Regulation of calcium content in bovine spermatozoa.

Plasma membrane vesicles isolated from bovine epididymal and ejaculated spermatozoa have widely different capabilities for transporting Ca2+. Spermatozoa were ruptured by nitrogen cavitation, and the plasma membrane fraction was harvested after low speed and sucrose gradient centrifugation; purity was assessed by marker enzyme analyses, electron microscopy, and sedimentation properties. Plasma membrane vesicles isolated from epididymal sperm accumulate Ca2+ passively at a faster rate and to a greater extent than vesicles prepared from ejaculated sperm. Ca2+ transport across bovine sperm plasma membranes is an ATP-independent, Na+-dependent process that obligatorily exchanges intravesicular Na+ for external Ca2+. The rate of Na+/Ca2+ exchange is significantly lower in ejaculated sperm vesicles than in those of epididymal sperm. Bovine plasma membranes contain little or no Ca2+-dependent ATPase activity. It is suggested that, at the time of ejaculation, calcium flux into bovine sperm is prevented by the interaction of the plasma membrane with putative factors in seminal fluid that specifically interfere with Na+/Ca2+ exchange. We have isolated a protein from seminal plasma that prevents calcium accumulation by bovine epididymal sperm (Rufo, G. A., Jr., Singh, J. P., Babcock, D. F., and Lardy, H. A. (1982) J. Biol. Chem. 257, 4627-4632). A protein with properties resembling those of the seminal calcium transport inhibitor is found on the membrane vesicles from ejaculated sperm but not on membranes from epididymal sperm. We conclude that this protein binds strongly to the plasma membrane of bovine sperm and is responsible for preventing calcium uptake by ejaculated sperm.

Plasma membrane vesicles isolated from bovine epididymal and ejaculated spermatozoa have widely different capabilities for transporting Ca2+. Spermatozoa were ruptured by nitrogen cavitation, and the plasma membrane fraction was harvested after low speed and sucrose gradient centrifugation; purity was assessed by marker enzyme analyses, electron microscopy, and sedimentation properties. Plasma membrane vesicles isolated from epididymal sperm accumulate Ca2+ passively at a faster rate and to a greater extent than vesicles prepared from ejaculated sperm.
Ca2+ transport across bovine sperm plasma membranes is an ATP-independent, Na+-dependent process that obligatorily exchanges intravesicular Na+ for external Ca2+. The rate of Na+/Ca2+ exchange is significantly lower in ejaculated sperm vesicles than in those of epididymal sperm. Bovine plasma membranes contain little or no Ca2+-dependent ATPase activity.
It is suggested that, at the time of ejaculation, calcium flux into bovine sperm is prevented by the interaction of the plasma membrane with putative factors in seminal fluid that specifically interfere with Na+/Ca2+ exchange. We have isolated a protein from seminal plasma that prevents calcium accumulation by bovine epididymal sperm ( 257, 4627-4632). A protein with properties resembling those of the seminal calcium transport inhibitor is found on the membrane vesicles from ejaculated sperm but not on membranes from epididymal sperm. We conclude that this protein binds strongly to the plasma membrane of bovine sperm and is responsible for preventing calcium uptake by ejaculated sperm.
In most cells, intracellular calcium is maintained at concentrations orders of magnitude lower than those found in the surrounding extracellular fluid via the so-called calcium pump or (Ca2+,Mg2+)-ATPase (1)(2)(3)(4)(5). While the literature is replete with information regarding the control of calcium extrusion and the voltage-sensitive calcium inflow channels of many excitable cells (1, 2), relatively little is known regarding control of calcium flow into nonexcitable cells.
Sperm plasma membranes have only recently been utilized in the study of calcium transport phenomena. Plasma mem-* This work was supported in part by Grant AM-10334 from the National Institutes of Health and by the Mobil Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed. branes prepared from boar sperm (6) have been shown to bind calcium with high affinity, especially in the presence of sodium or potassium. In addition, a Na+/Ca2+ antiporter has been demonstrated in boar (7) and ram (8) sperm plasma membranes. The studies outlined in this report have been designed to make intraspecies comparisons of calcium transport properties between plasma membrane vesicles isolated from bovine epididymal and ejaculated sperm.
