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Biology of Reproduction 67, 1058-1066 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Identification of Ras and Its Downstream Signaling Elements and Their Potential Role in Hamster Sperm Motility1

Subir K. NagDas2,a, Virginia P. Winfreya, and Gary E. Olsona

a Department of Cell Biology, Vanderbilt University, Nashville, Tennessee 37232


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ras, a member of the small G-protein family, regulates multiple signaling pathways in somatic cells. The objectives of the present study included the characterization and localization of Ras and the identification of its downstream effectors in hamster spermatozoa. Immunoblot analysis with a pan-Ras monoclonal antibody localized Ras to the particulate fraction of sonicated testicular and caput and cauda epididymal spermatozoa. However, Ras was present in both the particulate and soluble fractions of spermatocytes and round spermatids, suggesting that its membrane recruitment is completed during spermiogenesis. Immunoblots of plasma membrane fractions demonstrated that hamster spermatozoa express both N-Ras and K-Ras. Indirect immunofluorescence with pan-Ras antibody localized Ras to the flagellum. Immunoblot analysis of sperm plasma membrane fractions demonstrated the presence of phosphatidylinositol 3-kinase (PI3-kinase) and protein kinase C {zeta} (PKC{zeta}), the downstream targets of Ras, and coimmunoprecipitation analysis demonstrated their interaction with Ras. Inhibitors of PI3-kinase (wortmannin and 2-(4- morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) and PKC{zeta} (staurosporine) inhibited the hyperactivation of sperm motility during capacitation in a dose-dependent manner, indicating that both PI3-kinase and PKC{zeta} are associated with development of this motility pattern. The interaction of Ras with both PI3-kinase and PKC{zeta} suggests that Ras may regulate several signaling pathways in spermatozoa.

gamete biology, signal transduction, sperm maturation, sperm motility and transport, spermatogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ras, a member of the small G-protein family, acts as a molecular switch in several signaling pathways to regulate an array of intracellular functions. Membrane bound Ras cycles between an inactive GDP-bound and an active GTP-bound state. Guanine nucleotide exchange factors (GEFs) promote the conversion of the GDP-bound state to the active GTP-bound conformation. GTPase-activating proteins (GAPs) stimulate the intrinsic GTP hydrolytic activity of Ras to promote the formation of the inactive, GDP-bound form of Ras. Thus, the Ras GDP/GTP cycle is controlled by the balanced activities of GEFs and GAPs. GTP-Ras interacts with downstream effectors resulting in their activation and function in signaling pathways [1–5].

The Ras family comprises H-Ras, N-Ras, and two K-Ras isoforms, 4A and 4B [4–6]. Ras becomes anchored to the plasma membrane after a complex series of posttranslational modifications. The first modification is the addition of a 15-carbon farnesyl group to the cysteine residue of the C-terminal CAAX sequence (where C is the cysteine, A is an aliphatic or linear amino acid, and x is either serine or methionine) by the cytosolic enzyme farnesyltransferase. Subsequently, the AAX residues are proteolytically cleaved, and the C-terminal farnesylcysteine is methylated. H-Ras, N-Ras, and K-Ras 4A are further modified by the covalent addition of palmitic acid to cysteine residues located immediately upstream of the CAAX motif, whereas a tri-lysine motif of K-Ras 4B is required for transit to the plasma membrane [7–11]. These different processing mechanisms may result in the recruitment of Ras to distinct microdomains of the plasma membrane.

Northen blot analyses have demonstrated the presence of H-, N-, and K-Ras in spermatogonia, spermatocytes, round spermatids, and residual bodies [12–14]. However, neither the function nor the protein interactions of Ras in spermatozoa are well established. During posttesticular maturation in the epididymis, the sperm surface undergoes extensive remodeling, including the binding of epididymal secretory proteins to restricted surface domains and the lateral redistribution of preexisting proteins, frequently accompanied by their proteolytic processing, to a final residence domain [15–18]. How most of these changes result in a mature functional spermatozoon are poorly understood. Because spermatozoa do not synthesize new proteins [19, 20], the recruitment of cytoplasmic proteins including signaling elements to specific membrane domains could also represent an important step in posttesticular sperm maturation, resulting in motile spermatozoa that can interact with the egg. Ras may regulate distinct domain-specific signaling pathways in the sperm head or tail. In somatic cells, GTP-Ras mediates several signaling pathways by interacting with multiple downstream effector molecules, including Raf-kinase, phosphatidylinositol 3-kinase (PI3-kinase), protein kinase C {zeta} (PKC{zeta}), and RalGDS [3]. However, Ras-mediated downstream signaling pathways in mammalian spermatozoa are not well characterized. The present study was focused on the identification and localization of Ras and the characterization of its downstream effectors in hamster spermatozoa.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies

