Regulations of Microtubule Sliding by Ca²⁺ and cAMP and Their Roles in Forming Flagellar Waveforms

Abstract

The function of Ca²⁺ and cAMP in extruding doublet microtubules from sea urchin sperm axoneme and generating flagellar waves was investigated in order to clarify the regulatory mechanism of microtubule sliding and the formation mechanism of beating patterns of cilia and flagella. Almost all potentially asymmetric spermatozoa that were demembranated with Triton in the absence of Ca²⁺ and reactivated with MgATP²⁻ (Gibbons, B.H. and Gibbons, I.R. (1980). J. Cell Biol., 84: 13–27), beat with planar waves closely resembling those of the intact spermatozoa, whereas potentially symmetric spermatozoa, in which axonemal calmodulin was removed by detergent extraction in the presence of millimolar Ca²⁺ (Brokaw, C.J. and Nagayama, S.M. (1985). J. Cell Biol., 100: 1875–1883), beat with three-dimensional waves if they were reactivated with low MgATP²⁻. At a high MgATP²⁻, almost all demembranated spermatozoa beat with planar waves. cAMP enhanced the three-dimensionality of the flagellar waves at a low Ca²⁺. These changes in the flagellar waves were caused by different regulations of the microtubule sliding by calcium, cAMP, and MgATP²⁻.

Introduction

Cilia and flagella have the same 9+2 axonemal structure, but they generate different beating patterns to properly perform various functions (Sleigh, 1974). Therefore, their different beating patterns must be formed by different behaviors of localized active sliding between the doublet microtubules in the axoneme, although little experimental evidence has been reported on the relationship between the beating pattern of cilia and flagella and the regulation of the microtubule sliding. A detailed analysis of the rotational movements of a spermatozoon from various animals revealed that there were various degrees of three-dimensionality in the flagellar beating and they had two different chiralities (Ishijima and Hamaguchi, 1992; Ishijima et al., 1992). These chiralities were regulated by the Ca²⁺ concentrations in the sea urchin sperm flagella (Ishijima and Hamaguchi, 1993). Furthermore, different chiralities of ciliary beating have been reported; namely, the cilia of the Paramecium living in freshwater generate left-handed waves while those of the Mytilus living in seawater generate right-handed waves (Machemer, 1977; Ishijima et al., 1992). No difference in their function seems to be found between these chiralities. In contrast, nodal cilia have a counterclockwise rotation when viewed from the base (Hirokawa et al., 2006) and this rotational movement produces a unidirectional nodal flow that triggers the L-R axis determination in the mammalian development. Careful observations of the effect of increased viscosity on the flagellar waves of tunicate and sea urchin spermatozoa revealed that the planar waves on the sperm flagella convert into the helical waves in seawater with an increased viscosity (Woolley and Vernon, 2001; Ishijima, 2012). This conversion seems to be related to the Ca²⁺ and cAMP in the cell (Ishijima, 2012).

It is well known that Ca²⁺ and cAMP change the ciliary and flagellar beating. Ca²⁺ increases the asymmetry of the flagellar waves of the spermatozoa from both invertebrates and mammals (Brokaw et al., 1974; Gibbons and Gibbons, 1980; Lindemann and Goltz, 1988; Ishijima and Hamaguchi, 1993; Ishijima et al., 2006). On the other hand, the effect of cAMP on the sperm motility is rather complicated; that is, cAMP triggers the sperm motility (Morisawa and Okuno, 1982) and enhances the flagellar beating (Lindemann, 1978; Tash et al., 1984; Kinukawa et al., 2006), but does not change the percentage of motile spermatozoa and the beat frequency (de Lamirande et al., 1983; Ishijima and Witman, 1987; Ho et al., 2002). To resolve this apparent conflict among these studies and to clarify the effect of Ca²⁺ and cAMP on the ciliary and flagellar beating and the regulation of microtubule sliding, the effect of Ca²⁺ and cAMP on the behavior of microtubule sliding and their roles in forming flagellar waves were examined in the present study.

