Hyperactivated is Induced by Reagents Depressing the Function of Calcium in Mouse Sperm

Fugaku AOKI1, Senkiti SAKAI1 and Kaoru KOHMOTO1,2

1 Graduate School of Agriculture and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8756, Japan 2 Faculty of Veterinary and Animal Science, Nihon Veterinary and Animal Science University , Musashino-shi 180-8602, Japan

(Received December 10, 1998; Accepted January 19, 1999) Abstract To investigate the role of Ca2+ on the regulatory mechanism of hyperactivation, mouse sperm were treated with a calcium ionophore, ionomycin and a calmodulin inhibitor, W-13, and were analyzed for swimming pattern and flagellar bending. On the successive frames of photographs, the linearity of swimming and the bend angle were measured. Both reagents induced a non-linear swimming pattern as is observed in spontaneously hyperactivated sperm . However, changes in flagellar bending occurred in a different manner. After the occurrence of hyperactivation, the bend angle in the midpiece region was increased to the same direction as the curve of hook-shaped heads. Treatment with W-13 or ionomycin plus EGTA also increased the bend angle to that direction, whereas ionomycin plus Ca2+ increased it to opposite direction. The analysis for asymmetry of flagellar bending also revealed that the treatment with W-13 increased asymmetry to the same direction as the curve of head as is observed in spontaneously hyperactivated sperm and that ionomycin plus Ca2+ increased it to opposite direction. These results suggest that the depression of function of machinery controlled by Ca2+ is involved in the mechanism by which the sperm begin hyperactivated motility. Animal Science Journal 70 (2): 53-60, 1999 Key words: Sperm, Hyperactivation, Flagellar bending, Ca2+

Mammalian sperm undergo a change in the pattern increased Ca2+ concentrations induced asymmetry of of motility, termed hyperactivation, during the course flagellar bending in the demembranated sea urchin of capacitation33). The hyperactivated sperm in- sperm2,7,21) and that calmodulin is involved in the crease the amplitude and asymmetry of flagellar bend- regulation of asymmetry in flagellar bending in rat16,17) ing and reduce the linearity of swimming5,8,9,25,27). and sea urchin3) sperm. However, our recent work in Although the mechanism of hyperactivation is not mouse and hamster sperm showed that the flagellar known, many reports suggest that the increase of bending in hyperactivated sperm was asymmetric to intracellular free Ca+2 concentration ([Ca2+]i) is in- the same direction as the curve of their hook-shaped volved in the occurrence of hyperactivation: heads1). Lindemann and Goltz15) reported that the hyperactivation did not occur in mouse5,6,20)or guinea increase of flagellar curvature to that direction was pig sperm33) in the medium without Ca2+, and sperm induced by decreasing the concentration of Ca2+ in treated with calcium ionophore showed asymmetric the demembranated rat sperm. flagellar bending and non-progressive movement To clarify the role of Ca2+ in the regulatory mech- resembling those in hyperactivated sperm of anism of hyperactivated motility, we examined the mouse4, 26),ram23), hamster28) and guinea pig28). Con- effect of calcium ionophore and calmodulin inhibitor sistent with these results are the observations that on the flagellar bending in mouse sperm. Corresponding: Fugaku AOKI (fax: +81 (0) 3-5800-6988, e-mail: aokif@ hongo.ecc.u-tokyo.ac.jp)