Mouse monoclonal antibodies included pan-ras (Ab-3, catalog no. OP40; Oncogene Research Products, Calbiochem, Cambridge, MA), c-N-ras (Ab-1, no. OP25; Calbiochem), c-H-ras (Ab-1, no. OP23; Calbiochem), and c-K-ras (Ab-1, no. OP24; Calbiochem). A mouse monoclonal anti-PI3-kinase anitibody (no. P13020; Transduction Laboratories, Lexington, KY) and rabbit polyclonal antibodies to PKC{zeta} (no. 13199-013; Gibco Life Technologies, Grand Island, NY) were also used. Horseradish peroxidase-conjugated, affinity-purified, goat anti-mouse IgG and goat anti-rabbit IgG were from Jackson ImmunoResearch Laboratories (West Grove, PA). Other chemicals were from Sigma Chemical Company (St. Louis, MO).

Sperm Preparation

Mature male golden hamsters were housed in the university animal care facility on a 14L:10D cycle and given free access to food and water. Care and use of animals conformed to NIH guidelines for humane animal care and use in research, and all protocols were approved by the institutional animal care committee. Animals were killed with CO2. Caput and cauda epididymides were minced at 37°C in calcium-free Tyrode medium, and the sperm suspensions were centrifuged at 100 x g for 1 min to sediment epididymidal tubule fragments. The supernatants were recentrifuged at 1500 x g for 10 min at 4°C to obtain sperm pellets.

To prepare testicular spermatozoa, two testes were minced in 10 ml of Tyrode medium, incubated for 5 min at 37°C, and centrifuged at 100 x g for 5 min. The supernatant solution was mixed with 40 ml of 65% Percoll (Amersham Pharmacia Biotechnology, Piscataway, NJ) in Tyrode medium and then centrifuged at 23 000 rpm for 15 min in a 60Ti rotor (Beckman, Fullerton, CA). Two bands were present after centrifugation. The lower band, containing spermatozoa and some red blood cells, was diluted with Tyrode medium, layered on a discontinuous Ficoll (Sigma) density gradient containing 2 ml each of 10% (w/v), 20% (w/v), and 40% (w/v) Ficoll in Tyrode medium, and centrifuged at 1500 x g for 10 min at 4°C. The testicular spermatozoa at the 10%-20% interface were examined for acrosomal integrity by phase contrast microscopy, diluted with Tyrode medium, and pelleted by centrifugation at 1500 x g for 10 min at 4°C.

Isolation of Plasma Membrane from Cauda Epididymal Spermatozoa

Spermatozoa were resuspended in TNI (150 mM NaCl, 25 mM Tris-HCl, pH 7.5, 2 mM benzamidine, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 0.05% sodium azide), disrupted by nitrogen cavitation at 400 psi for 10 min, and pelleted by centrifugation at 1500 x g for 10 min at 4°C. Aliquots of the supernatant fluid (8 ml) containing plasma membrane vesicles were layered on discontinuous sucrose gradients composed of 2 ml 20% sucrose and 2 ml 50% sucrose; all sucrose solutions contained 150 mM NaCl and 25 mM Tris-HCl, pH 7.5. The gradients were centrifuged at 25 000 rpm for 60 min in an SW41 rotor (Beckman). The plasma membranes at the 20%-50% interface were collected, diluted with TNI, and pelleted by centrifugation at 100 000 x g for 30 min in a TL55 rotor (Beckman) [21]. Protein was estimated using the procedure of Bradford [22].

Spermatogenic Cell Isolation

Spermatogenic cells were purified by unit gravity sedimentation [23, 24]. Testes were minced, rinsed twice in Krebs-Henseleit buffer (KHB), and then incubated for 30 min at 33°C with gentle oscillation in KHB containing 1 mg/ml collagenase (Sigma). After three rinses with KHB, tissue was incubated with KHB containing 1 mg/ml trypsin and 0.1 U/ml micrococcal nuclease (Sigma) for 10 min and gently pipetted to disperse single cells. The cells were rinsed twice in KHB containing 0.1 U/ml micrococcal nuclease, resuspended in KHB containing 0.5% BSA, and then filtered through a 40-µM mesh sieve (Falcon 2340; Falcon Plastics, Los Angeles, CA) to obtain a cell suspension. Cells were counted, and samples exhibiting >90% viability by Trypan blue dye exclusion were used for unit gravity sedimentation. Cells were separated for 2 h at 4°C on a 2–4% gradient of BSA in KHB medium using a Celsep apparatus (Brinkman Instruments, Westbury, NY). Fractions containing >80% spermatocytes and round spermatids, as determined by phase contrast microscopy, were separately pooled and pelleted by centrifugation at 500 x g for 10 min.