Materials and Methods

Sperm preparation

Concentrated spermatozoa of the sea urchin Hemicentrotus pulcherrimus were obtained by intracoelomic injection of 1.0 mM acetylcholine dissolved in artificial seawater (Jamarin U, Jamarin Laboratory, Osaka, Japan) and placed in a plastic culture dish (35 mm×10 mm) kept in a refrigerator until used. A 20 μl sample of spermatozoa was diluted with 0.2 ml Ca²⁺-free artificial seawater.

Demembranation and reactivation

Demembranation and reactivation of the sea urchin spermatozoa were similar to those previously reported (Ishijima et al., 1996). To remove the sperm plasma membrane with Triton X-100 and millimolar calcium (potentially symmetric condition, under this condition some axonemal calmodulin was removed, Brokaw and Nagayama, 1985), 10 μl of the sperm suspension was placed in a well of a 24-well tissue culture plate containing 0.25 ml of extraction solution (0.15 M potassium acetate, 10 mM Tris buffer, 1 mM DTT, 0.2 mM EGTA, 0.05% (w/v) Triton X-100, 2 mM MgSO₄, and 2 mM CaCl₂, pH 8.2). The suspension was then gently stirred for approximately 30 seconds after which time 10 μl of the mixture was transferred to another well containing 0.25 ml of reactivation solution (0.25 M potassium acetate, 10 mM Tris buffer, 1 mM DTT, 2 mM EDTA or EGTA, and various concentrations of MgSO₄, CaCl₂, and ATP to obtain the desired Ca²⁺ or MgATP²⁻ concentrations without changing the concentrations of the other species in the solutions, pH 8.2). In the experiments to examine the effect of cAMP, various concentrations of cAMP were added to the reactivation solution. To remove the sperm plasma membrane with Triton in the absence of Ca²⁺ (potentially asymmetric condition), 10 μl of the sperm suspension was placed in a well of a 24-well tissue culture plate containing 0.25 ml of extraction solution (0.15 M potassium acetate, 10 mM Tris buffer, 1 mM DTT, 2 mM EGTA, 0.05% (w/v) Triton X-100, and 1 mM MgSO₄, pH 8.2). The suspension was then gently stirred for approximately 30 seconds after which time 10 μl of the mixture was transferred to another well containing 0.25 ml of the reactivation solution. Using this method, the percentage of motility in reactivated preparations was more than 95% (95.1±1.4%, mean of 728 sperm in five different experiments).

Sliding disintegration

Sliding of the doublet microtubules was induced as follows. Approximately 20 μl of the reactivated sperm suspension was transferred to a glass slide in an approximately 0.12 mm deep trough formed using a bilayer of transparent mending tape (Scotch Magic Tape No. 810, 3 M Corp., St. Paul, MN, USA) attached to the slide in two parallel strips; the trough was then covered with a glass coverslip. Microtubule sliding was achieved by applying a small volume of the reactivation solution containing 20 μg/ml elastase (E-0127; Sigma Chemical Co., St. Louis, MO, USA) to one end of the chamber while the excess fluid was drained from the opposite end with small pieces of filter paper. Exposure of the demembranated spermatozoa to MgATP²⁻ and elastase caused microtubule sliding disintegration after a certain period of beating of the reactivated sperm flagella. Therefore, the waveform of the reactivated sperm flagella and the pattern of microtubule sliding disintegration were examined using the same sperm flagella. The percentage of sliding disintegration of the sperm flagella was close to 100% (97.7±2.1%, mean of 693 sperm in five different experiments).

Recording and data analysis

The movement of the sperm flagella was observed and recorded using a Nikon Eclipse E600 microscope (Nikon Corp., Tokyo, Japan) equipped with a phase contrast condenser and a 40x BM objective. The disintegration of the axoneme was observed using dark field microscopy. The obtained images were directly stored on disk by a computer using a Panasonic CCD video camera (WV-BL 730, Matsushita Communication Industrial Co., Ltd., Yokohama, Japan) at the rate of 60 images per second. The shutter speed was 1/1000 s for the phase contrast microscopy and 1/100 s for the dark field microscopy. The beat frequency was determined from the period required for one complete propagation of the planar waves at the lower frequencies or by matching the strobe at the higher frequencies. All experiments and measurements were done at 20°C.

