Caltrin, the Calcium Transport Regulatory Peptide of Spermatozoa, Modulates Acrosomal Exocytosis in Response to the Egg’s Zona Pellucida*

Acrosomal exocytosis is initiated in mammalian sperm by stimulatory agonists in the zona pellucida. Recently, it was shown that exocytosis is modulated in bovine sperm by extrinsic factors present in seminal fluids (Florman, H. M., and First, N. L. (1988) Dev. Biol. 128, 464-474). Fractionation of bovine seminal fluids yields a M, = 6,500, basic (PI = 8.5) peptide that accounts for the positive modulation of zona pellucida- induced acrosome reaction (ED60 and maximal response at 0.2 and 1 pg/ml, respectively). In addition, application of purified peptide to fura 2-loaded, cauda epididymal sperm supported zona pellucida-promoted elevations of Ca2+i equivalent to those observed with sperm treated with unfractionated seminal fluids in vitro or in vivo. Finally, peptide treatment regulated gamete interaction in a manner consistent with the distinct behaviors of cauda epididymal and ejaculated bovine sperm. This purified seminal peptide was iden- tified by sequence analysis as caltrin, a previously characterized regulator of Ca2+ transport in bovine sperm. We therefore propose that caltrin is a regulator of sperm signal transduction pathways activated by zona pellucida binding.

Sperm of many animal species contain a single secretory vesicle, or acrosome, in the apical portion of the head. Exocytosis (the acrosome reaction, AR),' with an associated release of vesicular contents and presentation of new membrane domains at the cell surface, is an essential prerequisite for fertilization (Florman and Babcock, 1990;Kopf and Gerton, 1990).
In mammals, acrosome reactions may be initiated following sperm adhesion to the egg's extracellular coat, or zona pellucida (ZP). ZP from several species have been characterized * This work was supported by the National Institutes of Health and by the United States Department of Agriculture. Additionally, the Protein Chemistry Laboratory at the Worcester Foundation is supported by a grant from the W. M. Keck 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 "aduertkement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Internal Ca2+ (Ca2+i) is an essential effector of ZP3 signaling. Single, living sperm that have incorporated fluorescent ionic indicator dyes display Ca2+i elevations that are promoted by ZP agonist and that precede exocytosis (Florman et al., 1989;Storey et al., 1992). The ionic and exocytotic responses are further associated as both are attenuated by prior application of PTx (Florman etal., 1989(Florman etal., ,1992. Finally, ZP agonistinduced exocytosis is inhibited by several different classes of Ca2+ entry blockers (Florman et aZ., 1992). Ca2+; is also an important regulator of the AR produced in mammalian sperm by alternative agonists obtained from the female reproductive tract Meizel, 1988,1989;Blackmore et aZ., 1990Blackmore et aZ., , 1991Meizel and Turner, 1991) or in sea urchin sperm by the fucosylsulfated glycoconjugate obtained from egg jelly (Schackmann et al., 1978;Schackmann and Shapiro, 1981;Trimmer et al., 1986Trimmer et al., , 1987Guerrero and Darszon, 1989). As a result, it is generally believed that Ca"; elevation and the consequent activation of downstream effectors provide a final common pathway for the induction of exocytosis.
In order to coordinate exocytosis with the availability of eggs, it is necessary that Ca2+; be strictly regulated during the prolonged periods of gametogenesis, epididymal storage, and migration to the site of fertilization. This regulation is accomplished, at least in part, through poorly understood mechanisms that maintain sperm in an infertile state within the epididymis. Sperm undergo a cluster of physiological and biochemical alterations as a consequence of release into female reproductive tract secretions or into appropriate in uitro environment; these alterations are referred to collectively as "capacitation" (Florman and Babcock, 1990). One aspect of 5309 this process is the functional maturation of ZP3 signal transduction pathways.' ZP agonists promote neither Ca'+i elevations (Florman et al., 1989) nor exocytosis (Florman and First, 1988b) in bull sperm obtained from cauda epididymides, whereas both responses are displayed in sperm recovered from seminal secretions. Regulatory factor(s) that account for this functional maturation of agonist signal transduction are present in seminal fluids (Florman and First, 1988b;Miller et al., 1990).