Anim. Sci. J. 70 (2): 53-60, 1999 53 AOKI, SAKAI and KOHMOTO

active analogue, N-(4-aminobutyl)-2-naphthalene sul- Materials and Methods fonamide (W-12), were purchased from Seikagaku Sperm preparation Kogyo Co. (Tokyo). Stock solutions of W-12 and Male ICR mice were killed by cervical dislocation. W-13 (10mM) were diluted to 1mM with medium The cauda epididymides were removed and placed in and 20μl of each diluted solution was added to 180μl the medium (pH 7.4) consisting of 114mM NaCl, 2.7 sperm suspensions. After incubation for 5min, the mM KCl, 1.8mM CaCl2, 0.49mM MgCl2, 0.36mM sperm were photographed for analysis of motility. NaH2PO4, 25mM NaHCO3, 20mM HEPES, 5.5mM Analysis for swimming pattern and flagellar bending glucose, 0.1mM Na pyruvate, 100U/ml penicillin, 0.1 of sperm mg/ml streptomycin and 3mg/ml bovine serum albu- Swimming pattern and flagellar bending of sperm min (BSA). In the experiment using ionomycin, were analyzed by using successive frames of photo- CaCl2 was omitted and the concentration of BSA was graphs. Sperm samples for analysis were prepared as reduced to 0.3mg/ml. The sperm were released into follows. A 12-μl volume of sperm suspension was the medium by incising the epididymides longitu- placed on a glass slide and covered with an 18×24mm dinally and allowed to disperse for 5min at 37℃. To cover slip. The surrounding of cover slip was cov- obtain motile sperm, the sperm suspension was passed ered with a mounting agent (Diatex, Matsunami Inc., through a glass beads column19). The fraction of Tokyo). The glass slides and cover slips had been motile sperm was diluted with the medium to give a coated with poly glutamine-lysine (D-glutamine: D- concentration of 2×106 sperm/ml and incubated at lysine copolymer, 6:4; Sigma) to protect sperm 37℃. from sticking to the glass surface24). The sperm Treatment with Ca2+ ionophore samples thus prepared were photographed under a The procedure of treatment with calcium ionophore phase contrast microscope equipped with heating is similar to that described by Suarez et al.27) except stage set at 37℃. Five successive frames of photo- that ionomycin was used instead of A23187. A stock graphs were taken for each sample at a rate of 5 solution of ionomycin (5mM), dissolved in ethanol, frames/sec in an exposure of 1/2,000sec, was diluted with medium. A 10μl volume of diluted In analysis of swimming pattern, the swimming ionomycin (20μM) plus CaCl2 (36mM) or EGTA (2 track of head-midpiece junction of sperm was traced mM) was added to 190μl of sperm suspension, and 5 sequentially. To assess the linearity of swimming, min later 50μl of 50mg/ml BSA was added. BSA is the linear index (LI) was measured as follows. LI thought to bind to ionophore22) and decreases its con- was derived as the net velocity (Vn) divided by the centration to reduce the toxicity to sperm after long curvilinear velocity (Vc) after Vn and Vc were deter- time incubation23,26) After 5min of incubation, the mined by measuring the distance between the first and sperm were photographed for analysis of motility. In last position of head-midpiece junction, and the sum the experiment for ionomycin plus EGTA, the sperm of the distance between adjacent head-midpiece junc- were observed 5min after the addition of the reagents tions on five successive frames, respectively, per unit without subsequent addition of BSA. In the control time. experiment, 10μl medium containing 36mM CaCl2 In analysis of flagellar bending, the curvature of and 0.2% ethanol instead of ionomycin was added to midpiece region (the first 18μm of ) was the sperm suspension. To observe the hyperactivated measured using protractor (θmid). The bend in the motility that spontaneously occurred in control same direction as the curve of head was given a medium, the sperm were incubated for 80min after positive value and opposite direction was negative addition of BSA. value. Treatment with calmodulin inhibitor The asymmetry of flagellar bending was determined A calmodulin inhibitor, N-(4-aminobutyl)-5- as the previous work1). Since flagellar waveform chloro-2-naphthalene sulfonamide (W-13), and its in- consists of several circular bends connected with

Anim. Sci. J. 70 (2): 53-60, 1999 54 Regulation of Hyperactivated Motility by Ca2+

straight regions, the angle, the position of center and averaged to give the values of θp and θR for P-bend

the direction were determined for each bend in analy- and R-bend, respectively. To assess the degree of

sis for flagellar bending. Lines tangent to either side asymmetry, the value of θR-θP tivas calculated. of bend were drawn and the angle between lines was Results measured. The center position of the bend was deter- mined on the flagellum as the point on which the Changes in swimming pattern after treatment with bisector of the angles of bend crossed over. To locate calcium ionophore and calmodulin inhibitor each bend on the flagellum, the C-J distance, defined In the presence of Ca2+, the treatment with as the distance of center position of the bend (C) from ionomycin reduced the linearity of swimming. As head-midpiece junction (J), was measured by curvi- shown in the previous report26), the sperm reduced meter (Koizumi Inc., Tokyo). The direction of bend their motility to only vibratory movement within 5 was determined by using the asymmetry of hook- min after addition of ionomycin plus Ca2+ and then shaped heads as an indicator. The bend which resumed active motility after subsequent addition of curved to the same direction as the curve of the head BSA. These sperm swam in irregular trajectories and was defined as reverse bend (R-bend) and opposite their activities appeared to be slightly retarded. In direction was principal bend (P-bend) as described by these sperm, the values of Vc, Vn and LI were Wooley32). Since in some sperm the flagella twisted significantly lower than those in control sperm (Table and changed the bending plane in the distal region, the 1). When ionomycin was used together with EGTA, bend which occurred in the distal region should be the sperm did not lose their motility without addition excluded from analysis to correctly determine the of BSA, but appeared to rather increase their activities angles and direction of the bends. Therefore, only and swam in irregular trajectories as is observed in the bends in which the C-J distances were less than 75 hyperactivated sperm. The value of LI was

μm (about two third of the total length of the fla- significantly lower than that in control sperm. In gellum) were analyzed. The data of the bend angles, hyperactivated sperm which had been incubated for measured separately for P-bend and R-bend, were 80min, the value of LI was also significantly lower grouped in accordance with the C-J distance and than that in control sperm.