Previous work has shown that washed ejaculated bovine sperm are incapable of accumulating calcium in uitro, whereas epididymal sperm take up calcium rapidly (9). Thus, bovine sperm represent a unique system in which to study the effects of surface modification on Ca2+ flux across the plasma membrane. We have isolated a protein from seminal plasma that inhibits calcium uptake by ejaculated sperm (IO), and it is of interest to determine the site of action of this inhibitor. The technique of nitrogen cavitation provides sealed, right-sideout plasma membrane vesicles (1 1) suitable for the study of calcium inflow channels without interference from other cellular organelles.
The studies reported here are the first to demonstrate a sperm plasma membrane Na+/Ca2+ antiporter that is amenable to regulation by extrinsic factors present in the seminal plasma.

EXPERIMENTAL PROCEDURES
Preparation of Spermatozoan Plasma Membrane Vesicles-Plasma membrane vesicles were prepared from bovine epididymal and ejaculated sperm essentially by the methods of Gillis et al. (12). Epididymides were obtained within 1 h of slaughter (Oscar Mayer Co., Madison, WI) and cooled to 4 "C, and the sperm were expressed according to previously described methods (13). Following two centrifugal washings (600 X g, for 10 min each) in 0.25 M sucrose (Schwarz/Mann), 0.2 mM MgCI,, 10 m M MOPS,' pH 7.4, the sperm were suspended in the same buffer to a final concentration of 1-5 X io9 sperm/ml. Bovine semen was the generous gift of American Breeders Service, DeForest, WI. Ejaculated sperm were isolated by centrifugation (700 X g for 15 min) at 20 "C, then subjected to three washings and diluted as described above. From this point, the procedure for isolating plasma membranes was the same for both types of sperm. All subsequent procedures were carried out a t 4 "C.
Five-ml aliquots of sperm suspension were placed in a Kontes cell disruption chamber and subjected to nitrogen pressure of 750 p.s.i. for 15 min. The suspension was then slowly extruded into 2.5 ml of buffer to give final concentrations of 0.25 M sucrose, 0.13 mM MgCI,, 1 mM EDTA, 10 mM MOPS, pH 7.4. The combined suspensions were centrifuged at 600 X g (Beckman JA-20 rotor) for 10 min, and the pellet was washed twice with an equal volume of the above buffer minus MgCI2. The combined 600 X g supernatant fraction was centrifuged a t 6,000 x g for 10 min, and the pellet was washed once with the above buffer. The combined 6,000 X g supernatant was then layered on a three-step sucrose gradient (w/v. d,,, = 1.23. 1.17. 1.05) and centrifuged at 100,000 X g (Heckman SW 27 rotor) for 2 h. I'lasma membrane vesicles were collected from the 1.17/1.05 interface hy aspiration, washed once with 0.25 M sucrose, 10 mM MOPS, pH 7.4, and collected by centrifugation at 100,000 X g for 1 h. The resulting pellet, consisting mainly of right-side-out plasma membrane vesicles ( 1 1 ), was resuspended in the above huffer to a final protein concentration of 3-5 mg/ml. Protein was determined by the method of Inwry (14) using bovine serum albumin as a standard. Typical yields of memhrane protein were 150-:300 pg from lo9 sperm.
fi;kctron MicnJ.sc~Jp?-PUrified plasma membranes isolated from epididymal sperm were fixed with 2% cacodylate-buffered glutaraldehyde, pH 7.0. and postfixed in osmium tetroxide. The precipitated membranes were dehydrated with graded ethanol washes, treated with propylene oxide, and embedded in Epon. Thin sections were stained with uranyl acetate, and electron micrographs were ohtained using a Hitachi H500 electron microscope.