These observations are extended herein by the demonstration that seminal fluids couple agonist signal to the pathways controlling sperm Ca2+i; that a peptide, shown subsequently to be caltrin, can be isolated from bovine seminal fluids and accounts for this activity; and that peptide-promoted modulation of ZP agonist signal transduction has functional consequences for fertilization in vitro.

EXPERIMENTAL PROCEDURES
Biological Samples-Bovine sperm were obtained from either from semen (EjSp) or by retrograde flushing of cauda epididymides (CESp), washed (200 X g; 10 min), and incubated in a modified Tyrode's solution (dmTALP medium; Florman et al. (1989)), under conditions previously shown to support functional maturation of sperm (Parrish et al., 1988;Florman and First, 1988a). All manipulations subsequent to washing were carried out at 39 "C (bovine core body temperature). Seminal plasma was obtained by sequential low speed (600 X g; 10 min) and high speed (10,000 X g; 20 min) sedimentation of semen. Oocytes were obtained from abattoir-derived ovaries. Fertilization and ZP isolation were carried out as described previously (Florman and First, 1988a).
Determinations of Ca*+,-Fura 2 was incorporated into bovine sperm by a "postloading" protocol, and fluorescence images were acquired, digitized, and further processed, as described previously (Florman et al., 1989). Sperm tolerate loading and immobilization, as indicated by the retention of dye by -75% of cells, and by the absence of dye effects on either the time course or the potency of ZP agonist action. Data are expressed as the dimensionless value of Ca2+;/KD (thereby accounting for uncertainties regarding KD of Ca2+-fura 2 complexes in intracellular environments), according to the algorithm given in Equation 1.
R, &IN, and R M~ are the emission ratios following excitation at 340 nm and 380 nm in samples and in minimal and saturating Ca2+ solutions, respectively, and B is a sensitivity factor (Grynkiewicz et al., 1985). To permit comparisons with other reports, fura 2 data are also presented as Caz+;, assuming K D = 224 nM (Grynkiewicz et al., 1985). Calibrations were carried out in 50-pl drops of buffered CaClz solution under oil.
Determination of "Ca2+ Sequestration-Sperm (5 X i07/ml) were collected from dmTALP by sedimentation (200 X g; 10 min) and resuspended at 10' cells/ml in a Ca2+ uptake buffer containing (mM): NaCl (100); KC1 (3.2); MgClz (0.5); CaC12 (2.1); NaHC03 (10); lactic acid (10); pyruvic acid (0.25); Na-Hepes (40, pH 7.4). Cells (5 ml) were incubated for 15 min in uptake medium prior to the addition of 'TaClZ (2 X 10' cpm). Aliquots (1 ml) were diluted with ice-chilled wash buffer (0.25 M sucrose, 5 mM CoClZ, 5 mM MgCL, 10 mM Na-Hepes, pH 7.4), collected by gentle filtration (-15 s for collection) on Whatman GF/C filters, washed twice with 5 ml of wash buffer, and the "Ca" retained on filters determined by liquid scintillation spectrometry. Less than 15% of the applied cells were broken during "Functional maturation" denotes only those alterations of mammalian sperm associated with the development of ZP agonist-sensitivity. Previously, it was demonstrated that agonist responsiveness and fertility are expressed in sperm populations with similar time courses and manifest identical modulation by stimulatory and inhibitory regulators (Florman et al., 1989;Ward and Storey, 1984). We have avoided the more general term of "capacitation" that typically describes these transitions, as that term also is applied to a range of functional modifications, including activation of motility and enhanced rates of agonist-independent exocytosis (Bedford, 1983;Yanagimachi, 1988) that may be separated from the regulation of zona agonist signaling pathways. filtration under these conditions, as determined by assay of the filtrate for esterolytic activity (derived from lysed sperm; data not shown).