Preparation of Cell Fractions for Western Blot Analysis

To prepare soluble and particulate fractions, spermatocytes, round spermatids, testicular spermatozoa, and caput and cauda epididymal spermatozoa were suspended in TNI and sonicated for four 10-sec intervals with a Branson sonifier at a medium power setting. The sonicated suspensions were centrifuged at 100 000 x g for 30 min in a TL55 rotor (Beckman).

A detergent-soluble fraction of spermatozoa was prepared by extraction with 0.1% Triton X-100 in TNI for 1 h at 4°C followed by centrifugation at 12 000 x g for 10 min.

Gel Electrophoresis and Western Blotting

SDS-PAGE was performed with 12.5% acrylamide gels [25]. Polypeptides were electrophoretically transferred to polyvinylidene difluoride membranes for Western analysis [26]. Immunoblots were blocked in TBS (100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.1% Tween-20) containing 1% BSA for 1 h, followed by incubation with primary antibodies or nonimmune IgG in blocking buffer for 1 h. After three washes in TBS, the blots were incubated in an affinity-purified horseradish peroxidase-conjugated secondary antibody diluted in TBS containing 5% nonfat dry milk. Immunoreactive bands were identified by enhanced chemiluminescence (SuperSignal; Pierce Chemical, Rockford, IL) on BioMax film (Kodak, Rochester, NY).

Immunofluorescence Microscopy

Spermatozoa were fixed for 60 min on ice with 4% formaldehyde and 0.25% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4, attached to poly-L-lysine-coated coverslips and then permeabilized in -20°C acetone for 10 min. Cells were rinsed in TNT (0.1% Tween-20, 150 mM NaCl, 20 mM Tris-HCl, pH 8.0) for 15 min and then incubated with 0.4 mg/ml Trypsin in TNT containing 0.1 mM CaCl2 for 5 min at room temperature. After one rinse in TNT, cells were incubated with 0.5 mg/ml soybean trypsin inhibitor in TNT for 5 min at room temperature. The cells were then blocked for 60 min at room temperature in TNT containing 5% donkey serum, 2.5% BSA, and 1% fish gelatin (blocking solution) and incubated with equal dilutions of anti-pan-Ras (Ab-3) or nonimmune mouse IgG in blocking solution overnight at 4°C. Coverslips were then rinsed three times in TNT containing 1% goat serum and incubated in Cy3-conjugated donkey anti-mouse IgG (Jackson) diluted in blocking solution for 2 h at room temperature. Following several washes in TNT, the slides were examined by epifluorescence and phase contrast microscopy.

Immunoprecipitation

For coimmunoprecipitation experiments, sperm plasma membranes were extracted in IP buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1% Triton X-100, 0.5% NP-40, 2 mM benzamidine, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM iodoacetamide, 1 mM PMSF, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 1 mM sodium vanadate) for 1 h at 4°C and then centrifuged at 40 000 rpm for 30 min in a TL55 rotor (Beckman). To remove endogenous IgG, the supernatant fraction was incubated with protein A-sepharose beads at 4°C and centrifuged at 12 000 x g for 1 min. The supernatant was then incubated with anti-Ras or with anti-PKC{zeta}, and immune complexes were recovered by incubation with protein A-sepharose beads for PKC{zeta} immunoprecipitation or with protein A-sepharose beads preincubated with rabbit anti-mouse IgG for Ras immunoprecipitation. The beads containing bound proteins were washed two or three times by centrifugation in IP buffer. Immunoprecipitated proteins were solubilized with SDS sample buffer using reducing conditions for Ras or nonreducing conditions for PKC{zeta} and analyzed by Western blotting to identify coprecipitating effector proteins.

