Hyperactivated Motility 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 flagellum) 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.