Determination of Positive Modulatory Activity-Positive modulatory activity is defined as the ability of seminal secretions to produce a state wherein CESp undergo ZP agonist-induced exocytosis. Briefly, CESp (lo' cells/ml) were incubated with crude or fractionated seminal fluids for 15 min at room temperature, washed (2 X 10 ml of dmTALP) by sedimentation (200 X g; 10 min), resuspended in dmTALP (5 X lo7 cells/ml), and subjected to the 5-h incubation essential for the development of fertilizing ability and for coupling of signal transduction pathways (Florman and First, 1988b). Aliquots (12.5 pl) were assayed at intervals for the ability to elevate Ca2+; or to initiate exocytosis in response to addition of 50 pg of ZP protein/ ml, a maximally stimulatory agonist concentration (Florman and First, 1988a). Exocytosis was determined by phase contrast microscopy of fixed sperm and confirmed by application of a differential staining protocol (Bryan and Akruk, 1977), as described previously (Florman and First, 1988a).
Fc represents the fluorescence of dns-CaMlpeptide complexes, F p is the fluorescence attributable to peptide alone, and FCAM is the fluorescence of dns-CaM alone. Kffp is estimated as the peptide concentration producing a half-maximal fluorescence enhancement at each dns-CaM concentration. KO, corrected for the influence of dns-CaM concentration, is obtained from families of binding curves by plotting W f P X [dns-CaM] and by extrapolating to limiting [dns-CaM]. Bovine seminal fluids were depleted of calmodulin-binding peptides by chromatography on CaM-Sepharose (Pharmacia LKB Biotechnology Inc.). Aliquots (100 pg of protein) of semen were diluted with five volumes of protein-free dmTALP, mixed batchwise with CaM-Sepharose (4 "C, 16 h), and eluted with protein-free dmTALP.
Protein Chemistry-Peptide synthesis and sequencing was carried out at the Worcester Foundation Protein Chemistry Facility, using Applied Biosystems Model 430A peptide synthesizer and model 477A protein sequenator. Cali"7, a synthetic caltrin, was synthesized from reported caltrin sequences (Lewis et al., 1985) and was confirmed by sequence analysis and mass spectroscopy.
Materials-Chemicals were obtained from the following sources. 3'-quinuclidinyl benzilate was the kind gift of Dr. Bayard Storey, University of Pennsylvania; the free acid and acetoxymethyl ester of fura 2 was from Molecular Probes, Eugene, O R 45CaC12 was from Amersham Corp.; (f)PN200-110 was from Sandoz Ltd.; (CH3)zSOd (spectrophotometric grade) was from Aldrich; PTx was from Calbiochem. Lots of PTx were screened for infectivity based on previous observations that 10 ng/ml toxin produces a -80% inhibition of the ZP agonist-induced acrosome reaction of bovine sperm (Florman et al., 1989). Histone (Sigma type 11s) and all other chemicals were obtained from Sigma.

RESULTS
Purification of Positive Modulatory Factor-Mammalian sperm display both spontaneous (agonist-independent) and agonist-stimulated acrosomal exocytosis. Positive modulators in seminal fluids act specifically on the latter process, with no apparent effect on spontaneous events (Florman and First, 198813). Operational criteria applied to identify the agonistsensitive pathway are: 1) stimulation by ZP agonists and 2) inhibition of ZP agonist response by preliminary treatment of sperm with PTx (Endo et al., 1987(Endo et al., , 1988Florman et al., 1989Florman et al., , 1992, which attenuated signal transduction mediated by some G proteins. These criteria were used in order to identify active factors (positive modulatory factors, or PMF).