Effect of PI3-Kinase and PKC{zeta} Inhibitors on Hamster Sperm Motility

To isolate a motile sperm population, 50 µl of epididymal contents was placed in a 12- x 75-mm tube and overlain with 2 ml of warm (37°C) Tyrode medium (TALP-7) containing 0.3% BSA (fraction V), 0.1 mM hypotaurine, 0.02 mM D-penicilamine and 1 µM epinephrine [27, 28]. After 5 min of incubation at 37°C, a highly motile swim-up sperm suspension (upper 0.5 ml) was collected, and the sperm concentration was adjusted to 106 sperm/ml by dilution with warm TALP-7. These spermatozoa were then used for the following inhibitor treatments. Wortmannin and 2-(4- morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) (PI3-kinase inhibitors) and staurosporine (a potent but nonselective inhibitor of PKC) were dissolved in dimethyl sulfoxide (DMSO). A 2x working solution of inhibitor or DMSO (for control treatment) in TALP-7 was prepared immediately before use. Spermatozoa were diluted with an equal volume of inhibitor or DMSO stock solution and capacitated for 3 h at 37°C in a 5% CO2:95% air atmosphere. The final concentrations were 100 nM and 10 µM wortmannin, 25 µM and 50 µM LY294002, and 250 nM, 500 nM, and 1 µM staurosporine; the final DMSO concentration in all incubations was 0.2%. Sperm motility pattern and acrosomal integrity were examined by phase contrast microscopy. Based on the motility pattern, the spermatozoa were categorized into two groups: motile spermatozoa exhibiting any flagellar movement and hyperactivated spermatozoa exhibiting a whiplashlike flagellar movement with large-amplitude, asymmetric flagellar bends [29, 30].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Membrane Anchoring of Ras in Spermatozoa and Spermatogenic Cells

Western blot analyses of detergent-soluble sperm fractions stained with pan-Ras monoclonal antibody demonstrated the presence of Ras in both caput (Fig. 1, lane 1) and cauda (Fig. 1, lane 2) epididymal spermatozoa. No band was seen with nonimmune mouse IgG (Fig. 1, lanes 3 and 4). To determine whether membrane anchoring of Ras occurs during posttesticular maturation of spermatozoa, immunoblots of the soluble and particulate fractions of sonicated caput and cauda spermatozoa were stained with anti-Ras. Ras localized to the particulate but not the soluble fraction of both caput (Fig. 2, lanes 1 and 2) and cauda (Fig. 2, lanes 3 and 4) spermatozoa. This result suggests that the anchoring of Ras to the membrane occurs prior to posttesticular sperm maturation in the epididymis.



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  		FIG. 1. Immunoblot of Triton X-100-soluble fraction of hamster caput (lanes 1 and 3) and cauda (lanes 2 and 4) epididymal spermatozoa. Lanes 1 and 2 stained with anti-pan-Ras exhibit the presence of a single band of 21 kDa; lanes 3 and 4 stained with nonimmune mouse IgG show no immunoreactive bands. Each lane was loaded with extracts representing 3 x 106 spermatozoa



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  		FIG. 2. Anti-pan-Ras-stained immunoblots of soluble (lanes 1 and 3) and particulate (lanes 2 and 4) fractions of hamster caput (lanes 1 and 2) and cauda (lanes 3 and 4) epididymal spermatozoa. Each lane represents cell fractions obtained from 4 x 106 spermatozoa. Ras is only associated with the particulate fraction of both caput and cauda epididymal spermatozoa

We next examined the anchoring of Ras in testicular spermatozoa and spermatogenic cells. Immunoblots of the total (Fig. 3, lane 1) and the soluble (Fig. 3, lane 2) and particulate (Fig. 3, lane 3) fractions of sonicated testicular spermatozoa revealed that Ras partioned to the particulate fraction (lane 3). No band was seen when an identical blot was stained with nonimmune mouse IgG (data not shown). Phase-contrast microscopy demonstrated the homogeneity of the spermatocyte and round spermatid fractions (Fig. 4A). Immunoblot analysis revealed the presence of Ras in total testicular spermatogenic cells (Fig. 4B, lane 1), isolated spermatocytes (Fig. 4B, lane 2), and round spermatids (Fig. 4B, lane 3). An identical blot stained with nonimmune mouse IgG showed no band (data not shown). The distribution of Ras in the soluble and particulate fractions of isolated spermatocytes and round spermatids was also examined by Western blot analysis. Ras was present in both the particulate (Fig. 5, lanes 2 and 4) and soluble (Fig. 5, lanes 1 and 3) fractions of spermatocytes (Fig. 5, lanes 1 and 2) and spermatids (Fig. 5, lanes 3 and 4). However, more Ras was present in the particulate fraction. These data suggest that the recruitment of Ras to the plasma membrane occurs by the completion of spermatogenesis.