V~&k+-Plasma membrane vesicles prepared from either eipididymal o r ejaculated sperm were incubated at : 3 0 "C for 5 min in 110 mM NaCI. 5 mM KCI, 10 mM MOPS, pH 7.4 (unless noted otherwise). MES replaced MOPS in reactions conducted at pH intervals helow 7.0. Uptake was initiated by the addition of CaCI, laheled with 4sCa'+ ( 2 0 pCi/pmol; Amersham Corp.) or hy dilution of vesicles into appropriate media containing "Ca2+. Reactions were terminated by collecting and washing 100 p1 (25 p g ) of the membrane suspension on Millipore filters (Type HA, 0.45-pm pore size). The filters were washed three times with 5-ml portions of 0.25 M sucrose, 0.1 mM I,aCL, 1.0 mM CaC12, 10 mM MOPS, pH 7.4, and analyzed for radioactivity as previously described (10). Na+/('a'+ I k h a n g e Studies-Vesicles were passively loaded with Na' by incubation in 150 mM NaCI, 10 mM MOPS, pH 7.4, at 4 "C for [16][17][18] h. The Na+-loaded vesicles (60 p l , 300 p g ) were incubated at 30 "C lor 10 min, then diluted to 1.2 ml with initial reaction medium containing 150 mM NaCI, 1 mM CaClz (40 pCi of "Ca2+/ pmol), I O mM MOPS, pH 7.4, at 30 "C. The "Ca content of the vesicles was monitored for 5 min, after which the initial reaction medium was diluted into K'-or choline-containing medium (see legend to Fig. 4) to promote release of internal Na'. The 45Ca content was monitored for the next several minutes of incubation. Control experiments where Na'or K+-loaded vesicles were diluted into Na' medium were conducted in order to examine the dependence of an outwardly directed Na' gradient on the uptake of CaY+ by sperm plasma membrane vesicles. 1)c~lc~rmination of A7'l'a.w A~LiL.it\.-Spermatozoan plasma membrane vesicles (50 p g ) were incubated for 5 min a t 37 "C in a reaction medium containing 80 mM NaCI. 15 mM KCI, 6 mM MgC12, and 50 mM Tris-HCI, pH 7.0. in a final volume of 1 ml. Reactions were then initiated by the addition o f 3 mM Na-ATP (P-L Hiochemicals) and terminated 10 min later hy the addition of 1 ml of 10% trichloroacetic acid. After I 5 min at 0 "C. the protein precipitate was collected by centrifugation at 10,000 X g for 1 0 min. The supernatant fraction was assayed for inorganic phosphate as described above. Ca"-independent ATPase activity was determined in the presence of 1.5 mM EGTA, while Mi"-ATPase activity was determined in the presence of 1.5 m M EGTA plus 1 mM ouabain (Sigma). (Na+,K')-ATPase activity was defined as the difference hetween Ca"-independent ATPase and M$'-ATl'ase activities. h. Molecular weight markers and an internal isoelectric focused standard were included in the gel. The slat) gels were equilihrated and silver-stained according to the method of Oakley et al. (19).

Preparation of Playma Membrane
Vesicles-Plasma membranes isolated from epididymal and ejaculated bovine sperm were enriched in 5'-nucleotidase activity by 12-and 6-fold, respectively (Table I). While the specific activity of ejaculated bovine sperm vesicle 5'-nucleotidase was twice that found in epididymal sperm vesicles, the apparent purification of this membrane marker enzyme in the former preparation was only half that in the latter. The most likely explanation for this disparity resides in the fact that bovine seminal fluid is a particularly rich source of soluble 5'-nucleotidase activity (20). Any residual seminal plasma remaining after cell washing or adherence of soluble enzyme to isolated membrane could interfere with the determination of cellular enzyme activity. T h e low levels of succinate dehydrogenase activity indicate that the final product was almost completely free of contaminating mitochondrial membrane.
Electron micrographs (Fig. 1) of fixed epididymal sperm plasma membranes illustrate the vesicular nature of the preparation, which is apparently devoid of contaminating organelles. T h e final product contains some fragmented material along with some vesicles within vesicles, which is character-  istic of similar preparations of plasma membrane from boar sperm (12,21). Kinetics of Calcium Uptake by Bovine Spermatozoan Plasma Membrane Vesicles-Rates of "Ca2+ uptake were measured in the absence of added energy sources using vesicles prepared from both epididymal and ejaculated bovine sperm. Epididymal membranes sequestered 4sCa2+ at a faster rate and to a greater extent than those prepared from ejaculated sperm (Fig. 2). Uptake followed saturation-type kinetics (Fig. 2 A ) , and maximal uptake for both types of membrane preparations occurred in 60 min a t 30 "C. The effect of varying external calcium concentration from 50-600 PM is shown in Fig. 28. The differences in Ca uptake between epididymal and ejaculated sperm plasma membranes are similar to those obtained using whole sperm (9) and support the contention that the plasma membrane plays a key role in preventing calcium flux into ejaculated sperm.