PMF activity of crude bovine seminal fluids is not retained by concanavalin-or wheat germ agglutinin-Sepharose or by DEAE-cellulose (at pH 7.4). Flow-through fractions were resolved on phenyl-Sepharose, with active pools subsequently fractionated on Bio-Gel P6 (Fig. lA) to yield an excluded volume peak of PMF activity (peak I) as well as a resolved peak (peak 11). It is likely that these peaks represent different aggregation states of a single peptide, since they both display a single major component of M, 6,500 on SDS-PAGE ( Fig. 2) and since peak I comigrated with peak I1 upon rechromatography under denaturing conditions (8 M urea; Fig. 1B). Factors controlling aggregation are not understood, since preliminary studies have failed to demonstrate aggregation of purified peak 11. Typically, we obtain 2-5 pg of PMF (peaks I + II)/mg of seminal fluid protein, with peak I1 accounting for 50-70% of this total (Table I). All subsequent characterizations were carried out using peak 11. Potency of isolated PMF was determined by treating CESp with ZP agonist following preliminary application of increas- Gel filtration of positive modulatory activity from bovine seminal fluids. PMF activity was isolated from bovine seminal fluids by lectin affinity, ion exchange, and hydrophobic chromatography (see "Experimental Procedures"). Peak fractions were desalted on Bio-Gel P2 columns developed in 50 mM NH4CH3COOH (pH 7), lyophilized, resuspended in 50 mM NH4CH3COOH (pH 7), and chromatographed on Bio-Gel P6 (1 X 40 cm). P6 columns were developed in 50 mM NH4CH3COOH (pH 7; 4 "C) at a flow rate of 10 ml/h, and 1-ml fractions were collected. A , aliquots (100 pl) of fractions were lyophilized, resuspended in dm-TALP, and assayed for PMF activity as described under "Experimental Procedures." B, peak I material was pooled, lyophilized, resuspended in 4 M guanidinium chloride, and applied to Bio-Gel P6. This column was developed in 4 M guanidinium chloride, and 1-ml fractions were collected. Aliquots (100 pl) were desalted on Bio-Gel P2 (0.5 X 7 cm; equilibrated and developed in 50 mM NH4CH&OOH, pH 7), lyophilized, resuspended in dmTALP, and assayed for PMF activity. Void and included volumes were determined using ferritin and Iz5I-N-acetyl-L-tyrosine, respectively.

Isolation of positive modulatory activity from bovine seminal fluids
Crude seminal fluids were diluted with nine volumes of 50 mM NH4HC03, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 mM dithiothreitol (DH 7.4) and fractionated as indicated.

~~~ ~~
Step" protein units' ' Results of a single purification that are typical of the five purifications reported herein.
*Fractions were assayed for PMF activity (as described under "Experimental Procedures"), pooled, and dose-response relationships were determined for the most active pool. Unit activity is defined as the concentration (pg of protein/ml) of that active pool that produces half-maximal response. Acrosome reaction assays were based on triplicate samples (>ZOO sperm examined/sample). e PMF activity was not retained by coupled concanavalin A-Sepharose/WGA-Sepharose columns or by DEAE-cellulose (pH 7.4; 150 mM NaCI).
Step IV activity is applied to columns of Bio-Gel P2 (1 x 45 cm). Excluded fractions, containing PMF activity, are concentrated and fractionated on Bio Gel P6 as described in the legend to Fig. 1. ing concentration of peptide (Fig. 3A, m). PMF produced a concentration-dependent enhancement of induced exocytosis, with EDso and maximal responses at 0.2 and 1 pg/ml PMF, respectively. PMF had no observable effects on sperm motility, as monitored subjectively by direct observation, and did not induce acrosome reactions in the absence of ZP agonist, even when present at 10-100 pg/ml (acrosome reaction levels in CESp treated with buffer and with 100 pg/ml PMF for 30 min were 16 * 7 and 18 k 5%, respectively; n = 3).
Caltrin Is a Positive Modulatory Factor-The electrophoretic profile of PMF is similar to that of caltrin, a M, 5,411 peptide that is synthesized and secreted by bovine seminal vesicles and that may comprise as much as 1% of total seminal fluid protein (Lardy and San Agustin, 1989). Moreover, caltrin binds to the acrosomal region of the sperm head, and to the distal flagellum, regulating 45Ca2+ sequestration (Babcock et  Rufo et al., 1982Rufo et al., , 1984San Agustin et al., 1987;Lardy and San Agustin, 1989; San Agustin and Lardy, 1990). As the acrosome reaction is a Ca2+-dependent process occurring in the anterior head, it was reasonable to hypothesize that caltrin may account for PMF activity in seminal fluids.
