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FEBS Letters 580 (2006) 1515–1520

Roles of cAMP in regulating sliding and flagellar bending in demembranated hamster spermatozoa

Masashi Kinukawaa, Shoji Odaa, Yoshiyuki Shirakuraa, Masaki Okabea, Junko Ohmurob, Shoji A. Babab, Masao Nagataa, Fugaku Aokia,* a Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Shinryoiki-Seimei, Building 302, Kashiwa City, Chiba 277-8562, Japan b Department of Biology, Ochanomizu University, Tokyo 112-0816, Japan

Received 4 November 2005; revised 9 January 2006; accepted 23 January 2006

Available online 2 February 2006

Edited by Michael R. Bubb

erated, it is important to examine both microtubule sliding and Abstract To understand the mechanism regulating spermato- zoa motility, it is important to investigate the mechanism regu- its conversion into flagellar bending. lating the conversion of microtubule sliding into flagellar The demembranated spermatozoa model is useful for inves- bending. Therefore, we analyzed microtubule sliding and its con- tigating the components required for the activation of the version into flagellar bending using a demembranated spermato- flagellar axoneme. In spermatozoa that have been demembra- zoa model in which microtubule sliding and flagellar bending nated using the nonionic detergent Triton X-100, flagellar could be analyzed separately by treating the demembranated movement comparable to that of intact spermatozoa can be spermatozoa with and without dithiothreitol, respectively. Using reproduced in the presence of Mg2+–ATP [1]. Furthermore, this model, we examined the roles of cAMP and its target mol- the individual microtubule sliding produced by ATPase ecules in regulating flagellar bending and microtubule sliding. activity has been demonstrated in the demembranated sperma- Although flagellar bending did not occur in the absence of tozoa model after partial digestion of the axoneme proteins cAMP, microtubule extrusion occurred without it, suggesting that cAMP is necessary for the conversion of microtubule sliding with proteases, such as trypsin [2–6] and elastase [7,8]. Treat- into flagellar bending, but not for microtubule sliding itself. The ment with these induced the extrusion of microtu- target of cAMP for regulating flagellar bending was not cAMP- bules from the flagellar axoneme in the demembranated dependent protein kinase (PKA), since flagellar bending was still spermatozoa, which seemed to be dependent on microtubule observed in the spermatozoa treated with a PKA-specific inhibi- sliding. However, it is not appropriate to use proteases to tor. Alternatively, the Epac/Rap pathway may be the target. investigate the mechanism of microtubule sliding, since it Epac2 and Rap2 were detected in hamster spermatozoa using seems likely that these enzymes degrade important regulatory immunoblotting. Since Rap2 is a GTPase, we investigated the components involved in flagellar bending, as well as the pro- flagellar bending of demembranated spermatozoa treated with teins connecting . Recently, we developed a meth- GTPcS. The treatment markedly increased the beat frequency od to induce microtubule sliding using treatment with a and bending rate. These results suggest that cAMP activates the Epac/Rap pathway to regulate the conversion of microtubule reducing agent, without any proteases; this treatment induces sliding into flagellar bending. minimal digestion of the axonemal proteins and allows inves- 2006 Federation of European Biochemical Societies. Published tigation of the molecular mechanism regulating microtubule by Elsevier B.V. All rights reserved. sliding [9]. Using this method, it is possible to analyze micro- tubule sliding and flagellar bending separately in the demembr- Keywords: Flagellar bending; Microtubule sliding; Motility; anated spermatozoa model with and without the reducing cAMP; Sperm agent, respectively. The molecular mechanism regulating the conversion of microtubule sliding into flagellar bending remains unclear. A number of reports suggest that cAMP is involved in regulating spermatozoa motility. The activation of cAMP-dependent pro- 1. Introduction tein kinase (PKA) and stimulation of protein phosphorylation are involved in the initiation and maintenance of motility [10– Flagellar bending generates the propulsive force for the pro- 13]. The initiation of motility is regulated by the tyrosine phos- gressive swimming in spermatozoa, and is regulated via activa- phorylation of a 15-kDa protein, which is stimulated by cAMP tion of the flagellar axoneme. The generation of flagellar in rainbow trout spermatozoa [14,15]. A-kinase anchoring pro- bending consists of two processes: individual doublet microtu- teins (Akaps) are scaffold proteins for PKA and the disruption bules slide via activation of the dynein arm and then the sliding of Akap4 causes defects in spermatozoa motility [16]. In addi- of microtubules is converted into flagellar bending. Therefore, tion, cAMP is required for the reactivation of demembranated to elucidate the mechanism by which flagellar bending is gen- spermatozoa motility in the boar [17,18], dog [19,20], and ham- ster [18]. Although many studies have demonstrated that cAMP and its target kinase are involved in flagellar motility, little is *Corresponding author. Fax: +81 471 36 3698. known about their precise roles in the mechanism regulating E-mail address: [email protected] (F. Aoki). microtubule sliding and its conversion into flagellar bending.

