Activation of Mammalian Sperm Motility by Regulation of Microtubule Sliding Via Cyclic Adenosine 5'-Monophosphate-Dependent Phosphorylation'

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1 BIOLOGY OF REPRODUCTION 53, (1995) Activation of Mammalian Sperm Motility by Regulation of Microtubule Sliding Via Cyclic Adenosine 5'-Monophosphate-Dependent Phosphorylation' Yuming Si 2 and Makoto Okuno Department of Biology, College of Arts and Sciences, The University of Tokyo Komaba, Meguro-ku, Tokyo 153, Japan ABSTRACT Bicarbonate was found to be essential for activating live mouse sperm motility. The activated sperm flagella exhibited high beat frequency, high swimming velocity, and large principal and reverse bends. To gain further insight into the bicarbonate-triggered activation mechanism, the microtubule sliding characteristics of the activated versus the nonactivated sperm flagella were compared by use of demembranated sperm. We found that the effects of bicarbonate on live sperm were identical with the effects of camp on demembranated sperm both in microtubule sliding velocity and in sliding disintegration pattern. Furthermore, autoradiography revealed that the activation of mouse sperm motility was associated with camp-dependent phosphorylation of a 65-kDa flagellar protein. The results demonstrated that bicarbonate-triggered activation of mouse sperm motility was closely coupled with the regulation of microtubule sliding via campdependent phosphorylation. INTRODUCTION Mammalian spermatozoa are produced in testes and then transferred to epididymides for maturation and storage. It has been considered that most mammalian spermatozoa in the epididymal fluids are mature but nonactivated [1-3]. The activation of motility of epididymal spermatozoa would not take place unless the spermatozoa were mixed with male accessory gland secretions at the time of ejaculation, or diluted into a buffer solution containing activating factor, depending on species. In hamsters and mice, the live spermatozoa taken from caudal epididymis exhibit little motility when they are diluted into an isotonic sucrose solution. However, when calcium (for hamsters) or bicarbonate (for mice) is added to the sucrose solution, the spermatozoa are activated and exhibit a vigorous motility [1, 3]. Calcium and bicarbonate, however, are not effective for the activation of demembranated immotile hamster and mouse spermatozoa, respectively [3, 4]. The effects of calcium and bicarbonate on activation of live sperm motility supposedly occur through stimulation of intracellular camp synthesis from ATP via activation of sperm adenylate cyclase [1, 3, 5, 6]. In other animals, camp, as an intracellular second messenger, has been reported to play a pivotal role in governing sperm motility activation [7-9]. It is generally assumed that camp-induced activation of flagellar or ciliary motility in many animals has arisen from phosphorylation of some "motility-related proteins" by the camp-dependent protein kinase [10-14]. However, the question of how the protein phosphorylation leads to sperm Accepted May 24, Received March 8, 'This work was supported by scholarships from the people of China and the Ishizaka Foundation (to Y.S.), and by a Grant-in-Aid from the Ministry of Education, Science and Culture ofjapan (to M.O.). Y.S. is a postdoctoral fellow of thejapan Society for the Promotion of Science. 2Correspondence. FAX: motility activation and bending wave generation or propagation remains unanswered. To explore further the relationship between camp-dependent phosphorylation and sperm motility activation, we examined the sliding properties of microtubules in demembranated spermatozoa. Our study shows the change in sliding properties of microtubules during the transition from nonactivated to activated mouse spermatozoa and demonstrates that camp activates sperm motility through regulating microtubule sliding. In addition, a "motility-related" camp-dependent phosphoprotein is also reported. Reagents MATERIALS AND METHODS ATP, camp, EGTA, DTT, and trypsin were purchased from Sigma Chemical Co (St. Louis, MO), and [y- 32 P]ATP (3000 Ci/mmol) was from Du