Proc. Natl. Acad. Sci. USA Vol. 90, pp. 4660-4664, May 1993 Cell Intracellular calcium increases with hyperactivation in intact, moving hamster sperm and oscillates with the flagellar beat cycle (indo-1) SUSAN S. SUAREZ*, STEPHEN M. VAROSI, AND XIAOBING DAI Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610-0144 Communicated by Henry A. Lardy, February 4, 1993 (receivedfor review November 21, 1992)

ABSTRACT At some time before fertilization, mammalian (7, 8). The [Ca2+]1i increase is initiated by an influx of sperm undergo a change in movement pattern, termed hyper- extracellular Ca2+, apparently via an L-type voltage- activation. There is evidence that hyperactivation offers an activated calcium channel (9). The mouse sperm acrosome- advantage to sperm for detaching from the oviductal mucosa, reaction receptor has been identified as a galactosyltrans- for penetrating viscoelastic substances in the oviduct, and for ferase that is activated by the glycoprotein penetrating the zona pellucida. Hyperactivation is known to ZP3 (10). On the other hand, all that is known about hyper- require extracellular calcium, but little else is known about the activation is that it may be induced by calcium ionophore mechanisms by which calcium affects sperm movement. The A23187 (11, 12) and reversed by the removal of ionophore or calcium-sensitive fluorescent dye indo-1 was used to follow extracellular calcium (1, 11) and that demembranated sperm intracellular calcium levels ([Ca2+]O) in individual moving exhibit hyperactivated flagellar-bending patterns in solutions sperm. Sperm were loaded with 10 IAM of the acetoxymethyl containing 1 mM Ca2+ (13). On the basis of this information, ester form of the dye and then rinsed. The dye was excited at it has been assumed that a rise in [Ca2+]i initiates and, 340 nm by using a fitered xenon stroboscope, and images at the perhaps, also maintains hyperactivation. Hyperactivation 405-nm and 490-nm excitation maxima were simultaneously and the can occur independently (14); digitized at 30 per sec for 2.1 sec. [Ca2+], was significantly therefore, either the two events are driven by different higher in the acrosomal and postacrosomal regions of the head calcium regulatory pathways and/or they are separated by and in the flagellar midpiece (the principal piece could not be subcellular compartmentalization. The latter hypothesis is measured) in hyperactivated than in nonhyperactivated sperm possible for sperm because the acrosome reaction occurs on (P < 0.0001). [Ca2+]J oscillations were detected in the proximal the rostrum of the head and hyperactivation occurs in the half of the midpiece that were identical in frequency to the of these highly polarized cells. flagellar-beat-cycle frequency in 12 of 17 hyperactivated sperm The following experiments were done to determine (median, 3.5 Hz). Rapid [Ca2+]J oscillations were also detected whether hyperactivation entails an [Ca2+]1 increase limited to in the acrosomal and postacrosomal regions, as well as in the the region of the sperm flagellum and whether [Ca2+]i in distal midpiece. Oscillations were not elininated by dampening intact, moving sperm is related to flagellar bending. The cellulose. The oscil- calcium-sensitive fluorescent probe indo-1 was used to mon- the flagellar bending with methyl [Ca2+1, itor [Ca2+], in individual moving sperm, using a high- lations detected in sperm are significantly more rapid than temporal-resolution dual-image capture and analysis system oscillations detected in other cell types. developed for this purpose. Extracellular calcium ions regulate mammalian fertilization by participating in several events, including sperm- EXPERIMENTAL PROCEDURES hyperactivation, the acrosome reaction, sperm/egg fusion, Loading Sperm with Indo-1. Sperm were collected from the and egg activation (1). For sperm, millimolar levels of extra- caudal epididymides of sexually mature Syrian Golden ham- cellular calcium are required for two processes that precede sters, as