up, the spermatozoa become responsive to 15 µM progesterone after 2 additional hours incubation in capacitating conditions (7) . Inhibitors or stimulants were added as indicated in the legends to the figures. In experiments consisting of a series of successive treatments, new reagents were added to the preexisting mix. At the end of the experiment, 10 µl of each sample was spotted on slides and fixed/permeabilized in ice-cold methanol. Acrosomal status was evaluated by staining with fluorescein isothiocyanate-coupled Pisum sativum lectin according to ref. 21 . Briefly, spermatozoa that have preserved an intact acrosome show strong label with the fluorescent lectin at the acrosomal region. Cells that have undergone acrosomal exocytosis show no labeling at the acrosomal region or labeling limited to the edge of the granule (equatorial staining). At least 200 cells were scored using a Nikon microscope equipped with epifluorescence optics. Negative (no stimulation) and positive (stimulated with A23187 or progesterone) controls were included in all experiments. For each experiment, acrosomal exocytosis indexes were calculated by subtracting the number of reacted spermatozoa in the negative control (range 6–25%) from all values and expressing the resulting values as a percentage of the AR observed in the positive control (range 25–40%). The average difference between positive and negative controls was 14% (experiments where the difference was <10% were discarded). When specified, sperm viability was assessed by staining with 0.08% eosyn Y (at least 200 cells were scored). Motility was determined according to the WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction criteria (at least 200 cells were scored). Sperm were considered motile when showing at least a slow progressive pattern of movement.

Recombinant proteins
The plasmids encoding GST-R, His6-R-Rab3A, and His6-Rab3A were transformed into Escherichia coli strain BL21(DE3)pLysS, and GST-Rab3A was transformed into BL21. Protein synthesis was induced overnight at 22°C with 0.5 mM IPTG. Bacteria were centrifuged and lysed by sonication and proteins were purified under native conditions. GST-R and GST-Rab3A were purified with glutathione-Sepharose 4B (Amersham) and eluted with 20 mM glutathione in 0.2 M NaCl, 50 mM Tris-ClH buffer pH 8. His6-Rab3A and His6-R-Rab3A were purified with Ni-NTA agarose (Qiagen) and eluted with 500 mM imidazole in 300 mM NaCl, 50 mM NaH2PO4 pH 8. GST and GST-R were labeled with tetra-methyl-rhodamine iodoacetamide or Oregon Green iodoacetamide according to standard procedures. Except when specified, GST-Rab3A, His6-Rab3A, and His6-R-Rab3A were prenylated in vitro, as described (22) . Just before use, aliquots of the prenylated protein were loaded with the nonhydrolyzable nucleotide guanosine GTP{gamma}S or GDPβS as described (20) .

GST and GST-R permeability assays
Sperm cells were adjusted to 7 x 106 cells/ml with HTF and incubated 1–3 h at 37°C with 2–4 µM GST or GST-R labeled with tetra-methyl-rhodamine or Oregon Green 488. Cells were either washed with PBS and fixed with 3% paraformaldehyde or observed alive. Fixed cells were mounted with 1% propyl-gallate 50% glycerol in PBS and observed with a Nikon C1 laser scanning confocal unit (Nikon D-Eclipse C1) attached to an upright fluorescence microscope (Nikon Eclipse E600).

In other experiments, 5 x 106 sperm cells were incubated with either 1 µM GST or GST-R. After 1 h incubation, cells were treated with 1 mg/ml trypsin for 20 min and enzyme activity was stopped with 2 mg/ml trypsin inhibitor. Cell proteins were extracted to perform SDS-PAGE and Western blots; a sample of each condition had been separated and fixed earlier in 1% paraformaldehyde to perform indirect immunofluorescence against GST (see next section). Briefly, after removing the HTF medium by washing twice with PBS, sperm pellets (0.5x107 cells) were extracted in sample buffer without disulfide-reducing agents. Proteins were extracted by heating twice to 95°C for 6 min. Extracts were centrifuged (12,000 g) for 10 min and the supernatants were adjusted to 5% β-mercaptoethanol, boiled for 3 min, and used immediately or stored at –20°C. Proteins were separated on 15% polyacrylamide gels and transferred to nitrocellulose membranes. Purified GST-R (150 ng) was used as a control. Equal protein loads were systematically confirmed in all lanes by Ponceau red staining of the transferred membrane. Nonspecific reactivity was blocked by incubation with 1% fat-free milk dissolved in washing buffer (0.1% Tween 20 in PBS) for 1 h at room temperature. Blots were incubated with a rabbit anti-GST antibody (diluted 1:1000) in blocking solution for 2 h at room temperature. HRP-conjugated goat anti-rabbit IgG was used as secondary antibody with 1 h incubation. Excess first and second antibodies were removed by washing 5 x 10 min in washing buffer. Detection of Western blots was accomplished with an enhanced chemiluminescence system (ECL; Amersham Biosciences, Buenos Aires, Argentina) and subsequent exposure to Kodak XAR film (Eastman Kodak, Rochester, NY, USA).