Pont-New England Nuclear (Boston, MA). Other chemicals were of reagent grade from Wako Pure Chemicals Co. Ltd. (Osaka, Japan). Observation of Live Sperm Movement in Sucrose and Bicarbonate Solutions Caudal epididymides from sexually mature ICR mice were excised and kept in an incubator at C until use. Thirty to 40 Al of mineral oil was placed on a glass slide laid on a thermostatically controlled warm plate that was set on the microscope stage to keep the temperature constant (37 + 1C). Twenty microliters of a sucrose solution containing 310 mm sucrose, 1 mm EGTA, and 10 mm HE- PES-NaOH (ph 7.4 at 37 1 C), or a bicarbonate-containing sucrose solution (hereafter referred to as bicarbonate solution) consisting of 290 mm sucrose, 1 mm EGTA, 10 mm NaHCO 3, and 10 mm HEPES-NaOH (ph 7.4 at 37 ±+ 1C) was injected into the center of the oil drop. Then, a very 1081

2 1082 SI AND OKUNO TABLE 1. The motility parameters of live mouse spermatozoa in sucrose and bicarbonate solutions (mean + SD, n = 10-30). Sliding velocity + Solution PB* (rad) RB* (rad) (pm/s) Sucrose Bicarbonate 1.83 ± ± *PB, maximal bend angle of the principal bend. RB, maximal bend angle of the reverse bend (see Figure 1E and F). +The sliding velocity of microtubules in live sperm flagella is estimated based on the formula: V = 2df (O + Or), where V represents microtubule sliding velocity, d represents the distance between adjacent doublet microtubules [341, f represents the maximal beat frequency, and 0, and 8, representthe bend angles of the maximal principal and reverse bends, respectively. small amount of spermatozoa was taken from the epididymis and injected into the sucrose or the bicarbonate solution. A cover glass was placed on the slide immediately, and sperm movement was observed and recorded within 5 min through use of a phase contrast microscope (Optiphoto; Nikon, Tokyo, Japan) equipped with a stroboscopic illumination (Chadwick #236 and #271; Tokyo, Japan) and a video system (NEC T125A camera [Tokyo, Japan] and Panasonic NV-DS1 VTR [Tokyo, Japan]). Preparation of Mitochondria-Free Spermatozoa The sperm plasma membrane and the mitochondrial sheaths (MS) were removed with Triton X-100 and dithiothreitol (DTT) according to the method of Lindemann and Gibbons [15] with minor modifications [16]. Namely, one volume of the live spermatozoa in the sucrose or the bicarbonate solution was transferred to 9 volumes of an extraction solution, consisting of 200 mm sucrose, 25 mm glutamic acid, 25 mm KOH, 1 mm DTT, 0.1% (w/v) Triton X-100, and 20 mm Tris- HCl buffer (ph 9.0), and incubated for 5 min at room temperature. Then, one volume of the extracted sperm suspension was transferred for perfusion to 9 volumes of a working solution containing 200 mm sucrose, 25 mm glutamic acid, 25 mm KOH, and 20 mm Tris-HCl buffer (ph 7.9). Measurement of Microtubule Sliding Velocity One drop of the Triton-DTT extracted sperm suspension was injected into a perfusion chamber on a glass slide that was laid on a thermostatically controlled warm plate ( C) on the microscope stage. The chamber was made by supporting a coverslip on strips of double-sided Scotch (3M, St. Paul, MN) tape. The sperm head that attached to the surface of the glass slide was focused with a 40 X phase contrast objective, and the chamber was perfused with the working solution supplemented with 2 jig/ml trypsin and various concentrations of Mg-ATP. The sliding disintegration process of microtubules was recorded on video tape. Velocity of microtubule sliding disintegration was determined by measuring the change in length of microtubules extruding from the axoneme per second on thermal paper from a video copy processor (Mitsubishi SCT-P61; Tokyo, Japan). Flagellar Preparation To obtain activated sperm flagella, caudal epididymal spermatozoa were diluted (1:200) into the bicarbonate solution at 37 1C. The spermatozoa were activated and allowed to swim in the solution for 5-10 min; then those spermatozoa in the upper portion of the solution were taken and pelleted by centrifugation at 4000 rpm for 5 min. The pellet was diluted (1:20) with the working solution supplemented with protease inhibitors, 2 mm