described (15). These sperm were suspended in a fertilization, motility hyperactivation and the acrosome re- medium containing 110 mM NaCl, 5 mM KCI, 2.4 mM CaCl2, action. Flagellar movement is activated when mature sper- 0.49 mM MgCl2, 0.36 mM NaH2PO4, 24.9 mM NaHCO3, 5 matozoa are released from the caudal epididymis. Hyperac- mM glucose, 6.26 mM lactate, 0.125 mM pyruvate, 1.2% tivation (Fig. 1A) occurs sometime after insemination into the bovine serum albumin (fraction V; Calbiochem), and 0.006% female reproductive tract and has been associated with penicillin G (pH 7.5, 290-310 mOsm). Hypotaurine (100 ,uM), attaining the capacity to fertilize in vitro (2, 3). This hyper- epinephrine (1 ,uM), and penicillamine (20 ,uM) were added to activation entails an increase in flagellar-bend amplitude and the medium just before each experiment, according to the flagellar-beat asymmetry, similar in pattern to the chemotac- method of Bavister (16). Sperm numbers were adjusted to 5 tic response ofsome marine invertebrate spermatozoa to eggs x 106 sperm per ml, and the sperm were loaded with 10 ,.M (4). The acrosome reaction involves the exocytotic release of of indo-lAM (Molecular Probes) for 40 min at 37°C. Extra- enzymes that aid in the penetration of the zona pellucida and cellular dye was removed by centrifugation for 5 min at 135 prepare the sperm plasma membrane for fusion with the x g. Sperm were resuspended in medium to a concentration oocyte plasma membrane (5, 6). of 2 x 106 sperm per ml. The sperm were incubated an More is known about the role of Ca2+ in the acrosome additional 20 min at 37°C to allow deesterification of the reaction than in hyperactivation. The reaction has been indo-lAM to its calcium-sensitive form indo-1. Complete shown to involve an increase in intracellular calcium con- deesterification was confirmed by quenching the fluorescent centration ([Ca2+i) downstream from the receptor-mediated signal with Mn2+ (17). There was no detectable signal in the activation ofa guanine nucleotide inhibitorfactor-like protein presence of Mn2+ or in the absence of Mn2+ and indo-1;

The publication costs of this article were defrayed in part by page charge Abbreviations: [Ca2+]i, intracellular calcium concentration; FCR, payment. This article must therefore be hereby marked "advertisement" flagellar curvature ratio. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

4660 Downloaded by guest on October 2, 2021 Cell Biology: Suarez et al. Proc. Natl. Acad. Sci. USA 90 (1993) 4661

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FIG. 2. The digital-imaging and analysis system for determining [Ca2+]i in sperm using fluorescent probe indo-1. (a) Xenon strobo- scope, 2.5 J per pulse (modified Chadwick Helmuth Strobex 8490, El Monte, CA). (b) Quartz collector lens on Zeiss Axiovert microscope. (c) Thirty-nanometer-wide 340-nm-bandpass excitation filter (all filters and dichroic mirrors are from Omega Optical, Brattleboro, FIG. 1. (A) Patterns of activated (at left) and hyperactivated (at VT). (d) Three hundred and sixty-nanometer dichroic mirror right) movement in hamster sperm. The principal bend of the (360DCLP). (e) Zeiss Achrostigmat oil-immersion objective (x40/ flagellum is indicated by a solid line, and the reverse bend is indicated 1.3 numerical aperture). (f) Specimen on inverted slide with no. 1.5- by a dashed line. (B) The four regions ofhamster sperm analyzed for thick glass coverslip. (g) Dichroic mirror (455 nm) in Nikon Multi [Ca2+]i: acrosomal (a), postacrosomal (pa), proximal midpiece (pm), Image Module. (h) Thirty-five-nanometer-wide 405-nm-bandpass and distal midpiece (dm). (C) Flagellar curvature ratio (FCR), filter. (i) Twenty-two-nanometer-wide 490-nm-bandpass filter. (j) defined as the straight-line distance in ,um from the head/midpiece Gen II microchannel plate image intensifiers (Dage-MTI, Michigan junction to the first inflection point along the flagellum (dashed line), City, IN). (k) Charge-coupled device cameras (modified Dage