Indirect immunofluorescence
Sperm cells were adjusted to 7 x 106 cells/ml in HTF medium supplemented with 0.5% albumin and incubated with 100 µM 2-APB for 15 min (to prevent exocytosis), then with 1 µM His6-tagged {alpha}-SNAP or R-Rab3A for 30 min at 37°C. To detect His6-tagged proteins by immunofluorescence, sperm were spotted on round coverslips, washed with PBS, and fixed in 2% paraformaldehyde. After fixation, sperm were incubated in 50 mM glycine-PBS for 30 min at room temperature and blocked for 30 min in 5% bovine serum-PBS. Sperm cells were permeabilized or not with 1% Triton X-100 for 10 min. Then spermatozoa were labeled with a mouse anti-His6 antibody (1 µg/ml in 1% bovine serum-PBS) for 1 h at 37°C, followed by a rhodamine-labeled anti-mouse antibody (10 µg/ml in 1% bovine serum-PBS) for 1 h at room temperature.

To test GST and GST-R permeability, cells were treated as indicated above and immunolabeled with a rabbit anti-GST antibody (diluted 1:100 in 1% bovine serum-PBS) for 3 h at room temperature. Alexa Fluor 488-labeled anti-rabbit IgG was used as a secondary antibody (4 µg/ml in 1% bovine serum-PBS) for 1 h at room temperature. Coverslips were washed with PBS between incubations and mounted in 1% propyl-gallate/50% glycerol in PBS. The samples were examined with an Eclipse TE-300 Nikon microscope or Nikon Eclipse TE-2000 equipped with a Plan Apo 63/1.40 oil objective and a Hamamatsu (Bridgewater, NJ, USA) Orca 100 camera operated with MetaMorph 6.1 software (Universal Imaging, Downingtown, PA, USA). When indicated, confocal microscopy with a Nikon C1 laser scanning confocal unit attached to Nikon Eclipse E600 microscope was used.

Statistical analysis
Data were evaluated using 1-way ANOVA. Conditions used for data normalization (0% and 100%) were not included in the analysis. Two different post hoc tests were performed: Tukey-Kramer for pairwise comparison of the means and Dunnett for comparison of the means with a control condition. In the first case, the same letter was assigned to conditions that do not differ significantly (i.e., all groups that share a letter do not differ significantly, and if two groups do not share a letter they do differ significantly); in the second case the differences with a control group were classified as significant (s) or not significant (ns); 95% confidence limits for the means were used to estimate significant differences from conditions used for normalization (0% and 100%). Differences were considered significant at the P < 0.05 level.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Proteins containing an arginine-rich peptide can permeate into sperm
Sperm are transcriptionally and translationally inactive cells; hence, overexpression experiments to assess the function of a protein cannot be performed. An attractive approach to get past this limitation is the use of membrane-permeant proteins. It is well documented that arginine-rich peptides can confer this property to a large variety of proteins. At present, most studies indicate that endocytosis is part of the internalization process, although they do not rule out a simultaneous translocation from the plasma membrane (18 , 19 , 23 , 24) . Therefore, it is controversial whether endocytosis is strictly necessary for translocation. Sperm are an ideal system to address this question. To the best of our knowledge, there is no report of endocytosis in mammalian sperm, and careful electron microscopy studies show no evidence for endocytic organelles in these cells (25 , 26) . To assess whether proteins with an arginine-rich region can permeate into sperm, a polybasic peptide RRRQRRKRRRQ was engineered in the C terminus of GST (GST-R, Fig. 1 A). The protein was purified, and live sperm and CHO cells were incubated with rhodamine-labeled protein. GST-R was efficiently translocated into the nucleus and cytoplasm of CHO cells (results not shown). Sperm also incorporated GST-R that accumulated in the postnuclear area of the head and in the flagella (Fig. 1C ) but did not incorporate GST (Fig. 1B ). To rule out that the translocation of the protein was a fixation artifact, sperm were treated with trypsin before fixation. Under these conditions, GST was not detected by immunofluorescence (Fig. 1D ) or Western blot (Fig. 1F , ext GST), whereas the treatment did not abrogate the signal for GST-R (Fig. 1E, F , ext GST-R). Finally, Oregon Green–labeled GST-R was visualized in unfixed, live sperm (see Supplemental Movie 1).