Table 1. Swimming pattern of sperm treated with ionomycin and W-13

Vc: curvilinear velocity; Vn: net velocity; LI: linear index. a,b The sperm were incubated for 5min in control medium containing 1.8mM Ca2+ and then BSA (final concentration: 10mg/ml) was added as described in Materials and Methods. After incubation for 5min (a) or 80 min (b), the sperm were examined for swimming speed. After incubation for 5min in control medium, the sperm were examined for swimming speed. d Values are expressed as means±standard deviations . * Significantly different from corresponding controls (*: P<0.05, **: P<0.01, by Student's t test).

Anim. Sci. J. 70 (2): 53-60, 1999 55 AOKI, SAKAI and KOHMOTO

Treatment with W-13 reduced the linearity of swim- sperm treated with W-12. ming as well as with ionomycin. Immediately after The analysis for asymmetry of flagellar bending addition of W-13, the sperm showed the pattern of showed that the treatment with ionomycin increased movement like hyperactivated sperm: they appeared the asymmetry of bending to the direction of P-bend. to move vigorously and swam in irregular trajectories. Although both of the values of θP and θR increased by After prolonged incubation more than 10min, a large treatment with ionomycin in almost all the C-J dis- part of sperm swam in a small circular trajectory in a tances, the degree of the increase in θP was higher than plane. However, W-12, an analogue of W-l3 with that in θR (Fig. 1). Therefore, the value of θR-θP in low calmodulin inhibiting activity, did not show any the sperm treated with ionomycin was lower than that appreciable effect on the swimming pattern. The in the control sperm in almost all the C-J distances value of LI was significantly reduced by treatment (Fig. 2). In the hyperactivated sperm, the degree of with W-13 when compared to that of control, but was asymmetry was also increased but the direction of not significantly reduced by W-12 (Table 1). asymmetry was opposite to that in the sperm treated Changes in flagellar bending after treatment with with ionomycin. After incubation for 80min, both calcium ionophore and calmodulin inhibitor θP and θR increased and the degree of increase in θR In control sperm incubated for 5min in the presence was higher than that in θP opposed for those in of Ca2+ and BSA, the value of θmid was negative ionomycin-treated sperm (Fig. 1). Therefore, the

(Table 2). After incubation for 80min, θmid was value of θR-θP increased (Fig. 2), indicating that the significantly increased to near zero. In the sperm asymmetry to the direction of R-bend increased in the treated with ionomycin plus EGTA, θmid was also hyperactivated sperm. The pattern of flagellar bend- significantly increased, whereas it was significantly ing in the sperm treated with W-13 was similar to that decreased in the sperm treated with ionomycin plus in hyperactivated sperm. The value of θR promi-

Ca2+. Treatment with W-13 significantly increased nently increased and that of θP slightly increased in the value of θmid as well as ionomycin plus EGTA, almost all the C-J distances (Fig. 3), which resulted in although no significant change was observed in the the increase in the value of θR-θP (Fig. 4). Although

Table 2. Measurement of flagellar curvature in midpiece region (θmid)

a,b The sperm were incubated for 5min in control medium containing 1.8mM Ca2+ and then BSA (final concentration: 10mg/ml) was added as described in Materials and Methods. After incubation for 5min (a) or 80min (b), the sperm were analyzed for flagellar bending. After incubation for c 5min in control medium, the sperm were analyzed for flagellar bending.

d Values are expressed as means±standard deviations. * Significantly different from corresponding controls (P<0 .01, by Student's t test).

Anim. Sci. J. 70 (2): 53-60, 1999 56 Regulation of Hyperactivated Motility by Ca2+

Fig. 2. Effect of calcium ionophore (ionomycin) on asymmetry of flagellar bending. The sperm were analyzed for flagellar bending 5min after addition of BSA (final concentration: 10mg/ml) following treatment with 1μM ionomycin (■) or 0.02%

ethanol as a control (●). Thc hyperactivated sperm

were analyzed 80min after addition of BSA (final Fig. 1. Effect of calcium ionophore (ionomycin) concentration: 10mg/ml) following treatment with on bend angle of flagellum. The sperm were O.02% ethanol (○). The bend angles measured analyzed for flagellar bending 5min after addition of separately for P-and R-bend, were averaged for each BSA (final concentration: 10mg/ml) following C-Jdistance and the value of θR-θP was determined treatment with 1μM ionomycin (■) or O.02% by subtraction of an averaged angle of P-bend (θP) ethanol as control (●). The hyperactivated sperm from that of R-bend (θR). were analyzed 80min after addition of BSA (final