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  		FIG. 3. Western blot immunostained with pan-Ras antibody showing total hamster testicular sperm lysate (lane 1) and the soluble (lane 2) and particulate (lane 3) fractions obtained from testicular spermatozoa. Ras is only associated with the particulate fraction



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  		FIG. 4. A) Phase-contrast microscopic examination of hamster spermatocytes and round spermatids isolated by unit gravity sedimentation. B) Immunoblot analysis of total hamster testicular spermatogenic cells (lane 1), spermatocytes (lane 2), and round spermatids (lane 3) stained with anti-pan-Ras reveal the presence of Ras in all fractions (40 µg protein loaded in each lane)



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  		FIG. 5. Western blot showing the soluble (lanes 1 and 3) and particulate (lanes 2 and 4) fractions of hamster spermatocytes and spermatids immunostained with anti-pan-Ras. Ras is present in both the particulate and the cytosolic fractions of spermatocytes and spermatids, but the major portion of Ras is present in the particulate fraction of spermatocytes (lane 2) and spermatids (lane 4)

Identification of Ras Subtype(s) in the Plasma Membrane Fraction of Hamster Cauda Spermatozoa

Western blots of the enriched plasma membrane fraction of cauda spermatozoa showed the presence of Ras (Fig. 6, lane 1). Type-specific monoclonal antibodies against H-, K-, and N-Ras were tested by Western blot analyses to define Ras subtype(s) expressed in the hamster cauda sperm plasma membrane. Only K-Ras (Fig. 6, lane 3) and N-Ras (Fig. 6, lane 4) antibodies recognized a single polypeptide of predicted molecular mass. No band was seen when an identical blot was stained with nonimmune mouse IgG (data not shown).



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  		FIG. 6. Immunoblot of plasma membrane fractions stained with anti-pan-Ras (lane 1), anti-H-Ras (lane 2), anti-K-Ras (lane 3), and anti-N-Ras (lane 4); 15 µg protein was loaded in each lane. Pan-Ras, K-Ras, and N-Ras antibodies recognize a 21-kDa polypeptide, and no H-Ras is detectable

Immunofluorescence Localization of Ras

Cauda epididymal spermatozoa immunostained with pan-Ras (Ab-3) monoclonal antibody exhibited specific staining of the flagellum (Fig. 7A'). Sperm stained with nonimmune mouse IgG exhibited no fluorescence of the flagellum (Fig. 7B').



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  		FIG. 7. Matched phase contrast (A and B) and fluorescence (A' and B') microscopic examination of hamster cauda epididymal spermatozoa immunostained with pan-Ras (Ab-3) monoclonal antibody (A') and with nonimmune mouse IgG (B'). Note the specific fluorescence of the sperm flagellum stained with anti-pan-Ras (A'). Sperm incubated with nonimmune IgG exhibited no fluorescence (B'). One spermatozoon in B has tail angulation

Identification of Downstream Effectors of Ras

Immunoblots of an enriched plasma membrane fraction of cauda spermatozoa showed the presence of the 85-kDa regulatory subunit of PI3-kinase (Fig. 8, lane 1) and the 72-kDa PKC{zeta} (Fig. 8, lane 3); no stained bands were present in identical lanes probed either with nonimmune mouse IgG (Fig. 8, lane 2) or with nonimmune rabbit IgG (Fig. 8, lane 4). These data demonstrate the presence of PI3-kinase and PKC{zeta}, the downstream targets of Ras, in hamster cauda epididymal spermatozoa.



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  		FIG. 8. Immunoblot analysis of plasma membrane fraction separated by reducing SDS-PAGE on a 12% gel (lanes 1 and 2) and by nonreducing SDS-PAGE on a 7.5% gel (lanes 3 and 4); 15 µg protein was loaded in each lane. Lane 1, 85-kDa regulatory subunit of PI3-kinase, immunostained with anti-PI3-kinase; lane 2, no immunoreactive bands, immunostained with mouse nonimmune IgG; lane 3, 72-kDa PKC{zeta} polypeptide band, immunostained with anti-PKC{zeta}; lane 4, no immunoreactive bands, immunostained with nonimmune rabbit IgG