Figure 1
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  		Figure 1. Proteins containing arginine-rich peptide can permeate into sperm. A) Schematic representation of the permeant proteins used. B, C) Live sperm were incubated with 1 µM rhodamine-tagged GST (GST) or rhodamine-tagged GST with a polyarginine carboxyterminal tail (GST-R) for 3 h at 37°C. The cells were fixed and observed by confocal microscopy (a single plane is shown). Asterisks show the position of unstained spermatozoa. The inset is a region with increased brightness to demonstrate the presence of cells. D, E) Live sperm were incubated with 1 µM GST or GST-R for 1 h at 37°C and treated with 1 mg/ml trypsin for 20 min at 37°C. Trypsin activity was stopped with 2 mg/ml trypsin inhibitor and aliquots of the samples were then fixed and immunostained with an anti-GST antibody and observed by fluorescence microscopy. F) The rest was used to generate a protein extract that was analyzed by SDS-PAGE and Western blot using the same antibody (ext GST-R, extract from 0.5x107 cells incubated with GST-R; ext GST, extract from 0.5x107 cells incubated with GST; GST-R, 150 ng of purified GST-R). Molecular weights are indicated on the left. G) Live sperm were incubated with 1 µM His6-{alpha}SNAP (His-{alpha}SNAP) or H) His6-R-Rab3A (His-R-Rab) for 30 min at 37°C. Aliquots of the samples were fixed and immunostained with an anti-His6 antibody and observed by confocal microscopy (a single plane is shown). I, J) Sperm were or were not permeabilized with Triton X-100 after fixation, and were immunostained as stated previously (a single plane of confocal microscopy is shown). Bars, 10 µm.

The observations with GST indicate that proteins with arginine-rich regions can be incorporated into the cytoplasm of sperm cells. A new construct was generated (Fig. 1A ), now coding for Rab3A, a protein with a central role in exocytosis that can trigger secretion in permeabilized sperm (8 , 20) . His6-R-Rab3A contains the R peptide between an His6 moiety and Rab3A. The translocation of His6-R-Rab3A is shown in Fig. 1H . The protein was present in the head and tail of the sperm, indicating there was not a preferred region for translocation. Notice that His6-{alpha}SNAP, a His6-tagged protein used as control, was not incorporated into sperm (Fig. 1G ). Another potential artifact for arginine-tagged proteins is that these molecules bind avidly to membranes. Therefore, the protein detected by immunofluorescence can be bound externally to the plasma membrane. However, confocal planes of sperm cells show that the His6-R-Rab3A was not restricted to the plasma membrane (Fig. 1H, J ). Moreover, when immunofluorescence was carried out in nonpermeabilized cells to detect exclusively externally bound His6-R-Rab3A, the signal was significantly lower than when Triton was used (compare Fig. 1I , His-R-Rab – Triton and Fig. 1J , His-R-Rab+Triton). Quantification showed that the percentage of cells that incorporated His6-R-Rab was in the 60–95% range, depending on the experimental conditions.

These results indicate that the arginine-rich peptide strategy can be used to make proteins permeate the sperm plasma membrane and that endocytosis is not an absolute requirement for translocation of polyarginine-containing proteins across membranes.

Membrane-permeant Rab3A triggers acrosomal exocytosis in intact sperm
In permeabilized sperm, addition of GST-Rab3A prenylated, and loaded with GTP{gamma}S initiates sperm acrosomal exocytosis (8 , 20) . To assess whether the membrane-permeant protein was functionally active, His6-R-Rab3A was prenylated, loaded with GTP{gamma}S, and added to intact capacitated spermatozoa. The protein induced acrosomal exocytosis to an extent similar to that of the Ca2+ ionophore A23187 (Fig. 2 , His-R-Rab; A23187). Exocytosis was not induced by prenylated, GTP{gamma}S-activated GST-Rab3A or His6-Rab3A, two proteins that do not permeate through membranes (Fig. 2 , GST-Rab, His-Rab), or by GST-R, a membrane-permeant protein without function in secretion (Fig. 2 , GST-R). These controls indicate that the R peptide is necessary for translocation into the sperm but that the R-moiety without activated Rab cannot promote secretion. His6-R-Rab3A did not affect the sperm viability (control, 77.4%±6.4%; 1 µM R-Rab, 83.8%±1.0%; mean ± SE, n=5) or motility (control, 66.7%±7.3%; 1 µM R-Rab, 75.6%±1.5%; mean±SE, n=5). In conclusion, recombinant Rab3A with the addition of a polyarginine peptide can permeate into sperm, is not toxic for the cells, and is functionally active in secretion. The potency of this protein (ED50 ~0.5 µM; see Fig. 5B for an example of a dose-response curve) was slightly lower than that observed using GST-Rab3A in permeabilized sperm (ED50~0.15 µM) (7) .