concentration: 10mg/ml) following the treatment changes in flagellar bending occurred in a different with 0.02% ethanol (○). The bend angles, measured separately for P-and R-bend, were plotted manner among the treatments. Ionomycin plus versus C-J distance. For instance, the far left points EGTA and W-13 increased θmid and the asymmetry to in the figure indicate the average of angles of bends the same direction as the curve of head, whereas

whose centers locate at the position 0-15μm apart ionomycin plus Ca2+ decreased θmid and increased the from head-midpiece junction. All points shown are asymmetry to the opposite direction. averages of more than 7 data. Total number of 91, When considering the mechanism of hyperactiva- 90 and 77 figures of ionomycin-treated, control and tion, the direction of the changes in flagellar bending hyperactivated sperm, respectively, were analyzed on should be noted. It is well established that Ca2+ photographs which were taken from 3 independent affects the asymmetry of flagellar bending2,7,21) and experiments. exerts its effect at least in part via calmodulin3,16,17). the treatment with W-12 also increased the values of In the demembranated rat sperm, the bend angle of θR and θR-θP, the degree of increase was relatively low midpiece region in flagellum was a continuous func- when compared to that of W-13. tion of free calcium concentration ([Ca2+]) in the extraction buffer and the angle was increased to the Discussion direction of R-bend when [Ca2+] was decreased15). After treatment with ionomycin plus EGTA or W- It is possible in our study that the treatment with 13 as well as with ionomycin plus Ca2+, the sperm ionomycin plus Ca2+ increased the intracellular free showed a pattern of movement similar to those in calcium concentration ([Ca2+]i) to decrease θmid and hyperactivated sperm. All treatments rapidly induce the asymmetry to the direction of P-bend, that reduced the linearity of swimming. However, the ionomycin pflus EGTA decreased [Ca2+]; to increase

Anim. Sci. J. 70 (2): 53-60, 1999 57 AOKI, SAKAI and KOHMOTO

Fig. 4. Effect of calmodulin inhibitor (W-13) on asymmetry of flagellar bending. The sperm were analyzed for flagellar bending 5min after treatment with 0.1mM W-13 (○) or W-12 (■), or without

reagents as controls (●). The bend angles

measured separately for P- and R-bend, were Fig. 3. Effect of calmodulin inhibitor (W-13) on averaged for each C-J distance and the value of θR-θP bend angle of flagellum. The sperm were analyzed was determined by subtraction of an averaged angle for flagellar bending 5min after treatment with 0.1 of P-bend (θP) from that of R-bend (θR). mM W-13 (○) or W-12 (■), or without reagents as controls (●). Total number of 137, tal and 165 appreciable change in [Ca2+]i was observed around figures of the sperm treated with W-13, W-12 and the time of hyperactivation18). Thus, the change in without reagents, respectively, were analyzed on photographs which were taken from 4 independent [Ca2+]idoes not seem to be involved in the occurrence experiments. of hyperactivation. On the other hand, there are some indications that the function of calmodulin is θmid and that W-13 inhibited the function of reduced around the time of the onset of hyperactiva- calmodulin to increase θmid and induce the asymmetry tion. In bovine sperm, proteins bind to calmodulin to R-bend. Consistent with this interpretation is the and the binding decreases around the time of facts in the intact rat sperm that the treatment with capacitation11,12). In the presence of W-13, the de- procaine-HCl or 8-(N, N-diethylamino)-octyl-3,4, 5,- crease of the binding also occurred12),suggesting that trimethoxybenzoate, which can lower the [Ca2+]i,in- the sperm treated with W-13 in our experiment under- creased the curvature in midpiece region to the direc- went the events similar to those occurring in the tion of R-bend14). Such effects of these reagents were spontaneously hyperactivated sperm. In addition, also observed in mouse sperm (unpublished data). Leclerc et al.13) reported that the concentration of Since the hyperactivated sperm showed the increase in calmodulin decreased during the course of capacita- the curvature to the direction of R-bend, it is possible tion in bovine sperm. Such a slow decrease in the that hyperactivation is induced by the decrease of concentration of the proteins would explain why it [Ca2+]i or the reduction of function of machinery takes long time before the occurrence of hyperactiova- controlled by Ca2+. tion. Published results on the [Ca2+]i in the The role of extracellular Ca2+ on the mechanism of hyperactivated sperm of several species are contradic- hyperactivation is not clear, although Ca2+ is required tory. In the porcine sperm, [Ca2+]i increased around in the medium to induce of hyperactivation5,6,20,33). the time of the onset of hyperactivation35), whereas it It does not seem that the influx of extracellular Ca2+ decreased in hamster sperm31). 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