0014-5793/$32.00 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.01.078 1516 M. Kinukawa et al. / FEBS Letters 580 (2006) 1515–1520

In this study, to clarify the mechanism by which flagellar curvature of R- and P-bends was assigned a positive and negative va- bending is generated, we analyzed microtubule sliding and fla- lue, respectively. The curvatures were measured at positions 30, 60, gellar bending using the demembranated spermatozoa model and 90 lm from the head–midpiece junction (D30, D60, and D90, respectively). in which they could be analyzed separately by treating the demembranated spermatozoa with and without a reducing agent, respectively. Using this method, we examined the roles 2.5. PKA assay A1-ll aliquot of the mass of spermatozoa was suspended in 200 ll of cAMP and its target kinase in regulating flagellar bending of demembranation medium consisting of 1 mM EDTA, 50 mM N-2- and microtubule sliding. hydroxyethyl-piperazine-N-2-ethane sulfonic acid (HEPES, pH 7.9), 0.2% (w/v) Triton X-100, and a protease inhibitor cocktail that con- tained 1 lg/ml aprotinin, 2 lg/ml leupeptin, 1 lg/ml pepstatin, and 100 lg/ml phenylmethyl sulfonyl fluoride. The suspension was incu- 2. Materials and methods bated for 30 s with gentle stirring at 37 C to dissolve the plasma mem- brane and mitochondrial sheath. Then, 10 ll of the extracted 2.1. Spermatozoa preparation spermatozoa suspension was transferred to 90 ll of kinase reaction Sexually mature hamsters were killed by chloroform inhalation. The buffer consisting of 100 lM kemptide [24], 1 mM EDTA, 5 mM cauda epididymis was removed promptly. After removing blood from MgCl2, and 50 mM HEPES (pH 7.9), the protease inhibitor cocktail, the epididymal surface with physiological salt solution, the distal tu- and various concentrations of cAMP. Assays were initiated by adding bules were punctured with an 18-gauge needle in 5–10 places, and a 2 ll of 16.5 lM[c-32P] ATP containing 133 KBq/ml (Amersham Phar- mass of spermatozoa was squeezed out with forceps into a plastic petri macia Biotech, Buckinghamshire, UK). After incubation for 10 min at dish (35 · 10 mm). The mass of spermatozoa was covered with mineral 37 C, the suspensions were centrifuged at 12000 · g for 1 min. Twenty oil that had been pre-warmed to 37 C, in order to prevent evaporation microliters of the supernatant were then spotted on a P81 paper before use. square. After sitting at room temperature for 30 s, the papers were washed with 0.75% phosphoric acid three times. Then, the papers were washed with acetone, air-dried, and analyzed using Cerenkov count- 2.2. Procedure for reactivation and the induction of microtubule ing. The kinase activity was expressed as cpm of 32P incorporated into extrusion in the demembranated spermatozoa kemptide. Spermatozoa were demembranated and reactivated using a modifi- cation of the method developed by Ishijima and Witman [21].A1-ll aliquot of the mass of spermatozoa was suspended in 100 ll of dem- 2.6. Immunoblotting embranation medium that contained 1 mM EDTA, 50 mM N-2- Each 200 ll of the spermatozoa suspension was centrifuged at hydroxyethyl-piperazine-N-2-ethane sulfonic acid (HEPES, pH 7.9), 3000 · g for 5 min and the pellets were added to 80 ll of extraction and 0.2% (w/v) Triton X-100. The suspension was incubated for 30 s buffer consisting of 50 mM Tris–HCl, 150 mM NaCl, 1% Nonidet P- with gentle stirring at 37 C to dissolve the plasma membrane and 40, 0.5 mM EDTA, 100 lM sodium orthovanadate, 0.1% SDS, mitochondrial sheath. The demembranated spermatozoa so obtained 1 mM DTT, 1 lg/ml aprotinin, 2 lg/ml leupeptin, 1 lg/ml pepstatin, were reactivated by transferring 10 ll of the suspension of demembra- and 100 lg/ml phenylmethylsulfonyl fluoride (pH 8.0), and frozen at nated spermatozoa to 100 ll of reactivation medium containing 1 mM 20 C. To examine the binding of Epac2 to the flagellum, the suspen- EDTA, 1 mM ATP, 5 mM MgCl2, and 50 mM HEPES (pH 7.9). Fifty sion of demembranated spermatozoa with demembranation medium, micromolar cAMP was included in the reactivation medium if not described above, was centrifuged at 18000 · g for 3 min. After remov- specified. To induce microtubule extrusion, a protease inhibitor cock- ing the supernatant, the pellet was resuspended in the original volume tail that contained 1 lg/ml aprotinin, 2 lg/ml leupeptin, 1 lg/ml pep- of demembranation medium. These supernatant and pellet fractions statin, and 100 lg/ml phenylmethyl sulfonyl fluoride was added to were frozen