Figure 2
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  		Figure 2. Membrane-permeant Rab3A triggers acrosomal exocytosis in intact sperm. Human sperm were incubated for 15 min at 37°C without any stimulus (control) or stimulated with 10 µM A23187 (A23187). Some aliquots were incubated under the same conditions in the presence of 1 µM of the following recombinant proteins: GST with an arginine-rich peptide in the C terminus (GST-R), prenylated and GTP{gamma}S loaded GST-Rab3A (GST-Rab), His6-Rab3A (His-Rab), and His6-R-Rab3A (His-R-Rab). After incubation, the samples were fixed and the number of acrosomal-reacted cells was evaluated and expressed as explained in Materials and Methods. Values represent the mean ± SE of 4–8 independent experiments. The means of groups that share a letter do not differ significantly (Tukey-Kramer, P>0.05).


Figure 5
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  		Figure 5. The action of His6-R-Rab3A is modulated by cholesterol. A) Human sperm were incubated for 15 min at 37°C without any stimulus (control) or stimulated with 10 µM A23187 (A23187) or 1 µM His6-R-Rab3A (R-Rab). When indicated, 1 mM methyl-β-cyclodextrin (CyD), 1 mM CyD loaded with cholesterol (CyDC), or 12.5 µg/ml amphothericin (Amph) was added to the assay 15 min before stimulation. B) Samples were incubated in the presence (+CyD) or absence (control) of 1 mM CyD and stimulated with increasing concentrations of His6-R-Rab3A (R-Rab). The samples were fixed and the number of acrosomal-reacted cells was evaluated and expressed as explained in Materials and Methods. Values represent the mean ± SE of 3–6 independent experiments. The means of groups that share a letter do not differ significantly (Tukey-Kramer, P>0.05).

Membrane-permeant Rab3A must be prenylated and activated to trigger acrosomal exocytosis
Rab proteins act as molecular switches changing from an active conformation when loaded with GTP to an inactive conformation when loaded with GDP (27) . In addition, the GTP-bound form associates with membranes by means of geranylgeranyl residues present in the C terminus of the protein. Prenylation is essential for the normal function of Rab proteins (28) . To determine whether prenylation and activation of Rab3A were required for the effect of the membrane-permeant protein, His6-R-Rab3A, prenylated or unprenylated, was loaded with GTP{gamma}S or GDPβS (see Materials and Methods and legend to Fig. 3 ) and incorporated into the assay. The results in Fig. 3 show that unprenylated His6-R-Rab3A cannot induce acrosomal exocytosis even when loaded with GTP (Fig. 3 , uR-RabGTP{gamma}S, uR-RabGDPβS) and that only prenylated and GTP{gamma}S-loaded His6-R-Rab3A promoted secretion (Fig. 3 , R-RabGTP{gamma}S vs. R-RabGDPβS). These observations indicate that His6-R-Rab3A must be in the same functional configuration as endogenous Rab proteins (i.e., prenylated and in the GTP-bound form) to be active in exocytosis.


Figure 3
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  		Figure 3. Membrane-permeant Rab3A promotes acrosomal exocytosis only when prenylated and loaded with GTP. Human sperm were incubated for 15 min at 37°C without any stimulus (control) or stimulated with 10 µM A23187 (A23187). Some aliquots were incubated under the same conditions in the presence of 1 µM His6-R-Rab3A prenylated and loaded with GTP{gamma}S (R-RabGTP{gamma}S), prenylated and loaded with GDPβS (R-RabGDPβS), unprenylated, and loaded with GTP{gamma}S (uR-RabGTP{gamma}S), or unprenylated and loaded with GDPβS (uR-RabGDPβS). After incubation, the samples were fixed and the number of acrosomal-reacted cells was evaluated and expressed as explained in Materials and Methods. Values represent the mean ± SE of 3–6 independent experiments. The means of groups that share a letter do not differ significantly (Tukey-Kramer, P>0.05).