Four types of evidence suggest that caltrin and PMF are related peptides. First, direct amino acid sequence was obtained for a large fragment of PMF (Table 11). This is identical to caltrin sequences obtained either directly (Lewis et al., 1985) or deduced from cDNA (Wagner et al., 1990). This extensive similarity at the level of primary structure is consistent with the observed comigration on SDS-PAGE of purified PMF and of a synthetic full-length caltrin (Fig,  2). Second, both peptides have positive modulatory activity; ED50 values for PMF and caltrin were 0.2 k 0.1 and 0.5 f 0.1 pg/ml, respectively (Fig. 3A). Given estimates of both the potency (ED50 = 50-60 pg/ml; Fig. 3A) (Florman and First, 1988b) and the peptide concentration (Table I)  Third, both caltrin and PMF inhibit 45Ca2+ uptake by CESp, a signature characteristic of caltrin (Lardy and San Agustin, 1989). The effects of caltrin on Ca2+; compartmentation and the mechanism of impaired isotope accumulation are not well understood. Fluorescence of intracellular fura 2, attributable to Ca2+i, is not significantly altered in a sustained fashion following treatment of CESp with seminal fluids in vivo (Florman et al., 1989) or in uitro, or by application of purified caltrin (see below). It has been suggested that the inhibitory effects of caltrin on 45Ca2+ uptake reflect a primary alteration at the level of plasma membrane transport, with subsequent and indirect effect on mitochondrial sequestration (Singh et al., 1978;Rufo et al., 1984;Breitbart et al., 1990b).
PMF preparations had caltrin-like activity by producing inhibition of 45Ca2+ sequestration by CESp (Fig. 3B). Isotope accumulation was inhibited by 60-70% following application of seminal fluids either during natural mating or in uitro (69 f 11 and 62 f 10% inhibition, respectively; n = 3). Isolated PMF and caltrin both could account for the effects of crude seminal fluids, producing ~6 0 % inhibitions of 45CaZ+ uptake with similar potencies (EDsO and maximal response at 5-25 and at =lo0 gg/ml, respectively).
Fourth, the previously demonstrated affinity of caltrin for calmodulin (Comte et al., 1986;Milos et al., 1988) was used in two types of experiments in order to examine the relationship between caltrin and PMF in greater detail. In the first, bovine seminal fluids were fractionated by CaM-Sepharose chromatography. PMF is retained on these matrices, as demonstrated by the loss of PMF activity after affinity chromatography and by its recovery upon readdition of CaM-Sepharose-retained fractions (Table 111). Caltrin accounts for at least a portion of PMF activity, as demonstrated by the effects of a synthetic, full-length caltrin peptide Addition of to CaM-Sepharose depleted seminal fluid partially restored PMF activity (Table 111). In addition, a peptide of molecular mass similar to PMF and caltrin is the major (but not the only) species retained on CaM-Sepharose (not shown). In a second series of experiments, it was determined that caltrin and PMF had similar affinities for dns-CaM (KD values of 0.9 f 0.2 and 1.2 f 0.3 nM, respectively; n = 3).