model those two points, CCD 72). (1) Intensifier and camera controls. (m) Host processor divided by the curvilinear distance between (486) with 16 megabyte video memory in four digitizing boards (Epix measured along the flagellum. The more sharply curved the flagel- Silicon Video Mux, Northbrook IL). Custom software was by lum, the lower the FCR. Elektron Systems and S. Varosi). therefore, the signals analyzed are mainly attributable to the response of indo-1 to [Ca2+]i. mined according to the method of Tsien (22). Imaging System. A xenon stroboscope was used as the FCR. Measurement of FCRs (2) was made from the digi- fluorescence excitation source to limit sperm exposure to UV tized images (Fig. 1C). This measure was found to be more light and to provide sharp images of rapidly moving flagella precise (repeatable) than flagellar amplitude because the (Fig. 2). Sixty-four sequential paired images of 15-,usec flagellar bend does not form a perfect sine wave, especially exposures were collected and digitized at 30 Hz. [Ca2W]i was in hyperactivated sperm. estimated for four regions of the sperm (Fig. 1B). The fluorescent emission for each region was measured by placing 1 a box over the region and setting a threshold to eliminate extracellular signals. A second threshold was set to delimit the background signals surrounding the image within the box region (Esimage; Elektron Systems, St. Petersburg, FL). The corrected mean fluorescent signal (F) was derived by sub- tracting the mean background signal within the box from the Co mean signal within the region of the sperm. The fluorescence ratio (R) was calculated as F2/F1, where F1 was obtained t -l from the image collected at 490 nm and F2 was obtained from a the image collected at 405 nm. [Ca2+]i was calculated by using the formula [Ca2+], = KdB[(R-Rmin)/(Rma,-R)], where Kd is the dissociation constant for indo-1 and Ca2+, and B = -2 Fi,min/Fi,max (18). B and Kd were determined for the head and midpiece regions by adding bromo-A23187 to sperm in solu- log measured Ca2+ tions of known (19-21). Only R could be determined [Ca2W] FIG. 3. The image-analysis system produces a linear relationship for each individual sperm, whereas the other variables were between measured Ca2+ and known Ca2+ for the acrosomal region of determined for each experimental condition. the head (a) and the flagellar midpiece (v). The postacrosomal region The analysis system produced a linear relationship within yielded the same regression line as the acrosomal region; the principal the physiological range of [Ca2+], for solutions of CaCl2 in and end pieces ofthe flagellum were too narrow to measure accurately. indo-1 (regression R2 = 0.98) and also for sperm treated with The nonfluorescent ionophore bromo-A23187 was used to equilibrate the nonfluorescent ionophore bromo-A23187 in known solu- extracellular Ca2+ concentration with [Ca2+]i in solutions of known tions of EGTA-buffered Ca2+ (Fig. 3); [Ca2W]i was deter- Ca2+ buffered by EGTA. Each point represents the mean of 10 sperm. Downloaded by guest on October 2, 2021 4662 Cell Biology: Suarez et al. Proc. Natl. Acad. Sci. USA 90 (1993) Table 1. [Ca2+]i in nM for five experiments [Ca2+]j, nM Acrosomal region Postacrosomal region Proximal midpiece Distal midpiece Exp. Activated Hyper Activated Hyper Activated Hyper Activated Hyper 1 41 ± 10.0 132 ± 47.7 41 ± 13.2 217 ± 75.6 11 ± 8.4 162 ± 37.7 17 ± 7.9 185 ± 39.4 2 37 ± 9.0 122 ± 50.4 34 ± 11.7 219 ± 112.1 5 ± 3.1 181 ± 68.2 9 ± 5.6 197 ± 54.6 3 39 ± 7.8 153 ± 60.1 38 ± 8.6 280 ± 72.4 7 ± 4.5 172 ± 51.3 14 ± 6.6 214 ± 64.7 4 35 ± 7.9 107 ± 35.4 27 ± 8.1 206 ± 62.9 6 ± 3.1 159 ± 46.3 10 ± 4.5 192 ± 43.7 5 41 ± 9.3 109 ± 40.9 39 ± 11.2 225 ± 53.6 9 ± 4.4 132 ± 44.6 12 ± 6.1 167 ± 44.2 Mean 38.5 ± 9.05 124.6 ± 49.70 35.7 ± 11.63 229.2 ± 80.72 7.7 ± 5.48 161.2 ± 52.34 12.5 ± 6.74 190.9 ± 51.49 Twenty activated and 20 hyperactivated sperm were analyzed per experiment (Exp.). One image, the one in which FCR was at its minimum, was analyzed per sperm. [Ca2+]i