Inhibitors that block the signaling pathway upstream of the opening of SOC channels do not affect His6-R-Rab3A-triggered exocytosis
Physiological inducers of the acrosome reaction, such as progesterone and ZP3, activate a complex signaling mechanism that ultimately opens SOC channels in the plasma membrane (29) . The entrance of Ca2+ through these channels triggers the exocytic process. In SLO-permeabilized sperm, Ca2+ can freely permeate into the cell, mimicking the opening of the SOC channels. Our observations with this model indicate that Ca2+ entrance into the cytoplasm activates Rab3A, which in turn triggers secretion (6) . Therefore, several steps of the signaling pathway activated by progesterone should be upstream of the activation of Rab3A. This prediction cannot be tested in SLO-permeabilized sperm, where ionic and electrical gradients across the plasma membrane have collapsed and progesterone cannot induce exocytosis. His6-R-Rab3A was used to test the prediction that activation of Rab3A is a relatively late event in the progesterone pathway leading to activation of acrosome reaction. Voltage-dependent Ca2+ channel inhibitors, such as verapamil and nifedipine, efficiently blocked progesterone-induced acrosome reaction (1) (Fig. 4 , Pg, Nf->Pg and Vp->Pg); however, they did not affect His6-R-Rab3A-induced exocytosis (Fig. 4 , R-Rab, Nf->R-Rab and Vp->R-Rab). Ni2+, an inhibitor of SOC channels, blocked progesterone but not His6-R-Rab3A-triggered exocytosis (Fig. 4 , Ni->Pg and Ni->R-Rab). Another important signaling molecule in acrosome reaction is cAMP. Progesterone-induced acrosome reaction depends on the activity of protein kinase A (PKA) (30) ; however, we have demonstrated that Ca2+-induced acrosomal exocytosis requires cAMP, but acting through a protein kinase A-independent, Epac (exchange factor activated by cAMP) -mediated pathway (31) . Consistent with the idea that Rab3A is a late-acting factor, H89, a PKA inhibitor, blocked progesterone but not His6-R-Rab3A-induced exocytosis (Fig. 4 , H89->Pg and H89->R-Rab). The results indicate that PKA and several Ca2+ channels are part of the signaling pathway initiated by progesterone upstream of the activation of Rab3A.


Figure 4
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  		Figure 4. Inhibitors that block the signaling pathway upstream of the opening of SOC channels do not affect His6-R-Rab3A-triggered exocytosis. Human sperm were incubated for 15 min at 37°C without any stimulus (control), or stimulated with 10 µM A23187 (A23187), 15 µM progesterone (Pg), or 1 µM His6-R-Rab3A (R-Rab). When indicated, the following inhibitors were added to the assay 15 min before stimulation: 120 µM nifedipine (Nf), 100 µM verapamil (Vp), 0.1 mM NiCl2 (Ni), 10 µM H89 (H89). After incubation, the samples were fixed and the number of acrosomal-reacted cells was evaluated and expressed as explained in Materials and Methods. Values represent the mean ± SE of 3 or 4 independent experiments. The mean of groups with inhibitors were compared with the corresponding group without inhibitor using the Dunnett's test and classified as nonsignificant (ns, P>0.05) or significant (s, P<0.01).

Progesterone and His6-R-Rab3A are in the same signaling pathway
All the above results are consistent with the hypothesis that Rab3A is a late-acting factor in the signal transduction cascade initiated by progesterone. However, an alternative explanation would be that progesterone and His6-R-Rab3A are triggering secretion by independent mechanisms. To address this possibility, we altered the cholesterol content of the sperm membranes. Changes in sterols affect progesterone-induced exocytosis at least in part by regulating Rab3A membrane association (7) . Therefore, His6-R-Rab3A-triggered exocytosis should also be modulated by the cholesterol content of the membranes. The results in Fig. 5 A show that His6-R-Rab3A-induced secretion (R-Rab) was enhanced by extracting cholesterol from the membranes with 1 mM methyl-β-cyclodextrin (CyD->R-Rab), inhibited by cholesterol-loaded cyclodextrin (CyDC->R-Rab), a complex that increases cholesterol in membranes, and blocked by amphotericin (Amph->R-Rab), a cholesterol binding antibiotic that forms a complex with cholesterol. Similar effects have been reported for progesterone-induced acrosomal exocytosis (7 , 32) . The effect of cyclodextrin was more evident at suboptimal concentrations of His6-R-Rab3A (Fig. 5B ), suggesting that cholesterol extraction from the membranes promoted a more efficient interaction of the recombinant GTPase with the fusion machinery, as we have shown for endogenous Rab3A and for GST-Rab3A in permeabilized sperm (7) .