PMSF, and 20 g/ ml leupeptine. To obtain nonactivated sperm flagella, caudal epididymal spermatozoa were directly diluted (1:20) into the working solution supplemented with 2 mm PMSF and 20 pg/ml leupeptine. The activated and nonactivated sperm suspensions were homogenized at 0 C with strokes of a Teflon-glass homogenizer to dissociate sperm head from tail. The homogenates were diluted (1:10) with the working solution containing 0.5 mm PMSF and 5 jg/ml leupeptine and subjected to centrifugation at 1500 rpm for 5 min to remove sperm heads. The sperm flagella in the supernatant were demembranated with the extraction solution and then pelleted at 4000 rpm for 5 min. The pelleted flagella were diluted into the working solution with a final protein concentration of 1.2 mg/ml [17]. SDS-PAGE and Autoradiography The procedure for investigating the incorporation of inorganic phosphate into nonactivated and activated spermatozoa was based on that of Morisawa and Hayashi [11]. Briefly, 200 jil (about 250 g of protein) of nonactivated and activated demembranated flagellar suspensions was incubated with 1 mm MgSO 4, 0.5 mm ATP, and 5 Ci [y- 32 P]ATP (3000 Ci/mmol), either with or without 50 M camp at room temperature for 30 sec, respectively. The phosphorylation reactions were terminated by adding trichloroacetic acid (TCA) to a final concentration of 10%. The TCA precipitates were extracted twice with ice-cold ethanol before being dissolved into SDS sample buffer. Samples were subjected to SDS-PAGE on 5% stacking and 15% separating gels [18]. For autoradiography of y 3 2 P-labeled proteins, the Coomassie Brilliant blue-stained gels were dried and then exposed to Kodak XAR-5 film (Eastman-Kodak, Rochester, NY) backed with Cronex (Wilmington, DE) intensifying screens for 36 h at C. RESULTS Bicarbonate-Induced Change in Flagellar Bending Waveform of Live Spermatozoa Caudal epididymal spermatozoa diluted into the sucrose solution exhibited a sluggish motility with an average swimming velocity and beat frequency of 13.5 pm/sec and 1.5 Hz [3], respectively, during the first 5 min upon dilution. The bending waveforms of sperm flagella in the sucrose

3 MICROTUBULE SLIDING 1083 FIG. 1. Comparison of flagellar bending waveforms of live mouse spermatozoa in sucrose and bicarbonate solutions (see Materials and Methods). A and B) Maximal principal and reverse bends during one beat cycle in middle piece of sperm flagella diluted in sucrose solution. C and D) Maximal principal and reverse bends in bicarbonate solution. E and F) Measurement of principal and reverse bend angles in middle piece. Ta and Tb, tangents drawn at concave sides of sperm head and a point 40 pm from the connecting piece. Op between Ta and Tb in E, principal bend angle. Or between Ta and Tb in F, reverse bend angle. Maximal Op and Or are obtained by tracing successive sperm movements from video records (60 frames/sec). Bend angle is represented as rad = tro/180 (0 in degree). Pictures from video records. Temperature, 37 1 C. Bar, 20 m. solution are shown in Figure 1, A and B, which shows that the distal part of the flagellar principal piece produced oscillatory bending with a large amplitude, whereas the middle piece and the proximal part of the principal piece provided bending with a small amplitude. The maximal bend angles of the principal bend (Fig. 1A) and reverse bend (Fig. 1B) in the middle piece were on average 1.29 and 1.11 rad, respectively (Table 1). In contrast, the epididymal spermatozoa diluted into the bicarbonate solution exhibited vigorous motility, with an average swimming velocity and beat frequency of 181 gm/sec and 11 Hz [3], respectively. The sperm flagella exhibited a large amplitude initiated and propagated from the proximal part of the middle piece distally without attenuation, as shown in Figure 1, C and D. The maximal bend angles of the principal bend (Fig. C) and the reverse bend (Fig. D) in the middle piece were on average 1.83 and 1.51 rad, respectively (Table 1). Cyclic AMP-Induced Microtubule Sliding in Nonactivated Demembranated Spermatozoa Epididymal spermatozoa diluted into sucrose solution were demembranated, denuded of MS, and exposed to trypsin and Mg-ATP as described in Materials and Methods. Within 20 sec of Mg-ATP perfusion, the microtubule sliding disintegration began with the extrusion from the axoneme