It should be noted that was less active than preparations of highly purified, native caltrin (>95% caltrin, as determined by silver-stained SDS-PAGE). In addition, this synthetic peptide bound dns-CaM (KO = 8.1 0.7 nM; n = 3) with lower affinity than either caltrin or PMF. These differences may reflect difficulties in activating particularly in light of the complex macromolecular requirements for activation of native caltrin (San Agustin et al., 1987; San Agustin and Lardy, 1990). Alternatively, other peptides that are present at low concentrations ( 6 % ) in native caltrin preparations may also contribute to PMF activity. Regardless, studies with support the hypothesis that caltrin is a positive modulator of sperm signal transduction.  Table I) and further purified by SDS-PAGE. The major band ( M , 6,500) was transferred to Primary structure of bovine caltrin, as determined by direct amino acid sequencing by Lewis et al. (1985), is shown. nitrocellulose, washed, eluted from gel slices, and used for direct sequencing. e Primary structure of bovine seminalplasmin, a peptide shown previously to be identical to caltrin (Sitaram et al., 1986), is shown. Amino acid sequence was deduced from cDNA (Wagner et al., 1990).  Caltrin Modulation of Zona Pellucida-induced Ca2+, Eleuations-Previously, it has been demonstrated that bovine sperm obtained from the cauda epididymis and from seminal fluids differ in the ability to undergo ZP agonist-promoted exocytosis (Florman and First, 1988b;Miller et al., 1990) and CaZci elevations (Florman et al., 1989), and that applications of seminal fluids (Florman and First, 1988b;Miller et al., 1990) or of seminal fluid peptides (this study) in vitro accounts for differences in these cells with regard to exocytosis. This provides the opportunity to explore the relationship between agonist-promoted Ca2+, responses and exocytosis, by examining the effects of seminal fluids and peptides on sperm Ca2+i in fura 2-loaded bovine sperm in a series of video imaging experiments. Previous studies established that sustained, agonist-dependent Ca2+j elevations are observed in the subpopulation of sperm that display ZP-induced exocytosis, but that the time course and magnitude of the Ca2+i response vary extensively between cells (Florman et al., 1989). To account for this heterogeneity, both agonist-independent (basal) and maximal agonist-stimulated Ca2+, are displayed for groups of single cells.
Application of ZP agonist (50 pg/ml) to EjSp results in a 2-3-fold elevation of dye emission and is associated with induction of exocytosis (Fig. a), as reported previously (Florman et al., 1989). In contrast, sperm obtained from the cauda epididymis show neither enhanced dye fluorescence or acrosome reaction following addition of agonist (Fig. 4B). Yet The effects of seminal fluid components on sperm signal transduction may be defined further through the application of known inhibitors acrosome reactions. ZP agonist-induced Ca2+i elevations and exocytosis are attenuated by PTx (Endo et al., 1987(Endo et al., , 1988Florman et al., 1989Florman et al., ,1992Lee et al., 1992), the ADP-ribosyltransferase inhibitor of some classes of G proteins (Moss and Vaughan, 1988); by 3'-quinuclidinyl benzilate (Florman and Storey, 1982;Endo et al., 1988;Vazquez et at., 1989;Storey et al., 1992), a competitive antagonist of muscarinic cholinergic signaling (Hille, 1991;Hosey, 1992); and by PN200-110 (Florman et al., 1992), a member of the dihydropyridine antagonists of the L-type voltage-sensitive calcium channel of somatic cells (Miller, 1992) and of sperm (Kazazogolou et al., 1985;Babcock and Pfeiffer, 1987;Cox and Peterson, 1989;Florman et al., 1992). Each of these reagents inhibited ZP-promoted exocytosis of EjSp while having no significant effects on spontaneous ARs of cells obtained either from seminal fluids or from the cauda epididymides (Table IV). Yet, when each of these inhibitors were applied to CESp treated preliminarily with either seminal fluids or caltrin, a pronounced attenuation of ZP agonist-induced exocytosis and Ca2'i elevations was observed. These findings provide further evidence that the modulatory effects of caltrin account for many of the differences between CESp and EjSp with regard to ZP agonist signal transduction.

Effects of Caltrin on the Development of Agonist Sensitiv-
ity-The expression of agonist-sensitivity in EjSp does not occur immediately following admixture with seminal fluids, but rather requires extensive (4-5 h, under culture conditions used here) preliminary incubation (Fig. 5 ) (Florman and First, 1988b). These sperm populations become fertile, as assayed in uitro, with approximately the same time course, leading to the suggestion that maturation of these signal transducing pathways is a late event in the "capacitation" process (Ward and Storey, 1984;Florman and First, 1988b;Florman et d., 1989). In order to identify some of the regulatory events that control positive modulation, we compared the time courses for the expression of ZP agonist sensitivity in caltrin-treated CESp with that of EjSp. CESp were treated with purified caltrin and subsequently with ZP-agonist. ZP-promoted exocytosis was not observed immediately following caltrin addition; however, in vitro treated cells manifest ZP-stimulated, PTx-sensitive ARs within 1 h. Unrelated proteins of low molecular mass and basic PI had no comparable effect: for example, treatment with cytochrome c or histones resulted in levels of agonist-induced exocytosis only 10-20% of those observed in caltrin-treated populations.