Results and Discussion

Effects of Ca²⁺ and cAMP on sliding disintegration of the axoneme

The pattern of microtubule sliding extruded from the sea urchin sperm axoneme was highly dependent on the Ca²⁺ and cAMP concentrations (Fig. 1). When the sea urchin spermatozoa were demembranated with Triton in the absence of Ca²⁺ (potentially asymmetric condition) and then they were exposed to MgATP²⁻ and elastase, a bundle of doublet microtubules slid off the sperm axoneme regardless of the Ca²⁺, cAMP, and MgATP²⁻ concentrations (Fig. 1A). On the other hand, the number of bundles of doublet microtubules extruded from the sperm axoneme increased with the decreasing Ca²⁺ concentrations in the reactivation buffer when the spermatozoa were demembranated with Triton and millimolar Ca²⁺ (potentially symmetric condition) (Fig. 1B and C). Furthermore, the axonemal disruption by the microtubule sliding occurred in different loci on the flagellum (Fig. 1C). This effect by Ca²⁺ diminished at low MgATP²⁻ concentrations (less than approximately 20 μM), where many bundles of the doublet microtubules were always observed (data not shown). cAMP enhanced the microtubule sliding at a low Ca²⁺ (Fig. 1D). These findings indicated that only a specific pair of doublet microtubules is allowed to slide under the potentially asymmetric condition, whereas almost all doublet microtubules are capable of sliding under the potentially symmetric condition (Table I). In fact, there are various reports on the pattern of the extrusion of the doublet microtubules; that is, the doublet microtubules of the axoneme independently slide (Summers and Gibbons, 1971; Brokaw, 1989; Ishijima et al., 1996; Nakano et al., 2003) and a bundle of doublet microtubules is extruded from the axoneme (Sale, 1986; Ishijima et al., 1996).

Fig. 1

Dark field micrographs showing effects of Ca²⁺ and cAMP on the microtubule sliding. The microtubule sliding was induced with 0.5 mM MgATP²⁻ and elastase. (A) The microtubule sliding of the potentially asymmetric spermatozoa. All disintegrated flagella (103 examples observed) broke into two bundles of doublet microtubules at 10⁻⁷ M Ca²⁺. (B–D) The microtubule sliding of the potentially symmetric spermatozoa. (B) Almost all disintegrated flagella (92.8±3.9%, mean±S.D. from 142 flagella in four different experiments) broke into two bundles of doublet microtubules at 10⁻⁴ M Ca²⁺. (C) All disintegrated flagella (115 examples observed) broke into more than two bundles of doublet microtubules at 10⁻⁹ M Ca²⁺. (D) All disintegrated flagella (152 examples observed) broke into more than two bundles of doublet microtubules at 10⁻⁹ M Ca²⁺ and 0.3 mM cAMP. Scale bar, 10 μm.

Table I Relationship between disintegration pattern of the sperm flagella and flagellar waveform under various concentrations of Ca²⁺, cAMP, and MgATP²⁻

Condition	Concentration		10−4 M Ca2+	10−7 M Ca2+	10−9 M Ca2+	10−9 M Ca2+ +0.3 mM cAMP
Potentially asymmetric	MgATP2−	Higha	2, 2Db	2, 2D	2, 2D	2, 2D
		Low	2, 2D	2, 2D	2, 2D	2, 2D
Potentially symmetric	MgATP2−	Higha	2, 2D	≥3, 2Dd	≥3, 2D	≥3, 2De
		Low	≥3, 3Dc	≥3, 3Dd	≥3, 3D	≥3, 3D

a  Approximately more than 20 μM MgATP²⁻ (see text for details).

b  2, 2D: 2, two bundles disintegration of the sperm flagella and 2D, planar waveform.

C  ≥3, 3D: ≥3, more than two bundles disintegration of the sperm flagella and 3D, three-dimensional waveform.

d  Data not shown.

e  Almost all spermatozoa beat with planar waves, but some spermatozoa beat with three-dimensional waves (see text for details).

The effect of Ca²⁺ on the microtubule sliding of the sperm axoneme has been previously observed using light and electron microscopes (Ishijima et al., 1996). Furthermore, similar effects of Ca²⁺ and ATP on the short segments of the potentially symmetric sperm axonemes have been reported (Nakano et al., 2003), although these authors examined neither the potentially asymmetric sperm axonemes nor the difference occurred in different loci on the axoneme.