at 20 C before use. After thawing, the samples were the demembranation and reactivation media; then 33 mM of dithio- denatured for SDS–PAGE as follows. The suspensions were added threitol (DTT) was added to the reactivation medium. In some exper- to 80 ll of extraction buffer, 50 llof4· SDS sample buffer [25], and iments, spermatozoa were treated with 2.0 U/ml cAMP 96 mg of urea, and then boiled for 1 min. These samples were subjected phosphodiesterase, 10 lM PKA inhibitor, N-[2-(p-bromocinnamyla- to electrophoresis on 10% SDS–PAGE gels. The proteins were electro- mino)ethyl]-5-isoquinolinesulfonamide (H89; Seikagaku, Tokyo, Ja- blotted onto a polyvinylidene difluoride membrane (Millipore, Bed- pan), 0.2 mM GTPcS, or 0.2 mM ATPcS. ford, MA, USA). Non-specific binding on the membrane was blocked with 5% BSA in Tris-buffered saline (TTBS; 150 mM NaCl, 20 mM Tris–HCl, and 0.1% Tween 20, pH 7.6) for 1 h at room temper- 2.3. Microscopic observation of spermatozoa ature. The blot was incubated overnight at 4 C with polyclonal anti- After the reactivated spermatozoa were incubated at 37 C for at Epac2 (sc-9384, Santa Cruz Biotechnology, Santa Cruz, CA, USA; least 10 min, 10 ll of each spermatozoa suspension was placed on a 2 lg/ml in TTBS) and anti-Rap2 antibody (R23020, Transduction glass slide pre-warmed to 37 C and covered with an 18 · 24-mm cov- Laboratories, Lexington, KY, USA; 2 lg/ml in TTBS). To confirm erslip. As soon as the sample was prepared, photographs were taken at the specificity, the primary anti-Epac2 antibody was neutralized with 60 frames per second and an exposure time of 1/1000 s, with a FAST- 10 lg/ml of antigen peptide (sc-9384s, Santa Cruz Biotechnology). CAM-Net high-speed camera (PHOTRON, Tokyo, Japan) under a The blot was then washed with TTBS three times for 10 min each phase-contrast microscope. The images obtained were recorded using and kept for 1 h at room temperature with horseradish peroxidase- Movie Ruler (PHOTRON). Spermatozoa were selected for the analysis conjugated donkey anti-goat IgG (Amersham) for anti-Epac2 anti- at random. body and sheep anti-mouse IgG (Amersham) for anti-Rap2 antibody at a 1:1000 dilution. After 1 h at room temperature, the membrane 2.4. Analysis of spermatozoa motility was washed with TTBS three times, for 20 min each, and the peroxi- The beat frequency and flagellar bending of spermatozoa were ana- dase activity was detected using LAS-1000plus (Fuji Photo Film, To- lyzed by taking photographs at 125 frames per second and an exposure kyo, Japan); the resulting images were recorded using Image Gauge time of 1/1000 s with a FASTCAM-Net high-speed camera (PHO- (Fuji). TRON) under a phase-contrast microscope. The images obtained were recorded and analyzed using the flagellar image analysis software Boh- boh (BohbohSoft, Tokyo, Japan). Curvature was measured as de- scribed previously [22] to determine the bending rate and maximum 3. Results curvature in a beat cycle. In the analysis, 60 successive frames were used to analyze the bending rate. The direction of each bend was deter- 3.1. The effect of cAMP on microtubule extrusion and flagellar mined using the direction of the hook-shaped projection of the head, as described by Woolley [23]. A bend in the same direction as the curve of bending the hook-shaped head was defined as a reverse bend (R-bend) and one Several studies have demonstrated that cAMP is necessary in the opposite direction was defined as a principal bend (P-bend). The for flagellar bending [17,19,20,26–28], while its necessity in M. Kinukawa et al. / FEBS Letters 580 (2006) 1515–1520 1517 microtubule sliding has not been examined. Consistent with re- ported results, flagellar bending was not observed in the dem- embranated spermatozoa without cAMP (Fig. 1). When 0.1 lM cAMP was added to the reactivation medium, flagellar bending was observed in 14% of the spermatozoa. At concen- trations greater than 1 lM, it was observed in 60% of the sper- matozoa. However, microtubule extrusion occurred in the reactivation medium without cAMP. Cyclic AMP did not have any effect on the extrusion. More than 80% of the spermatozoa always extruded microtubules at any concentration of cAMP in the reactivation medium. This occurred even in the cAMP-free medium containing cAMP phosphodiesterase, which degrades endogenous cAMP (Fig. 1). These results indi- cate that cAMP is not necessary for microtubule sliding and plays an essential role in the conversion of microtubule sliding into flagellar bending.