4 1084 SI AND OKUNO FIG. 2. Microtubule sliding disintegration patterns in mouse spermatozoa. Microtubule sliding disintegration pattern in: A) sucrose-treated spermatozoa, B) bicarbonatetreated spermatozoa, and C) sucrose-treated spermatozoa incubated with camp. Successive micrographs in each series from top to bottom in A, B, and C were taken at intervals of 0.25 sec from video record. D) End of disintegration in sucrose-treated spermatozoa. E) End of disintegration in bicarbonate-treated spermatozoa. Arrowheads indicate fibrous sheathes. Note that in mammalian spermatozoa, microtubules disintegrated together with their corresponding outer dense fibers. Temperature, 37 1C. Bar, 10 im. of one or two doublet microtubules together with their outer dense fibers, which looped out at the annulus as shown in Figure 2A. Then other microtubules, usually one or two, extruded sequentially from the axoneme in the same way. In many cases, the microtubules in the proximal region of middle piece were not able to disintegrate from the axoneme and were kept within the axonemal bundle even at the end of sliding disintegration (Fig. 2D). The microtubules in bicarbonate-activated sperm flagella exhibited a different sliding disintegration pattern, as shown in Figure 2B. One microtubule extruded from the axoneme preferentially and looped out at the connecting piece; this was followed by sequential extrusion of other microtubules in the same way. In addition, the headward sliding of the fibrous sheath through the axoneme to the middle piece associated with microtubule extrusion was observed in the activated sperm flagella, as shown in Figure 2E. This interesting finding has been presented elsewhere [16, 19]. The results described above suggested that bicarbonate was an extracellular factor responsible for changing the microtubule sliding disintegration pattern. Evidence that bicarbonate stimulated adenylate cyclase activity resulting in production of camp has been provided in mouse spermatozoa [20]. We therefore considered that the effect of bicar-

5 _Y _ MICROTUBULE SLIDING 1085 bonate on the change in the microtubule sliding disintegration pattern was due to the effect of intracellular camp. To substantiate this speculation, we investigated the direct effect of camp on the microtubule sliding disintegration pattern, using demembranated spermatozoa. Sucrose-treated spermatozoa were demembranated and incubated with 50 gm camp and 1.0 mm ATP before removal of MS and then denuded of MS and exposed to trypsin and Mg-ATP. Microtubules exhibited the same sliding disintegration pattern as those observed in the bicarbonate-activated spermatozoa; namely, microtubules extruded at the connecting piece (Fig. 2C), and the fibrous sheath also slid to the middle piece as in Figure 2E (data not shown). Meanwhile, when bicarbonate-activated spermatozoa were subjected to the extrusion test in the presence of camp, microtubules showed the same extrusion patterns as those observed in Figure 2, B and E (data not shown). Increase in Microtubule Sliding Velocity by camp Observing the microtubule sliding disintegration in nonactivated and activated sperm flagella, we also noticed that microtubule sliding velocities of sperm flagella were considerably different between activated and nonactivated types. Figure 3 shows the double reciprocal plots of microtubule sliding velocity and Mg-ATP concentration. The maximal sliding velocity ( V,,) and apparent Michaelis constant (K,) for Mg-ATP were calculated from the linear regression line of the plots. The V,, and K, of sucrose-treated spermatozoa were 8.3 jtm/sec and 0.04 mm, respectively. However, when sucrose-treated spermatozoa were incubated with camp, the Vm,, and K, changed to 14.2 gm/sec and 0.19 mm, respectively, which were almost equivalent to the V,,m (16.6 m/sec) and the Km (0.2 mm) obtained from the bicarbonate-activated sperm flagella E.q 0.2 0, 0.1 I/Mg-ATP [mm - 1 ] 4 - ' i 4i~ 5 1U FIG. 3. Double reciprocal