Coimmunoprecipitation analyses were performed to examine the interaction of Ras with the effector proteins PI3-kinase and PKC{zeta}. As shown in Figure 9A, Ras was completely recovered in the anti-Ras immunoprecipitation pellet (lane 3). A portion of the PI3-kinase coprecipitated with Ras (Fig. 9B, lane 2), and the remaining PI3-kinase was present in the unbound fraction (Fig. 9B, lane 1). No Ras or PI3-kinase bands were detected when an identical lane of the Ras immunoprecipitation pellet was stained with nonimmune mouse IgG. (Fig. 9A, lane 4). Only a portion of the PKC{zeta} was obtained in the anti-PKC{zeta} immunoprecipitation pellet (Fig. 9C, lane 3), and the remaining PKC{zeta} was present in the unbound fraction (Fig. 9C, lane 2). No PKC{zeta} band was seen when an identical lane of anti-PKC{zeta} immunoprecipitation pellet was stained with nonimmune rabbit IgG (Fig. 9C, lane 4). A portion of both Ras (Fig. 9D, lane 2) and PI3-kinase (Fig. 9E, lane 2) was present in the PKC{zeta} immunoprecipitation pellet, and the remainder was present in the unbound fractions (Fig. 9, D and E, lane 1).



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  		FIG. 9. Identification of Ras-associated proteins by coimmunoprecipitation analysis. Triton X-100-soluble lysates of the isolated plasma membrane fraction (~200 µg protein) were immunoprecipitated with anti-pan-Ras (A and B) and analyzed by reducing SDS-PAGE on a 12% gel or immunoprecipitated with anti-PKC{zeta} (C–E) and analyzed by nonreducing SDS-PAGE on a 7.5% gel. A) Immunoblot of Triton X-100-soluble plasma membrane fraction (lane 1), immunoprecipitation (IP) supernatant (lane 2), and anti-pan-Ras IP pellet (lane 3) immunostained with anti-Ras. Note that a complete recovery of Ras (21 kDa) was observed in the IP pellet (lane 3); both the heavy (50 kDa) and light (25 kDa) chains of IgG are evident. An identical lane (lane 4) of the anti-pan-Ras IP pellet stained with nonimmune mouse IgG (NI-IgG) shows only the heavy (50 kDa) and light (25 kDa) IgG chains and not the 21-kDa Ras polypeptide. B) Anti-pan-Ras IP supernatant (lane 1) and IP pellet (lane 2) immunostained with anti-PI3-kinase. The 85-kDa regulatory subunit of PI3-kinase is present in both the IP supernatant (lane 1) and the IP pellet (lane 2); the 50-kDa and 25-kDa IgG chains are also visible in the IP pellet. C) Immunoblot of Triton X-100-soluble plasma membrane fraction (lane 1), IP supernatant (lane 2), and anti-PKC{zeta} IP pellet (lane 3) immunostained with anti-PKC{zeta}. In the IP pellet (lane 3), partial recovery of 72-kDa PKC{zeta} is achieved and the 150-kDa IgG band is present. An identical lane of IP pellet stained with rabbit nonimmune IgG (lane 4) shows the presence of the 150-kDa IgG but not the 72-kDa PKC{zeta} polypeptide. D and E) Anti-PKC{zeta} IP supernatant (lane 1) and IP pellet (lane 2) immunostained with anti-Ras (D) and anti-PI3-kinase (E) reveal the presence of Ras and PI3-kinase molecules in both the IP supernatant and the IP pellet

Effect of Inhibitors of PI3-Kinase and PKC{zeta} on Hamster Sperm Motility

Control (DMSO only) and inhibitor-treated spermatozoa were capacitated for 3 h and were examined for motility using phase contrast microscopy. Approximately 60–70% of the sperm population in each treatment, except 10 µM wortmannin, exhibited some type of flagellar movement; in the 10 µM wortmannin-treated preparation, only ~40% of spermatozoa exhibited flagellar movement. The various treatment groups differed in the percentage of motile spermatozoa exhibiting hyperactivated motility (Fig. 10). Approximately 50% of motile spermatozoa in the vehicle control population exhibited hyperactivated motility; most of the remaining spermatozoa displayed a linear or circular motility pattern, and occasional spermatozoa displayed head-to-head agglutination. Approximately 15% and 5% of the motile spermatozoa exhibited hyperactivated motility in the presence of 100 nM and 10 µM wortmannin, respectively; the remaining spermatozoa exhibited flagellar quivering or a low-amplitude flagellar beat. In the presence of 25 µM LY294002, approximately 15% of the motile spermatozoa exhibited hyperactivated motility and many spermatozoa exhibited head-to-head agglutination and moved in a circular motion, whereas in the presence of 50 µM LY294002, ~10% hyperactivated spermatozoa were observed and the remaining spermatozoa exhibited quivering and lower amplitude flagellar beat. When capacitation was performed in the presence of 250 nM staurosporine, 50% of the motile spermatozoa exhibited hyperactivated motility, but in the presence of 500 nM or 1 µM staurosporine, only ~10–15% of the motile spermatozoa exhibited hyperactivated motility and the remaining spermatozoa displayed a circular motion or flagellar quivering. No changes in the acrosomal integrity of the capacitated spermatozoa were observed in the presence of these inhibitors. Hyperactivation of sperm motility during capacitation was inhibited by wortmannin, LY294002, and staurosporine in a dose-dependent manner, suggesting that the activities of PI3-kinase and PKC{zeta} are associated with hyperactivated motility.