is significantly higherforhyperactivated sperm thanforactivated sperm in all cell regions (ANOVA, P < 0.0001). Hyper, hyperactivated sperm. RESULTS AND DISCUSSION was identical to the frequency of the flagellar beat cycle (median frequency, 3.5 Hz; range, 2-4.5 Hz). Peak frequen- [Ca2+], was higher in both the head and tail regions of cies of [Ca2+]i oscillations in the acrosomal region, postac- hyperactivated than activated sperm (Table 1). rosomal region, and distal midpiece were identical to the The scatter of the [Ca2+]i data observed in all experiments led us to suspect that [Ca2+], oscillations could be occurring. Low-frequency oscillations (-1 per min) were first investi- o1000 gated because these were most often reported to exist in 0 nonexcitable cells (23-25). Paired images of individual hy- x 800 peractivated sperm were recorded at 4-sec intervals for a - period of 2 min. It was possible to accomplish this because u 600 hyperactivated hamster sperm swim in circles. No evidence could be found for such oscillations (data not shown). 'z 400 Next, high-frequency oscillations, on the order of the

I length ofthe beat cycle, were investigated. Paired images (64 + 200 per sperm) were collected at rates of 30 images per sec over U several beat cycles (2 sec). Oscillations in [Ca2+]i were apparent on the time plots ofthe data (Figs. 4 and 5). Fourier decomposition of the data into component oscillations re- 0 vealed that for 12 of 17 hyperactivated sperm, the peak 0 frequency of oscillations in the proximal flagellar midpiece x - A '0 800, - - 0 700 - 600 ' c

+ r 400- cU 300 h

+a

U4 0 I 2 Time, sec FIG. 5. (A) [Ca2+]j oscillations (low-amplitude tracing) and flagel- lar-bend oscillations (high-amplitude tracing) in a hyperactivated Time (sec) sperm. The slight dips in center of each FCR peak represent reverse bends. (B) [Ca2+]1 oscillations (lower tracing) and flagellar-bend 4. FIG. [Ca2+]i oscillations in four regions of a sperm. oscillations (upper tracing) in an activated sperm. In this case, [Ca2+]j were taken every 1/30th sec for 2.13 sec. Peak frequencies for [Ca2+]i oscillations are clearly seen for both principal and reverse bends. (C) oscillations are identical for all four regions, as determined by [Ca2+]i oscillations (lower tracing) and flagellar-bend oscillations analysis of Fourier decomposition of data. (A) [Ca2+]i oscillations in (upper tracing) in a hyperactivated sperm in 1.5% methyl cellulose. acrosomal and postacrosomal regions. (B) [Ca2+]j oscillations in Although the principal bends are highly dampened and the reverse proximal and distal midpiece. Amplitudes of the oscillations are bends are completely dampened, [Ca2+]i peaks that correspond with greater in the distal midpiece. both can be seen. Downloaded by guest on October 2, 2021 Cell Biology: Suarez et al. Proc. Natl. Acad. Sci. USA 90 (1993) 4663 flagellar-beat-cycle frequency for 6 of 17, 7 of 17, and 6 of 17 spermatozoa, respectively, and, thus, were not as closely tied to the flagellar beat as in the proximal midpiece. In the hyperactivated sperm, secondary frequency peaks could also be detected at double the frequency of the primary peaks for 12 of 17 sperm (median, 7 Hz), indicating that [Ca2+]i oscillates with the development of both the principal and reverse flagellar bends (Fig. 5). The flagellar beat frequencies are lower than previously reported by us (15); however, this difference was determined to be from the use of shallower chambers, which were required to keep the head and mid- piece in focus for [Ca2+]i determinations. Images derived from the paired images by calculating ratios on a pixel-by- pixel basis revealed consistent development of a [Ca2+]j peak during the reverse bend and frequent development of a peak during the principal bend (Fig. 6). Peak [Ca2+]i oscillation frequencies in the proximal mid- piece that were equal to the flagellar beat frequency were only detected for 8 of22 activated sperm; however, this result may have been due to decreased