plots of sliding velocity of microtubules against Mg-ATP concentrations (mean + SD, n = 15-20). Triangles, results obtained from sucrosetreated sperm flagella. Closed circles, results obtained from sucrose-treated sperm flagella incubated with camp. Open circles, results obtained from bicarbonatetreated sperm flagella. Temperature, 37 1C. that the bicarbonate-induced increase in intracellular camp introduced phosphorylation of a 65-kDa protein endogenously and activated sperm motility in vivo. Further phosphorylation of the 65-kDa protein in vitro by exogenous camp did not take place when endogenous phosphorylation was accomplished by intracellular camp in sperm flagella. Other proteins appearing below 50 kda (lanes A and C) were considered to have no direct correlation with activation of sperm motility, since they were phosphorylated by exogenous camp in vitro following in vivo activation of sperm motility. Identification of the Functional Target of camp-induced Activation of Sperm Motility Since camp has been known as a protein kinase activator that catalyzes the phosphorylation of a substrate protein, we investigated the incorporation of inorganic phosphate into the nonactivated and activated sperm flagella, using [y- 32 P]ATP to identify the functional target of camp-induced activation of sperm motility. As shown in lane C of Figure 4, when sucrose-treated spermatozoa were incubated with [y- 32 P]ATP in the presence of camp, the phosphorylation occurred in several proteins. In particular, a 65-kDa protein (indicated by an arrow) was strongly phosphorylated in the presence of camp. On the other hand, when spermatozoa were activated in vivo with use of bicarbonate and were incubated with [y- 32 P]ATP in the presence of camp, the 65- kda protein did not phosphorylate (Figure 4, lane A) compared with the protein in lane C. These results suggested FIG. 4. Autoradiogram showing camp-dependent phosphorylation of flagellar proteins in mouse sperm flagella. Lanes A and B: Bicarbonate-treated flagella were incubated for 30 sec with labeled ATP in presence (A) and absence (B) of camp. Lanes C and D: Sucrose-treated flagella were incubated for 30 sec with labeled ATP in presence (C) and absence (D) of camp. Molecular mass standards are shown (in kda) on right.

6 1086 SI AND OKUNO DISCUSSION The estimated sliding velocities of microtubules in live spermatozoa were on average 0.26 m/sec and 3.60 um/ sec in the sucrose and bicarbonate solutions, respectively (Table 1), which were much lower than those in the corresponding disintegrating flagella (Fig. 3). The lower sliding velocity of microtubules in live flagella than that in disintegrating flagella was also reported in sea urchins and Chlamydomonas [21, 22]. One possible explanation for the lower sliding velocity is that the load imposed upon microtubules in live sperm flagella was larger than that imposed upon microtubules in disintegrating ones because of the loss of bending resistance by digestion with trypsin. Another interpretation is that trypsinization contributed directly to the increase in microtubule sliding velocity and induced microtubule sliding disintegration, as reported in sea urchin and trout spermatozoa [23, 24]. However, under the same condition of demembranization and trypsinization, the sliding velocity of microtubules in nonactivated mouse sperm was lower than that in camp-activated sperm (Fig. 3), indicating that the effect of camp on the increase in microtubule sliding velocity could not be duplicated by trypsin, as discussed below. In the present study, we observed that the sucrosetreated sperm flagella showed smaller principal and reverse bends in the middle piece than did the bicarbonate-activated ones. The small bends were assumed to result either from the stiffness of flagellum itself or from the weak bending force. The force-velocity relationship obtained both from actin-myosin [25] and tubulin-dynein [26] systems indicated that the higher the sliding velocity, the lower the force. The high sliding velocity in the activated sperm flagella suggested that the active bending force was not presumedly larger than that in the nonactivated