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  		FIG. 10. Effect of inhibitors of PI3-kinase (wortmannin and LY294002) and PKC{zeta} (staurosporine) on hamster sperm motility. Highly motile cauda epididymal spermatozoa (5 x 105/ml) were capacitated in TALP-7 at 37°C in a humidified incubator with 5% CO2:95% air for 3 h in the presence of different concentrations of wortmannin (100 nM and 10 µM), LY294002 (25 µM and 50 µM), staurosporine (250 nM, 500 nM, and 1 µM), or vehicle (DMSO). The percentage of motile spermatozoa and the percentage of motile spermatozoa exhibiting hyperactivated forward motility were estimated using phase contrast microscopy. All data are expressed as mean ± SD of three separate experiments. The inhibitors of both enzymes inhibited the hyperactivation of sperm motility during capacitation in a dose-dependent manner


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Ras, a low-molecular-weight GTP-binding protein, plays a key regulatory role in many biochemical processes. In this study, we demonstrated the presence of Ras in detergent-soluble lysates of hamster caput and cauda epididymal spermatozoa. In somatic cells, Ras is recruited to the inner face of the plasma membrane following posttranslational lipidation at its C-terminus by the enzyme farnesyltransferase [31, 32]. Previously, we demonstrated the upregulated expression of farnesyltransferase in the cytoplasmic lobe of elongating hamster spermatids and in the cytoplasmic droplet of epididymal spermatozoa [33]. A requisite for Ras function is localization to the plasma membrane [5]. Our data demonstrate that in testicular and epididymal spermatozoa Ras localizes to the particulate cell fraction and that it is highly enriched in the plasma membrane fraction of cauda epididymal spermatozoa. However, in both spermatocytes and spermatids Ras is present in both soluble and particulate fractions, suggesting that the translocation of Ras from cytosol to the membrane occurs by the completion of spermatogenesis. Future studies will address the specific stage(s) of spermatogenesis where the posttranslational modification of Ras occurs.

N-Ras, H-Ras, K-Ras 4A, and K-Ras 4B are the four mammalian Ras isoforms [4–6]. These isoforms are identical in the 85 amino acids at the N-terminus and differ only at the carboxyl terminus, a region implicated in interaction with the plasma membrane and possibly with other proteins [1, 4, 34]. There is a distinct temporal differential regulation in the mRNA expression of the three members of the Ras gene family in murine male germ cells [13, 14]. A low level of H-Ras mRNA was detected in both meiotic and postmeiotic cells [13]. K-Ras mRNA was faintly detectable in spermatogonia and significantly reduced in round spermatids, and N-Ras mRNA transcript was present in all spermatogenic cell types [14]. However, the localization and the presence of different Ras isoforms in spermatozoa were not established in those previous studies. Ras has been reported to be localized to the extracellular surface of the acrosomal segment of human spermatozoa [35], but this assertion is inconsistent with the known intracellular localization of Ras [7–11]. Our data demonstrate that both N-Ras and K-Ras are associated with the plasma membrane of hamster spermatozoa, and immunocytochemical evaluation revealed Ras localization to the flagellum.