resolution. The activated sperm flagella beat considerably faster than the hyperacti- vated sperm flagella (median, 10 Hz; range, 5-14 Hz) and had lower average [Ca2+]j levels throughout the beat cycle. Furthermore, amplitudes of the oscillations were only on the order of tens of nM instead of hundreds of nM (Fig. 5). A few analyzable sets of images were collected at 60 Hz that gave stronger indication of oscillations because of increased tem- poral resolution (Fig. SB); however, the amplitude of the pulse from the xenon stroboscope had to be halved to collect images at this rate, and this subsequently reduced the bright- ness of the fluorescent signals and the measurement accu- racy. To determine whether the oscillations might be artifactual changes in the signal ratio produced by the bending of the sperm, [Ca2+]i was measured in hyperactivated sperm swim- ming in 1.5% methyl cellulose. The minimum FCR was dampened in these sperm from a range of 0.13-0.52 (median, 0.32; n=100) to a range of 0.56-0.99 (median, 0.95). In four out of five sperm, the Fourier-determined peak [Ca2+]1 os- cillation was the same as the beat frequency, and the range of the [Ca2+]i fluctuation was still =200 nM (Fig. SC). The rapid oscillations detected in could be attrib- [Ca2+]i FIG. 6. Sequential pseudocolor images of [Ca2+]i taken from uted to rapid opening and closing of calcium channels or to moving, intact activated (at left) and hyperactivated (at right) sperm rapid binding and release of calcium intracellularly by calci- loaded with the fluorescent calcium probe indo-1. The principal piece um-binding proteins. There is now strong evidence for the ofthe tail is not shown. Images were captured at 30 per sec in 15-ILsec existence of L-type calcium channels in sperm, channels that exposures. As the flagellar bend develops, [Ca2+]i increases through- open by membrane depolarization. These channels have been out the sperm. The approximate [Ca2+]j, based on ionophore treat- implicated in induction of the acrosome reaction in sperm at ment of sperm in known concentrations ofcalcium, is illustrated next fertilization (9). It is possible that sperm also possess other to the color spectrum. The signal intensity for each pixel on the sperm voltage-activated channels present in excitable cells, such as was averaged with its left and right neighbor before the ratio of the N- or T-type channels, that open and close more rapidly than two images was produced in order to reduce noise; however, there L-type channels (17). In combination with Ca2+-ATPase was no averaging of entire images. (26-28) and possibly a Na+/Ca2+ antiporter (29) in sperm that cell types, there would be a third possibility for the observed pump Ca2+ out ofthe cell (26-28), rapidly opening and closing and release vesic- channels could account for the rapid oscillations. The second [Ca2+]i oscillations-i.e., rapid uptake by possibility is that the oscillations represent the binding and ular intracellular stores. In sperm, however, there are no release of Ca2+ from calcium-binding proteins. Sperm have reticula or vesicles. an unusually small cytoplasmic compartment; therefore, Because increased [Ca2+]i levels were detected after hy- calcium-binding proteins may produce detectable effects on peractivation in the head of sperm as well as in the flagellum, the cytoplasmic Ca2+. The calcium-binding protein calmod- initiation of the acrosome reaction may require still higher ulin has been localized by antibodies to the inner surface of levels of [Ca2+]j. In preliminary experiments, additional the outer dense fibers ofthe tail and beneath the acrosome on [Ca2+]1 increases were detected in hyperactivated sperm in the head (30, 31), where it might produce the oscillations which the acrosome reaction was induced by lysophosphati- observed. Other calcium-binding proteins resembling calpac- dylcholine or zona pellucida components (data not shown); tin (32), calsequestrin (33), and calreticulin (34) have been therefore, [Ca2+]i can