Mechanism of Calcium Transport across the Spermatozoan Plasma Membrane-The proposed pathways of calcium inflow across the plasma membrane are thought to involve one of two exchange mechanisms: a H+/Ca2+ antiporter shown to be operative in the plasma membrane of Ehrlich ascites tumor cells (22) or a Na+/Ca2+ antiporter found in a number of membrane preparations ( 2 3 ) .
When bovine epididymal sperm plasma membranes, equilibrated at pH 7.4, were incubated in buffers of varying pH (5.5-7.9), no appreciable differences were observed in the rate of calcium uptake (Fig. 3). The protonophore carbonyl cyanide m-chlorophenylhydrazone did not affect calcium uptake at the various pH intervals tested (not shown).
Ca2+ efflux in exchange for Na+ has been demonstrated in several membrane types ( 2 3 ) including the plasma membrane of ram sperm flagella (24) and boar sperm (7). This type of exchange is also thought to operate in the direction of calcium influx (25). To test for the existence of a Na+-dependent calcium channel in bovine sperm, plasma membrane vesicles were loaded with 150 mM Na+ and subjected to conditions that would favor the release of intravesicular Na+. *Ta2+ uptake was simultaneously monitored using both types of  Fig. 4 reveals that, when diluted into either a K+or choline-containing medium, Na+-loaded epididymal sperm vesicles sequester external 45Ca2+ at a significantly greater rate than vesicles from ejaculated sperm. In control experiments, where Na+or K+-loaded vesicles were diluted into Na' medium (conditions expected to result in no net movement of Na+ or to promote inward Na+ movement, respectively), no stimulation of the low basal rate of calcium uptake was observed (not shown). Thus, it appears there is a strict requirement for outward movement of Na' as a prerequisite to calcium influx across sperm plasma membranes. These data distinguish the calcium transport process of sperm plasma membranes as being Na+-dependent and regulated by putative factors present in semen that react with the plasma membrane and serve to restrict the inward flux of calcium into ejaculated sperm. Epididymal sperm vesicles take in calcium at a faster rate in K+-containing media than when choline was the prevalent cation. This is probably a result of the greater permeability of plasma membranes to K+ compared to choline. An explanation of the influence of K+ is presented under "Discussion." ATPase Activities in Bovine Sperm Plasma Membranes-(Ca2+,Mg2+)-ATPase activity was determined in bovine sperm plasma membranes to assess the role of active transport on maintaining calcium homeostasis in bovine sperm. It has been shown that ram sperm flagellar plasma membranes contain a Ca2+-dependent ATPase that represents 14% of the total membrane ATPase activity (26). In contrast, bovine sperm plasma membranes were found to possess little or no Ca2+dependent ATPase activity. Data in Table I1 show that this activity represents 2% or less of the total membrane ATPase. This specific activity of Ca'+-dependent ATPase in bovine sperm plasma membranes is not more than 50% of that observed in ram sperm plasma membranes (26). The lesser activity was not the result of membrane damage, for the (Na+,K')-ATPase activity agrees closely with previous determinations (27), indicating that enzyme activity was not affected during isolation procedures. Rendering the membranes permeable with digitonin or Triton X-100 (28) to ensure that  C1 (A and A). "Ca2' content, was determined at the designated time points. The rate and extent of 4sCa2+ uptake following dilution were significantly higher in epididymal than in ejaculated sperm membrane vesicles (p s 0.05, n = 3).
The data are expressed as mean values f S.D. of duplicate analyses from three individual preparations of vesicles.
substrates were available to the enzyme resulted in no change in the enzyme activities reported above.