The results of a number of recent studies in somatic cells have indicated that the different Ras subtypes may mediate different functions. For example, gene targeting studies have shown that K-Ras is essential for mouse embryogenesis [36, 37]. Inhibitors of farnesyltransferase, used for cancer treatment, are effective inhibitors of H-Ras [38, 39]. The different Ras isoforms may participate in different signaling pathways in spermatozoa, and it is important to identify the upstream and downstream components of the pathways that function in spermatozoa. GTP-Ras binds to and activates a number of effector molecules, including the serine/threonine kinase c-Raf-1, the lipid kinase PI3-kinase, p120GAP, RalGDS and related proteins, AF6, RIN1, and PKC{zeta} [3]. The serine/threonine kinase c-Raf-1 is the most well-characterized downstream effector in somatic cells [40]. Two domains of Ras, termed Switch I (amino acids 32–40) and Switch II (amino acids 60–72) have been identified; these domains mediate the stable interactions between GTP-Ras and its downstream effectors [41]. Thus, in somatic cells Ras can interact with multiple effectors to generate more than one cellular signal. The effectors through which Ras mediates its downstream signals in spermatozoa have not previously been identified. In the present study, we demonstrated the presence of PI3-kinase and PKC{zeta}, which are potential downstream targets of Ras, in the plasma membrane fraction of hamster cauda epididymal spermatozoa.

PI3-kinase phosphorylates the 3'-OH group of the inositol ring to inositol phospholipids, and the enzyme has an 85-kDa regulatory subunit and a 110-kDa catalytic subunit [42–46]. In somatic cells, PI3-kinase coimmunoprecipitates with Ras [47, 48], and the activation of PI3-kinase occurs by direct interaction of the catalytic subunit of the enzyme with GTP-Ras both in vivo and in vitro [49–51]. In this study, we demonstrated that a significant fraction of sperm PI3-kinase is complexed to Ras. The remaining unbound fraction of PI3-kinase may participate in Ras-independent signaling pathways, suggesting that PI3-kinase is associated with multiple signaling cascades in hamster cauda epididymal spermatozoa. Future studies will determine whether some PI3-kinase associates with other effector molecules.

The zeta isoform of PKC belongs to the atypical PKC family. It does not respond to calcium or phorbol ester/DAG [52–54] and is stimulated by phospholipids [55]. Enzymatic activity and autophosphorylation of PKC{zeta} are stimulated by PIP3, the product of PI3-kinase [56]. In other cells, both in vitro and in vivo analyses have demonstrated the direct interaction of Ras with the regulatory domain of PKC{zeta} [57]. With coimmunoprecipitation analyses, we also demonstrated that a significant fraction of PKC{zeta} was associated with Ras and PI3-kinase in cauda sperm plasma membrane fractions. Presently, we cannot determine whether PKC{zeta}-Ras and PKC{zeta}-PI3-kinase are two independent signaling cascades in spermatozoa or whether the Ras-PI3-kinase-PKC{zeta} complex regulates signaling events through a single pathway.

PI3-kinase is potently inhibited by wortmannin [58, 59] and LY294002 [60]. Fisher et al. [61] showed that the induction of the human sperm acrosome reaction by mannose-BSA and by a polyclonal antibody to sperm zona pellucida receptor kinase was inhibited by wortmannin and suggested that PI3-kinase is involved in the human sperm acrosome reaction. Staurosporine, a microbial alkaloid, is a potent but nonselective inhibitor of PKC, which interacts with the kinase domain [52]. In the present study, we demonstrated that wortmannin, LY294002, and staurosporine inhibited the hyperactivation of sperm motility during capacitation of hamster spermatozoa in a dose-dependent manner, reflecting the association of the catalytic activities of PI3-kinase and PKC{zeta} with the development of this motility pattern. The concentrations of wortmannin and LY294002 used in the present study were comparable to those consistently demonstrated to inhibit PI3-kinase in other cell types [58, 60, 62].

We have described the association of Ras with the plasma membrane fraction of cauda epididymal spermatozoa, the localization of Ras to the flagellum, and the inhibition of hyperactivation of sperm motility during capacitation by wortmannin and LY294002, two PI3-kinase inhibitors, and by staurosporine, an inhibitor of PKC. The interaction of Ras with both PI3-kinase and PKC{zeta} indicates a diversity of Ras target proteins in spermatozoa. We hypothesize that one of the potential signal transduction pathways in spermatozoa is activation of PI3-kinase by GTP-Ras, and the catalytic products of PI3-kinase activate PKC{zeta}. PKC{zeta} as a kinase may phosphorylate flagellar proteins on serine and threonine residues or it may activate other signaling elements, and this pathway may function in the regulation of sperm motility.


   FOOTNOTES
 
1 This research was supported by NIH grants HD20419 and HD36824. Some of these data were presented in abstract form for the 34th Annual Meeting of the Society for Reproduction (Biol Reprod 2001; 64(suppl 1):195). Back

2 Correspondence. FAX: 615 343 4539; subir.nag-das{at}mcmail.vanderbilt.edu Back

Received: 9 January 2002.

First decision: 6 February 2002.

Accepted: 23 April 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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