increase beyond the levels attained found in mammalian sperm. This possibility, which presumes during hyperactivation. The higher levels of [Ca2+]j might be that the [Ca2W]i oscillations are the result of flagellar beat required by calcium-binding proteins forming the signal- initiations rather than the cause, would explain why demem- transduction cascade that produces the acrosome reaction. branated sperm beat normally in the continual presence of The L-type channels implicated in the acrosome reaction may constant amounts ofCa2+ in the medium (13, 35, 36). In other stay open long enough to reach the threshold required for the Downloaded by guest on October 2, 2021 4664 Cell Biology: Suarez et al. Proc. Natl. Acad. Sci. USA 90 (1993) reaction, whereas the channels involved in hyperactivation 5. Yanagimachi, R. (1988) in The Physiology of Reproduction, may only raise the calcium enough to increase flagellar eds. Knobil, E. & Neill, J. (Raven, New York), pp. 135-185. bending. Conversely, although [Ca2+]i increases associated 6. Wassarman, P. M. (1987) Science 235, 553-560. with hyperactivation may not initiate acrosome reactions, the 7. Florman, H. M., Tombes, R. M., First, N. L. & Babcock, [Ca2W]i spike associated with the acrosome reaction appar- D. F. (1989) Dev. Biol. 135, 133-146. movement. 8. Wilde, M. W., Ward, C. R. & Kopf, G. S. (1992) Gamete Res. ently affects flagellar Hyperactivated hamster 31, 297-306. sperm in which the acrosome reaction has been induced 9. Florman, H. M., Corron, M. E., Kim, T. D. H. & Babcock, exhibit greater flagellar-bend amplitude and asymmetry than D. F. (1992) Dev. Biol. 152, 304-314. uninduced hyperactivated sperm (37), which infers that the 10. Miller, D. J., Macek, M. B. & Shur, B. D. (1992) Nature [Ca2W]i increase that occurs during the acrosome reaction (London) 357, 589-593. also affects flagellar function. 11. Suarez, S. S., Vincenti, L. & Ceglia, M. W. (1987) J. Exp. The patterns of activation and hyperactivation resemble Zool. 244, 331-336. the two swimming patterns of some marine invertebrate 12. Suarez, S. S., Dai, X. B., DeMott, R. P., Redfern, K. & sperm as they respond to chemotactic signals from eggs (4, Mirando, M. A. (1992) J. Androl. 13, 75-80. 38). There have been some reports of chemotaxis in human 13. Lindemann, C. B. & Goltz, J. S. (1988) Cell Motil. Cytoskel. 10, 420-431. sperm (39, 40), but the phenomenon has not been demon- 14. Yanagimachi, R. (1981) in Fertilization and Embryo Develop- strated to be widespread in mammalian sperm. Regardless of ment in Vitro, eds. Mastroianni, L. & Biggers, J. D. (Plenum, whether hyperactivation is a form ofchemotactic response in New York), pp. 81-182. mammalian sperm, the underlying mechanisms regulating 15. Suarez, S. (1988) Gamete Res. 19, 51-65. this asymmetrical flagellar-movement pattern in marine in- 16. Bavister, B. D. (1989) Gamete Res. 23, 139-158. vertebrates and mammals are similar. Demembranated mod- 17. Tsien, R. W. & Tsien, R. Y. (1990) Annu. Rev. Cell Biol. 6, els of sea urchin and mammalian sperm respond in similar 715-760. fashion to calcium (13, 41). 18. Grynkiewicz, G., Poenie, M. & Tsien, R. Y. (1985) J. Biol. Although the role of hyperactivation in chemotaxis is Chem. 260, 3440-3450. 19. Owen, C. S., Sykes, N. L., Shuler, R. L. & Ost, D. (1991) debatable for mammalian sperm, there is evidence that this Anal. Biochem. 192, 142-148. movement pattern offers other advantages to sperm in the 20. Wahl, M., Lucherini, M. J. & Gruenstein, E. (1990) Cell oviduct. Hyperactivated sperm penetrate viscous and vis- Calcium 11, 487-500. coelastic media more effectively than activated sperm (42, 21. Bancel, F., Salmon, J. M., Vigo, J. & Viallet, P. (1992) Cell 43). Thus, hyperactivation may offer an advantage to sperm Calcium 13, 59-68. for penetrating mucus that occupies the oviductal lumen and 22. Tsien, R. Y., (1989) Methods Enzymol. 172, 230-262. the hyaluronate matrix of the cumulus oophorus. It has been 23. Devor, D. C., Ahmed, Z. & Duffey, M. E. (1991) Am. J. calculated that hyperactivation increases the amount of Physiol. 