3.2. The target of cAMP for regulating flagellar bending Although PKA is a dominant mediator of the cAMP signal in various cell types and we confirmed that PKA activity in- creased with the cAMP concentration in the suspension of demembranated spermatozoa (Fig. 2A), flagellar bending was still observed when 10 lM H89, a PKA-specific inhibitor, was added to the demembranation and reactivation media (Fig. 2B). Upon treatment with 50 lM cAMP plus 10 lM H89, the PKA activity was inhibited completely (Fig. 2C), but the percentage of spermatozoa that showed flagellar bend- ing did not change (Fig. 2B). Therefore, the target of cAMP that is involved in flagellar bending is not PKA activity in the demembranated hamster spermatozoa. To investigate targets of cAMP other than PKA, we exam- ined the Epac/Rap pathway, since recent reports found that this pathway is stimulated by cAMP in somatic cells [29,30]. In the immunoblotting analysis for Epac and Rap, specific Fig. 2. The involvement of PKA in flagellar bending and microtubule dense bands at 110 kDa for Epac2 and 22 kDa for Rap2 were extrusion in demembranated spermatozoa. (A) Effects of various concentrations of cAMP on PKA activity. Spermatozoa that had been detected in hamster spermatozoa. The 110-kDa band for demembranated and reactivated with various concentrations of cAMP Epac2 was specific, since it disappeared upon neutralization were examined for PKA activity. The experiment was performed twice, with similar results. Each value is the mean of two experiments. (B) Effects of a PKA inhibitor on flagellar bending. The demembranated spermatozoa were reactivated in reactivation medium containing 50 lM cAMP with or without 10 lM PKA inhibitor (H-89). The percentages of reactivated spermatozoa that showed flagellar bending were examined. The experiment was conducted three times. In each experiment, 100 spermatozoa were examined for each cAMP concen- tration. The values are expressed as the means ± S.E. (C) Effects of a PKA inhibitor (H-89) on PKA. Spermatozoa that had been dem- embranated and reactivated in medium containing 50 lM cAMP with or without PKA inhibitor (H-89) were examined for PKA activity. The experiment was performed twice, with similar results. Each value is the mean of two experiments.

with antigen peptide against anti-Epac2 antibody (Fig. 3). Fig. 1. The involvement of cAMP in flagellar bending and microtubule The specificity for the antibody against Rap2 was not exam- extrusion in demembranated spermatozoa. Demembranated sperma- ined because no antigen was available. Epac1 and Rap1 were tozoa were reactivated in reactivation medium with various concen- not detected (data not shown). These results suggest that the trations of cAMP. At 0 lM cAMP, 2.0 U/ml cAMP phosphodiesterase was added to the reactivation medium. In the analysis of microtubule Epac2/Rap2 pathway is present in hamster spermatozoa. extrusion, 33 mM DTT was added to the reactivation medium, but not In order to examine whether Epac2 is associated with the fla- in the analysis of flagellar bending. The percentages of reactivated gellum of demembranated spermatozoa, the suspension of spermatozoa that showed flagellar bending (solid column) and demembranated spermatozoa was centrifuged, and the result- microtubule extrusion (hatched column) were examined. The experi- ment was performed three times. In each experiment, 100 spermatozoa ing supernatant and precipitate were subjected to immunoblot- were examined for each cAMP concentration. The values are expressed ting with anti-Epac antibody. Epac2 was detected in the as the means ± S.E. precipitate, but not in the supernatant, suggesting that Epac2 1518 M. Kinukawa et al. / FEBS Letters 580 (2006) 1515–1520