Presence of Seminal Calcium Transport Inhibitor on Eja- preventing calcium uptake by ejaculated bovine sperm. In order to determine whether this protein is present on plasma membranes isolated from ejaculated sperm, two-dimensional gel electrophoresis was performed on samples of epididymal and ejaculated sperm membranes. The resulting chromatograms (Fig. 5) clearly show the presence of a low (about 15,000) molecular weight protein having a p1 of approximately 8.3 on the ejaculated sperm membrane preparations (Fig. 5B). These physical criteria coincide closely to that of pure seminal calcium transport inhibitor (10). No such protein was detected in the membrane preparations from epididymal sperm (Fig.  5A).

DISCUSSION
Plasma membrane vesicles have been prepared from bovine epididymal and ejaculated sperm using the method of nitrogen cavitation cell disruption. Membranes prepared by these procedures are thought to originate principally from the apical, or head, portion of the sperm (7). In support of this view is the observation that cavitation of bovine sperm labeled with fluorescent concanavalin A results in loss of fluorescence from the head region only.' Enrichment of 5'-nucleotidase activity and the lack of succinate dehydrogenase activity (Table I) indicate that vesicles prepared by the above procedures consist mainly of purified plasma membrane devoid of mitochondrial contamination. The specific activity of 5"nucleotidase in our membrane preparations agrees well with the value reported for boar sperm plasma membranes (12). The morphology (Fig.  1) and sedimentation properties of bovine sperm plasma membranes resemble similar preparations of plasma membrane from boar sperm (12,21).
Plasma membrane vesicles isolated from bovine spermatozoa are capable of accumulating exogenously added Ca'+, and the rate and extent of passive Ca2+ diffusion (Fig. 2) and Na+induced Ca2+ influx are significantly reduced in membrane preparations from ejaculated sperm as compared to epididymal sperm. Na+/Ca2+ exchange activity in bovine sperm plasma membranes is considerably lower than that observed in plasma membrane preparations from ram sperm flagella (8) but is comparable to the activity reported for boar sperm head plasma membranes (7). The pH-independent uptake of Ca2+ (Fig. 3)   cumulate 45Ca2+ at a faster rate and to a greater extent simply masking Cap+-binding sites on the surface of sperm following dilution into K+-containing media comp-red to cells and thus prohibiting access of Ca2+ to the plasma memcholine-containing media (Fig. 4), which indicates that charge brane. The absence of heat lability of Ca2-dependent ATPase compensation probably occurs, in vivo, by movement of K+ in activity in bovine sperm head plasma clearly distinguishes the same direction as Ca2+.
seminal calcium transport inhibitor from the calmodulin-like Bovine sperm plasma membranes contain high levels of component of human seminal plasma that stimulates M$+-dependent ATPase activity, less than 2% of which is (Ca2+,Mg2+)-ATPase of erythrocytes (29). Ca'+-dependent (Table 11). This finding contrasts with similar The sperm acrosome reaction is an absolute prerequisite measurements of (Ca'+,M$+)-ATPase activity in plasma for fertilization (30). It is induced rapidly by ionophoremembranes isolated from ram sperm flagella (26) and boar mediated calcium uptake (31) or more slowly in the presence sperm head (7) and suggests that active Ca2+ extrusion does of calcium but absence of ionophore (32). The failure of not play a significant role in preventing net accumulation of ejaculated spermatozoa to take UP calcium despite the high Ca2+ by bovine ejaculated sperm. concentration in seminal fluid (9) can be explained by the Previous studies demonstrating the capability of bovine presence of the calcium transport inhibitory protein. Capaciepididymal sperm, but not ejaculated sperm, to accumulate tation of spermatozoa in vi00 is known to involve the removal Caz+ in vitro (9) and the subsequent isolation of a Ca*+ or modification of plasma membrane proteins (33). That may transport-inhibiting factor from seminal plasma (lo), corn-release the inhibitory effect of the seminal protein, allow bined with the results of this study, lead us to conclude that calcium to enter the SPermatozoa, stimulate the ~.rosomal the Na+/Ca2+ exchanger is responsible for controlling Ca2+ reaction, and accomplish capacitation. uptake by bovine sperm. Furthermore, the plasma membrane contains the apparatus through which seminal calcium trans-