If progesterone and Rab3A were in the same pathway, inhibition of endogenous Rab3A would block the progesterone-induced acrosome reaction. To test this prediction, unprenylated His6-R-Rab3A loaded with GDPβS (uR-RabGDPβS) was added to cells in the hope it would compete with the endogenous protein for guanosine nucleotide exchange factors required for its activation. As control, membrane-impermeable GST-Rab3A and His6-Rab3A (uRabGDPβS) treated in the same manner were used. Recombinant proteins were incubated with sperm prior to the stimulus. As shown in Fig. 6 , unprenylated His6-R-Rab3A-GDPβS efficiently inhibited progesterone-induced exocytosis (uR-RabGDPβS->Pg) whereas unprenylated GST-Rab3A-GDPβS, a protein that does not permeate into the cell, had no effect (uRabGDPβS->Pg). These observations indicate that Rab3A is activated at a late event in the signaling cascade stimulated by progesterone.


Figure 6
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  		Figure 6. Progesterone- and Ca2+-induced exocytosis is inhibited by unprenylated His6-R-Rab3A loaded with GDP. Human sperm were incubated for 15 min at 37°C without any stimulus (control) or stimulated with 15 µM progesterone (Pg) or 10 µM A23187 (A23187). When indicated, 1 µM His6-R-Rab3A unprenylated and loaded with GDPβS (uR-RabGDPβS) or 1 µM GST-Rab3A (Pg stimulation) or His6-Rab3A (without the R peptide; A23187 stimulation) unprenylated and loaded with GDPβS (uRabGDPβS) was added to the assay 15 min before stimulation. After incubation, the samples were fixed and the number of acrosomal-reacted cells was evaluated and expressed as explained in Materials and Methods. Values represent the mean ± SE of 3 or 4 independent experiments. The means of groups that share a letter do not differ significantly (Tukey-Kramer, P>0.05).

According to our model, activation of Rab3A is also downstream of the increase in cytoplasmic Ca+2. To test whether unprenylated His6-R-Rab3A loaded with GDPβS (uR-RabGDPβS) inhibits Ca+2-induced acrosome reaction, we challenged spermatozoa that had been incubated with unprenylated His6-R-Rab-GDPβS or unprenylated His6-Rab-GDPβS with A23187. As shown in Fig. 6 , His6-R-Rab3A-GDPβS abrogated A23187-triggered exocytosis (uR-RabGDPβS->A23187), whereas the impermeable His6-Rab3A-GDPβS had no effect (uRabGDPβS->A23187). These results suggest that Ca2+ and progesterone converge in the same pathway leading to acrosomal exocytosis.

Acrosomal exocytosis triggered by His6-R-Rab3A does not require extracellular Ca2+ but depends on intracellular Ca2+ stores
A rise of Ca2+ in the cytosol initiates exocytosis in most secretory cells. However, in permeabilized sperm, Rab3A-triggered exocytosis can occur even in the presence of 5 mM BAPTA in the medium provided the acrosomal Ca2+ store is intact and IP3-sensitive channels are not inhibited (11) . The strong implication from these results is that the entrance of Ca2+ through SOC channels is necessary to activate Rab3A; once this GTPase is in the GTP-bound form, Ca2+ entry from the extracellular medium is dispensable. The permeant protein permits us to test this hypothesis in living sperm. As expected, progesterone and A23187 required extracellular Ca2+ to induce exocytosis and were inhibited by 5 mM BAPTA (Fig. 7 A, BAPTA->Pg and BAPTA->A23187). In contrast, His6-R-Rab3A induced exocytosis in the presence of the chelator (Fig. 7A , BAPTA->R-Rab). Moreover, secretion was observed in the presence of BAPTA-AM, a membrane-permeant Ca2+ chelator, which accumulates in the cytoplasm in nonpermeabilized sperm (Fig. 7A , BAPTA-AM->R-Rab). Note that 2.5 µM of this reagent were enough to completely block the progesterone-induced acrosome reaction (Fig. 7A , BAPTA-AM->Pg; Fig. 7B , triangles). In intact spermatozoa, thapsigargin triggers an acrosomal reaction by opening SOC channels, but addition of BAPTA to the medium inhibits replenishment of the acrosomal store by the Ca2+ ATPase present in the acrosome (Fig. 7A , BAPTA+Tg ->Pg) (11) . This treatment completely abrogated His6-R-Rab3A-elicited acrosomal reaction (Fig. 7A , BAPTA+Tg->R-Rab), suggesting that the acrosomal Ca2+ store is necessary for the His6-R-Rab3A-induced exocytosis. Furthermore, adding an excess of BAPTA-AM (which will eventually diffuse into the acrosome; ref. 33 ) inhibits His6-R-Rab3A-mediated secretion (Fig. 7B , circles). In addition, 2-APB and xestospongin C, two inhibitors of IP3-sensitive Ca2+ channels, blocked both progesterone- and His6-R-Rab3A-triggered secretion (Fig. 7A, Xp ->Pg, 2APB->Pg, Xp->R-Rab and 2APB->R-Rab).