Effects of Ca²⁺, cAMP, and MgATP²⁻ on flagellar waveform

Intact spermatozoa of the sea urchin H. pulcherrimus beat with asymmetrical planar waves and never generated the helical waves (Ishijima, 2012). All spermatozoa (85 examples observed) demembranated under the potentially asymmetric condition also beat with planar waves when they were reactivated with MgATP²⁻ regardless of the Ca²⁺, cAMP, and MgATP²⁻ concentrations (Fig. 2, Table I). On the other hand, all spermatozoa (103 examples observed) demembranated under the potentially symmetric condition beat with three-dimensional waves when they were reactivated with a low MgATP²⁻ (less than approximately 20 μM; beating of approximately 8 Hz) regardless of the Ca²⁺ and cAMP concentrations (Fig. 3, Table I), although Ca²⁺ and cAMP affected the detailed waveforms (Fig. 3). Especially, cAMP increased the three-dimensionality of the flagellar waves in a dose dependent manner at a low Ca²⁺ (Fig. 3C). However, the reactivation of the potentially symmetric spermatozoa with a high MgATP²⁻ produced planar waves (Fig. 4, Table I). In this case, at high cAMP and low Ca²⁺ concentrations, the three-dimensional waves were sometimes observed (9.4±3.2%, mean±S.D. from 68 sperm in four different experiments, Fig. 4C, Table I) probably because cAMP also increased the amount of sliding between all doublet microtubules and thus enhanced the three-dimensionality of the flagellar waves, and it broke the planar waves sustained by the rapid beating.

Fig. 2

Phase contrast micrographs of the planar waves of the potentially asymmetric spermatozoa. The spermatozoa were attached to the coverslip by their heads. Almost all parts of the flagellum are in focus. (A) The spermatozoa were reactivated with 5 μM MgATP²⁻ and 10⁻⁷ M Ca²⁺. The reactivated spermatozoa beat at 1.8±0.5 Hz (mean±S.D. from 18 sperm in three different experiments). (B) The sperm were reactivated with 5 μM MgATP²⁻, 10⁻⁹ M Ca²⁺, and 0.3 mM cAMP. (C) The sperm were reactivated with 0.5 mM MgATP²⁻ and 10⁻⁷ M Ca²⁺. The reactivated spermatozoa beat at 32.6±3.9 Hz (mean of 18 sperm in three different experiments). The time interval between successive images is 150 ms in (A), 170 ms in (B), and 15 ms in (C). Scale bar, 10 μm.

Fig. 3

Phase contrast micrographs of the three-dimensional waves of the potentially symmetric spermatozoa. The spermatozoa were reactivated with 5 μM MgATP²⁻ and attached to the coverslip by their heads. Several parts of the flagellum are out of focus. (A) Free-Ca²⁺ concentration was adjusted to be 10⁻⁴ M. Left-handed three-dimensional waves were determined by differential focusing; namely, the flagellar parts are focused on the lower focal plane. (B) Right-handed three-dimensional waves (the flagellar parts are focused on the lower focal plane) at 10⁻⁹ M Ca²⁺. (C) At 10⁻⁹ M Ca²⁺ and 0.3 mM cAMP. The time interval between successive images is 120 ms in (A) and (C) and 270 ms in (B). Scale bar, 10 μm.

Fig. 4

Phase contrast micrographs showing effects of Ca²⁺ and cAMP on the potentially symmetric sperm flagella waves. The spermatozoa were reactivated with 0.5 mM MgATP²⁻ and attached to the coverslip by their heads. (A) Free-Ca²⁺ concentration was adjusted to be 10⁻⁴ M. (B) 10⁻⁹ M Ca²⁺. (C) The spermatozoa usually beat with planar waves, but sometimes beat with three-dimensional waves at 10⁻⁹ M Ca²⁺ and 0.3 mM cAMP. The time interval between successive images is 15 ms. Scale bar, 10 μm.