4. Discussion

Many studies have described the various factors involved in the flagellar bending of spermatozoa [32–34]. However, few re- ports have analyzed the factors involved in regulating microtu- bule sliding, although microtubule sliding is a basal process that generates flagellar bending. Previously, we developed a method to efficiently induce microtubule extrusion in dem- embranated spermatozoa using treatment with reducing agents [9]. In this study, we analyzed microtubule sliding and flagellar bending separately by treating the spermatozoa with and with- out DTT, respectively. Using this method, we showed that

Fig. 3. Immunoblotting analysis of Epac/Rap in hamster spermato- zoa. (A) Spermatozoa were lysed in extraction buffer and the spermatozoa proteins were separated using SDS–PAGE and probed with anti-Epac2 (Epac2) and anti-Rap2 (Rap2) antibodies. Arrows indicate specific dense bands at 110 kDa for Epac2 and 22 kDa for Rap2, which correspond to their respective predicted molecular weights. Anti-Epac2 antibody was neutralized with antigen peptide, which is shown in the right column (peptide+). The experiment was performed three times, with similar results. Molecular mass standards are indicated to the right of the lanes. (B) Suspensions of demembr- anated spermatozoa were centrifuged to obtain the supernatant (sup) and precipitate (ppt). These two fractions and the initial suspension (whole) were immunoblotted with anti-Epac2 antibody. The arrow indicates Epac2. is a cytoskeletal flagellar protein that is not extracted with a detergent. To clarify the function of Rap2, which is a GTPase, we examined the effect of GTPcS treatment on the motility of demembranated spermatozoa. GTPcS is a non-hydrolyzable GTP analog, which is a GTPase activator [31]. As shown in Fig. 4A, the beat frequency increased markedly upon treat- ment with GTPcS; the frequency was significantly greater in the spermatozoa treated with GTPcS than those treated with ATPcS and no reagent. To determine which characteristics of flagellar bending GTPcS changed to increase the beat fre- quency, the bending rate and maximum curvature in a beat cy- Fig. 4. The involvement of GTPase in regulating flagellar bending in cle were measured. Either or both of these parameters could demembranated spermatozoa. The (A) beat frequency, (B) bending change to increase the beat frequency. Fig. 4B shows that rate, and (C) maximum values of the curvatures of the R- and P-bends GTPcS treatment increased the bending rate; it was higher in were analyzed in spermatozoa that had been demembranated and the spermatozoa treated with GTPcS than in those treated reactivated in medium containing no reagent (–), ATPcS, or GTPcS. with ATPcS and no reagent at all positions on the flagellum, The curvatures of the flagellum were measured at positions 30, 60, and 90 lm from the head–midpiece junction (D30, D60, and D90, although a significant difference was detected only at position respectively) to determine the bending rate and maximum value of D30. However, the maximum curvature did not change with the curvature. The curvature of the R- and P-bend was assigned a plus GTPcS treatment in any direction of the R- or P-bends at and minus value, respectively. The experiments were performed three any position on the flagellum (Fig. 4C). These results demon- times, with similar results. The data were pooled and expressed as the means ± S.E. (n = 18, 6, and 6 in the analysis of the beat frequency, strated that GTPcS changed only the bending rate, but not the bending rate, and maximum curvatures, respectively). Asterisks amplitude of the flagellum to increase the beat frequency in indicate significant differences from the value of control spermatozoa hamster spermatozoa. incubated with no reagent (Student’s t test; *P < 0.05 and **P < 0.01). M. Kinukawa et al. / FEBS Letters 580 (2006) 1515–1520 1519 cAMP is necessary for inducing flagellar bending, but not hamster spermatozoa. The results suggest that cAMP is an microtubule sliding, suggesting that cAMP is involved in the essential factor involved in the conversion of microtubule slid- mechanism by which microtubule sliding is converted into fla- ing into flagellar bending, but not microtubule sliding itself. In gellar bending. Interestingly, microtubule sliding alone is not addition, PKA activity is not required for mediating the cAMP sufficient to generate flagellar bending. Some other mechanism signal. The target of cAMP may be the Epac2/Rap2 pathway regulating the coordinated sliding is required to generate fla- and the GTP-binding form of Rap2 may be involved in regu- gellar bending. lating flagellar bending. To produce flagellar bending to generate the propulsive force, individual microtubule sliding must be converted into References the coordinated sliding of microtubules. According to the ‘‘switch-point’’ hypothesis, the active bundles of the dynein [1] Lindemann, C.B. and Gibbons, I.R. (1975) Adenosine triphos- arms switch, in an alternating fashion, from one side of the fla- phate-induced motility and sliding of filaments in mammalian gellum to the opposite side of the flagellum [35,36]. The coor- sperm extracted with Triton X-100. J. 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