Figure 7
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  		Figure 7. Only intraacrosomal Ca2+ is required for His6-R-Rab3A-triggered exocytosis. A) Human sperm were incubated for 15 min at 37°C without any stimulus (control) or stimulated with 15 µM progesterone (Pg), 10 µM A23187 (A23187), or 1 µM His6-R-Rab3A (R-Rab). When indicated, 5 mM BAPTA (BAPTA), 2.5 µM BAPTA-AM (BAPTA-AM), 2.2 µM xestospongin C (Xp), 100 µM 2-APB (2APB), or 1 µM thapsigargin simultaneously with 5 mM BAPTA (BAPTA+Tg) were added to the assay 15 min before stimulation. B) Sperm were incubated for 15 min at 37°C with increasing concentrations of the membrane-permeable Ca2+ chelator BAPTA-AM. 1 µM His6-R-Rab3A or 15 µM Pg were then added to the assay and the incubation was pursued for 15 min. In both panels, the samples were fixed after incubation and the number of acrosomal-reacted cells was evaluated and expressed as explained in Materials and Methods. Values represent the mean ± SE of 2–5 independent experiments. The mean of groups with inhibitors was compared with the corresponding group without inhibitor using the Dunnett’s test and classified as nonsignificant (ns, P>0.05) or significant (s, P<0.05).

In brief, these results indicate that Ca2+ influx from extracellular medium is necessary to activate Rab3A. Upon activation of this protein, cytoplasmic Ca2+ is dispensable but exocytosis still requires Ca2+ release from intracellular stores (i.e., the acrosome) through IP3-sensitive Ca2+ channels.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Several membrane-permeable peptides have been used as carriers to deliver macromolecules into living cells (for a review, see ref. 12 ). It has been shown that polyarginine peptides are particularly efficient in conferring membrane permeability to proteins (13) . The translocation mechanism of these molecules into the cell cytoplasm is not well understood and is the object of intense debate. Early reports indicated that internalization of these peptides was not significantly inhibited by incubation at low temperature, by depletion of the cellular ATP pool, or by inhibitors of endocytosis, suggesting a direct transport from the plasma membrane by an energy-independent mechanism (34 35 36) . However, some authors have criticized these observations, showing that at least part of the internalization reported was an artifact caused by a redistribution of the positively charged peptides to the nucleus during fixation (37) . In addition, some observations did not distinguish between internal and membrane-bound peptides. However, the translocation properties of polybasic peptides have been confirmed in carefully controlled experiments. Moreover, they have been used successfully to modulate different aspects of cell physiology (e.g., 17 , 38 , 39 ). At present, most studies indicate that endocytosis is part of the internalization process, although they do not rule out a simultaneous translocation from the plasma membrane (17 , 23 , 24) . Therefore, it is controversial whether endocytosis is strictly necessary for translocation. Sperm are an ideal system to address this question because they are one of the few cells that do not endocytose under physiological conditions (i.e., at 37°C without ATP depletion). Our morphological and functional results show that proteins containing a single R peptide are efficiently translocated into sperm. This observation strongly indicates that polyarginine-containing polypeptides can permeate directly from the plasma membrane and that endocytosis is not a strict requirement for translocation. The position of the R peptide within the protein was irrelevant for translocation. Similar results were observed when the R motif was at the C terminus (GST-R) or near the N terminus (His6-R-Rab3A) of the protein.