260, C598-C608. thrust that sperm can develop against the zona peliucida that 24. Berridge, M. (1990) J. Biol. Chem. 265, 9583-9586. 25. Miyazaki, S.-I., Yuzaki, M., Nakada, K., Shirakawa, H., surrounds the oocyte (44) and may, therefore, offer an Nakanishi, S., Nakade, S. & Mikoshiba, K. (1992) Science 257, advantage for penetration of the zona. In addition to increas- 251-256. ing the ability of sperm to penetrate various barriers to the 26. Babcock, D. F., First, N. L. & Lardy, H. A. (1976) J. Biol. oocyte, there is also evidence that hyperactivation aids sperm Chem. 251, 3881-3886. in remaining free of attachment to the oviductal wall and 27. Bradley, M. P. & Forrester, I. T. (1980) Cell Calcium 1, entrapment by mucosal folds in the oviduct (2, 45, 46). 381-390. Therefore, this phenomenon may have originally developed 28. Breitbart, H. & Rubinstein, S. (1983) Biochim. Biophys. Acta for locating the egg in species with external fertilization and 732, 464-468. then assumed new functions in species with internal fertili- 29. Bradley, M. P. & Forrester, I. T. (1992) FEBS Lett. 121, 15-18. 30. Gordon, M., Morris, E. G. & Young, R. J. (1983) Gamete Res. zation. 8, 49-55. In summary (Fig. 6), the indo-1 emission patterns indicate 31. Kann, M.-L., Feinberg, J., Rainteau, D., Dadoune, J.-P., that [Ca2+]i is increased in hyperactivated sperm, that the Weinman, S. & Fouquet, J.-P. (1991) Anat. Rec. 230, 481-488. increases occur throughout the sperm, and that [Ca2+]i os- 32. Berruti, G. (1988) Exp. Cell Res. 179, 374-384. cillates with the formation ofeach flagellar bend. The greatest 33. Berruti, G. & Porzio, S. (1990) Eur. J. Cell Biol. 52, 117-122. correlation between [Ca2+]i oscillation frequency and flagel- 34. Nakamura, M., Michikawa, Y., Baba, T., Okinaga, S. & Arai, lar-bending frequency was detected in the proximal flagellar K. (1992) Biochem. Biophys. Res. Commun. 186, 668-673. midpiece. Demembranated models of sperm have been used 35. Okuno, M. & Brokaw, C. J. (1981) Cell Motil. 1, 349-362. to ascertain that calcium can increase flagellar-bend ampli- 36. Tash, J. S., Krinks, M. Patel, J., Means, R. L., Klee, C. B. & tude our indicates that enters Means, A. R. (1988) J. Cell Biol. 106, 1625-1633. (13, 35); report calcium both the 37. Suarez, S. S., Katz, D. F. & Meizel, S. (1984) Gamete Res. 10, head and tail of sperm and that [Ca2+]i oscillates in both of 253-265. these regions during flagellar bending. 38. Cosson, M. P. (1990) in Controls ofSperm Motility, ed. Gag- non, C. (CRC, Boca Raton, FL), pp. 103-135. We thank Mr. John Zentmeyer (Z-Trends, Claremont, FL) and Mr. 39. Gnessi, L., Ruff, M. R., Fraioli, F. & Pert, C. B. (1985) Exp. Joe Malek (Elektron Systems) for technical assistance with devel- Cell Res. 161, 219-230. opment of the image-analysis system. This work was supported by 40. Ralt, D., Goldenberg, M., Fetterolf, P., Thompson, D., Dor, J., Grant HD19584 from the National Institutes of Health, and the Mashiach, S., Garbers, D. L. & Eisenbach, M. (1991) Proc. College of Veterinary Medicine, University of Florida Journal Series Natl. Acad. Sci. USA 88, 2840-2844. no. 330. 41. Brokaw, C. J. (1991) Cell Motil. Cytoskel. 18, 123-130. 42. Suarez, S. S., Katz, D. F., Owen, D. H., Andrew, J. B. & 1. Yanagimachi, R. (1982) Gamete Res. 5, 323-344. Powell, R. L. (1991) Biol. Reprod. 44, 375-381. 2. Suarez, S. S., Katz, D. F. & Overstreet, J. W. (1983) Biol. 43. Suarez, S. S. & Dai, X. B. (1992) Biol. Reprod. 46, 686-691. Reprod. 29, 1277-1287. 44. Drobnis, E. Z., Yudin, A. I., Cherr, G. N. & Katz, D. F. 3. Burkman, L. J. (1990) in Controls ofSperm Motility:Biological (1988) Dev. Biol. 130, 311-323. and Clinical Aspects, ed. Gagnon, C. (CRC, Boca Raton, FL), 45. Suarez, S. S. (1987) Biol. Reprod. 36, 203-210. pp. 303-329. 46. DeMott, R. P. & Suarez, S. S. (1992) Biol. Reprod. 46, 779- 4. Miller, R. L. (1985) J. Exp. Zool. 234, 383-414. 785. Downloaded by guest on October 2, 2021