ones. Thus, it seemed likely that the smaller bends in the nonactivated flagella arose from greater stiffness rather than from a weak bending force. The question, then, was how the stiffness was produced. One assumption is that the increase in stiffness is a result of some inter-doublet cross-bridge formation such as by dynein arms, which increases the secondary moment of the cross section of the axoneme [27]. In addition, the cross bridges supposedly offer resistance to microtubule sliding. As a result, the midpiece microtubules were not able to disintegrate from the axoneme in the nonactivated spermatozoa unless they were activated with bicarbonate extracellularly or with camp intracellularly. It is possible that bicarbonate-induced production of intracellular camp released the flagella from the stiff state via phosphorylation of the 65-kDa protein. One integrated hypothesis for explaining both the increase in microtubule sliding velocity and the change in disintegration pattern is that the 65-kDa protein is closely associated with dynein, e.g., is one component of dynein and regulates the dynein-based cross-bridge formation. If this is the case, it could be assumed that the mechano-chemical cycles of ATP hydrolysis by dynein were accelerated by shortening the time when dynein was attached to the adjacent doublet microtubules after the protein phosphorylation. The acceleration of ATP hydrolysis cycles was supposed to provide both the increase in the sliding velocity and the decrease in the stiffness of flagella due to a decrease in cross bridges between the doublet microtubules [27]. In Paramecium, a 29-kDa protein was shown to control, as a regulatory light chain of 22S dynein, the rate of microtubule translocation by changes in its phosphorylation state [28]. The interrelation between the 65-kDa phosphoprotein of mouse spermatozoa and dynein will be investigated in a future study. Mammalian sperm flagella contain some accessory cytoskeletal components that encircle the axoneme [16, 291. To determine whether the 65-kDa phosphoprotein was really an axonemal component, we investigated by autoradiography the localization of the protein in the sperm flagella. Nonactivated and activated sperm flagella were treated with a low concentration of SDS and DTT to selectively dissolve the axoneme [30]. Phase-contrast microscopy and SDS-PAGE showed that the axoneme was dissolved (dynein and tubulin were not seen), but the outer dense fibers and fibrous sheath remained (data not shown). When this fibrous sheath complex and these outer dense fibers were subjected to a camp-dependent phosphorylation assay, the phosphorylation of the 65-kDa protein was not observed. Thus, it is likely that the 65-kDa protein is an axonemal component, although an alternative possibility, that the protein is a component of the outer dense fibers or fibrous sheath and therefore sensitive to SDS and DTT digestion, could not be ruled out. Mouse caudal epididymal spermatozoa were nonactivated, probably because of the low concentration of bicarbonate in epididymis (2-4 mm) [6]. During ejaculation, the spermatozoa are activated by the high concentration of bicarbonate (20 mm) mainly from the seminal vesicles [6]. When spermatozoa are ejaculated to the female reproductive tracts, they are exposed to higher concentrations (35-90 mm) of bicarbonate [31]. High concentrations of bicarbonate might be essential for sperm hyperactivation [32], a phenomenon characterized by vigorous, large-amplitude, and whiplash-like flagellar beating [33]. On the basis of the present experiments, it is likely that the large amplitude of the hyperactivated sperm flagella was also derived from increased flexibility in the middle piece of the flagella by bicarbonate-induced production of intracellular camp. In conclusion, the present study demonstrated that bicarbonate triggered the production of intracellular camp, and the camp-dependent phosphorylation of a 65-kDa flagellar protein, assumed to be an axonemal component, was responsible for activation of mouse sperm motility through regulation of microtubule sliding.

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