The possibility of constructing membrane-permeant proteins is a powerful tool for basic science and therapeutics. In this report, we present data showing that membrane-permeant Rab3A can efficiently initiate acrosomal exocytosis in living cells. The observations that His6-R-Rab3A must be prenylated in the GTP-bound conformation is in complete agreement with the idea that the protein translocates into the cytoplasm and triggers secretion by activating downstream of the Rab3A effectors. In addition, the fact that unprenylated protein loaded with GDPβS (which would compete with the endogenous protein for guanine nucleotide exchange factors) inhibits progesterone and A23187-induced acrosome reaction is the first direct evidence that Rab3A is part of the secretion mechanism triggered by this physiological agonist of acrosome reaction. Cholesterol content of sperm membranes decreases during sperm capacitation, a poorly understood process that prepares spermatozoa to undergo acrosome reaction when stimulated (40) . His6-R-Rab3A-induced exocytosis showed the same pattern of modulation by reagents that alter membrane cholesterol as that observed in progesterone-stimulated sperm (7 , 32) . This observation provides additional support to the idea that Rab3A is part of the secretion mechanism initiated by the hormone. Consistent with this conclusion, Rab3A has been localized to the acrosomal region in other species (41 , 42) . Moreover, a peptide corresponding to the effector domain of Rab3A affects acrosome reaction in ram and rat sperm (43) .

Ca2+ plays a central role in regulated exocytosis in several systems (44) . In neurons and several neuroendocrinal cells, there is a tight temporal association between the increase in intracellular Ca2+ and exocytosis. In these cells, a pool of secretory vesicles is already attached to the plasma membrane and Ca2+ activates a molecular switch that is directly connected with the membrane fusion machinery responsible for exocytosis. Synaptotagmin is the most likely Ca2+ sensor that triggers secretion (45) . However, Ca2+ regulates several steps of the secretion process, and numerous Ca2+ binding proteins participate in regulated exocytosis (46) . In other secretory systems, the Ca2+ rise and exocytosis are not so tightly coupled in time, suggesting that this ion plays important roles besides triggering membrane fusion. Ca2+ is a central signaling molecule in acrosome reaction, and several channels for this ion are involved (2 , 29) . Voltage-dependent Ca2+ channels in the plasma membrane are probably responsible for an early increase of cytoplasmic Ca2+ after sperm stimulation with progesterone or ZP3. Channels in the acrosome membrane are then activated by IP3 generated by the activation of phospholipase C (PLC). In turn, SOC channels in the plasma membrane are opened by the efflux of Ca2+ from the acrosome causing a second, sustained increase of Ca2+ that triggers acrosome reaction (3) . Several results support the idea that this Ca2+ activates Rab3A, which in turn initiates secretion. Preactivated membrane-permeant Rab3A triggers exocytosis in the presence of 5 mM BAPTA in the extracellular medium or when Ca2+ in the cytoplasm was chelated by addition of BAPTA-AM. This observation is in complete agreement with previous reports using membrane-impermeant Rab3A in permeabilized sperm (11) . In contrast, Rab3A-triggered secretion is inhibited by conditions where intra-acrosomal Ca2+ is depleted or IP3-sensitive channels are inhibited, suggesting that an efflux of Ca2+ from intracellular stores is necessary at a step downstream of the activation of the GTPase, proximal to the membrane fusion process. In permeabilized sperm, we have shown that this Ca2+ efflux from the acrosome is required after trans SNAREs complexes have been assembled and likely activates synaptotagmin VI (6) . In particular, our results with membrane-permeant Rab3A indicate that progesterone initiates a signaling cascade that ultimately causes the opening of Ca2+ channels in the plasma membrane. The resulting increase in intracellular Ca2+ activates Rab3A. At this step, Ca2+ influx from the extracellular medium is dispensable; however, Ca2+ must be released from inside the acrosome to complete the secretory process.

In brief, our results indicate that polyarginine peptides are a suitable strategy to deliver macromolecules into the sperm cytoplasm and that endocytosis is not a strict requirement for translocation of these peptides. This strategy may become a powerful tool to study sperm physiology. Moreover, the development of permeant proteins involved in the fertilization process brings new perspectives into the field of in vitro fertilization.


   ACKNOWLEDGMENTS
 
We thank Marcelo Furlán and Alejandra Medero for technical assistance, and Claudia Tomes for critical reading of the manuscript and for providing His6-{alpha}SNAP. This work was supported by an International Research Scholar Award from the Howard Hughes Medical Institute and by grants from Consejo Nacional de Investigaciones Científicas y Técnicas and Agencia Nacional de Promoción de la Ciencia y la Tecnología (Argentina) to L.M.

Received for publication December 14, 2006. Accepted for publication May 31, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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