The three-dimensional waves had different chiralities at different Ca²⁺ concentrations (Fig. 3A and B). The right-handed waves were dominant at 10⁻⁴ M Ca²⁺ (78.4±3.9%, mean of 110 sperm in five different experiments) and the left-handed waves at 10⁻⁹ M Ca²⁺ (69.6±4.3%, mean of 126 sperm in five different experiments). The existence of two chiralities of flagellar waves and its Ca²⁺ dependency were consistent with the data from the previous studies (Ishijima and Hamaguchi, 1993; Ishijima et al., 1996).

A comparative study of the flagellar beating of the spermatozoa from American and Asian horseshoe crabs revealed that the central pair complex is not necessary for beating but plays a role in forming the planar waves because the 9+0 axoneme of the Asian horseshoe crab sperm that lacks a central pair complex generates helical waves while the 9+2 axoneme of the American horseshoe crab sperm generates planar waves (Ishijima et al., 1988). A more precise mechanism for the conversion of microtubule sliding into flagellar waves can be discussed on the basis of the pattern of microtubule sliding observed in the current study. The potentially asymmetric sperm axoneme behaved as if the axoneme consisting of two bundles of doublet microtubules (Fig. 1A, Table I) generated the planar waves (Fig. 2). On the other hand, the potentially symmetric sperm axoneme behaved as if it consisted of many bundles of doublet microtubules (Fig. 1C and D, Table I) that generated the three-dimensional waves at a slow beating (Fig. 3, Table I). These mechanisms for the conversion of the microtubule sliding into flagellar waves were previously pointed out without experimental evidence; namely, the localized active sliding discontinuously switching from one group of doublets to another generates the planar waves (Brokaw, 2002; Fig. 5A and A′) while the localized sliding between the doublet microtubules continuously propagating along and around the axoneme generates the three-dimensional waves (Ishijima and Hamaguchi, 1993; Fig. 5B and B′). The present study also proved the three-dimensional flagellar waves with different chiralities and their Ca²⁺ regulation. Therefore, the changes in the flagellar waves are caused by different regulations of the microtubule sliding by calcium, cAMP, and MgATP²⁻. Cilia and flagella, especially the cilia, must use these different patterns of microtubule sliding to generate complex three-dimensional waves by changing the Ca²⁺, cAMP, and MgATP²⁻ concentrations in the cell.

Fig. 5

Schematic diagrams explaining the relationship between the pattern of microtubule sliding and the flagellar waveform. Side views (A, B) and cross-sections (A′, B′) of the axoneme. The doublet microtubules are numbered individually from one to nine and c indicates the central pair microtubules. The sliding between doublets 7 and 8 (black arrows in A) induces the axonemal disintegration into two bundles of doublets (Sale, 1986), resulting in the planar waves because the bending direction (a white arrow in A′) is approximately parallel to the beating plane. On the other hand, the sliding between doublets 7 and 8 and, e.g., doublets 9 and 1 (black arrows in B) induces the axonemal disintegration into more than two bundles of doublets, resulting in the three-dimensional waves because the bending direction is not always parallel to the beating plane (white arrows in B′).

The planar waves of the sperm flagella are likely to be formed by a high level of calmodulin or rapid beating. Different types of evidence have been accumulated (Inaba, 2011; Gokhale et al., 2012), but further studies are needed to understand the role of calcium and calmodulin in activating the sliding between a specific pair of doublet microtubules. Furthermore, the planar waves were stable at a rapid beating. Experimental evidence supports this finding; the stiffness of the sea urchin sperm axoneme for bending in the plane perpendicular to the beating plane is more than twelve times greater than that for bending within the beating plane (Ishijima and Hiramoto, 1994), whereas that of the sea urchin motionless sperm flagella has at most a five times difference between the direction parallel to the beating plane and that perpendicular to it (Okuno and Hiramoto, 1979). Furthermore, the planar waves of the tunicate and sea urchin sperm flagella convert to the helical waves at a slow beating in high viscosity media (Woolley and Vernon, 2001; Ishijima, 2012). These facts suggest that even if the all doublet microtubules are capable of sliding each other, the microtubule sliding that is ineffective for rapid beating is skipped, so that the localized active sliding discontinuously switches from one group of doublet microtubules to another and thus forms the planar waves.

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