TABLE IV Effects of inhibitors on ZP agonist-induced acrosome reaction in sperm obtained from seminal fluids or in sperm'treated in vitro with unfractionated seminal fluids and with caltrin CESp were incubated preliminarily with dmTALP or with medium supplemented with either unfractionated seminal fluids or with caltrin, exactly as described under "Experimental Procedures." CESp and EjSp were cultured 5 h, permitting maturation of ZP agonist signal transduction pathways, aliquots (12.5 pl) mixed with ZP agonist, and exocytosis assessed after 30 min. Data represents the mean (+S.D.) of triplicate experiments, with duplicate assays per experiment and 200 sperm assayed/sample. QNB, 3'-quinuclidinyl benzilate.   (0) were washed and incubated, as described previously (Florman and First, 1988b). ZP agonist (50 pg/ml) was added to sperm aliquots and acrosomal exocytosis deter-  (Ball et al., 1983;First and Parrish, 1987;First, 1988a, 1988b), thereby raising questions of the functional significance of positive modulators in seminal fluids. In order to assess the effects of seminal fluid constituents on gamete interaction directly, we examined fertilization of bovine eggs in vitro as a function of sperm concentration (Fig. 6). CESp were less efficient in fertilizing eggs than were EjSp, as indicated by the requirement for higher concentrations of the former preparation. Treatment of CESp with seminal fluids or with caltrin were sufficient to shift the concentration-response relationship to that of EjSp. Thus, the positive modulatory activity of caltrin plays a functional role during gamete interaction in uitro. DISCUSSION Previously, we (Florman and First, 1988b;Florman et al., 1989) and others (Miller et al., 1990) observed that bull sperm obtained from cauda epididymal fluids and from seminal fluids differ in response to ZP agonists. The central finding of this study is that a basic peptide isolated from seminal fluids accounts for this difference in agonist sensitivity. Application of this peptide to cauda epididymal sperm produces efficient coupling between stimulus, provided by ZP agonist, and cellular regulatory systems controlling CaZti and exocytosis. In contrast, control peptides of similar basic PI (cytochrome c, histone) had no effect on sperm signal transducing cascades.

Effects of Caltrin on Gamete Interaction-Bovine eggs may be fertilized in vitro by both EjSp and CESp
Bovine seminal fluids were previously shown to contain a similar peptide isolated independently as an inhibitor of sperm 45Ca2' sequestration by sperm and termed caltrin (Rufo et al., 1982(Rufo et al., ,1984Lardy and San Agustin, 1989), and by virtue of bactericidal activity, and designated seminalplasmin (Reddy and Bhargava, 1979;Theil and Scheit, 1983). Several lines of evidence indicate that the positive modulatory peptide identified herein is caltrin. First, these peptides share biological activities, with both displaying an inhibition of 45Ca2' transport and a positive modulation of ZP agonist-induced exocytosis. Second, caltrin and PMF are structurally related peptides, as suggested by extensive sequence similarity. Available quantities of PMF only permitted 40 cycles of sequence analysis, but this region of PMF was identical to caltrin sequences determined by both direct and deduced methods. These two peptides differ somewhat in apparent molecular mass (6500 and 5411, respectively), possibly as a consequence of the known anomalous migration of caltrin on acrylamide gels (Rufo et al., 1982;Lewis et al., 1985). Additional structural relationships are indicated by examination of peptide interactions with calmodulin. Caltrin binds CaM with high affinity, with the likely binding domain consisting of the central region of the peptide (Comte et al., 1986). Although CaM recognition is attributed to conserved charge distribution and secondary structural motifs, rather than to specific sequences (O'Neil and DeGrado, 1990), these observations nevertheless point to similarities between PMF and caltrin. Third, seminal fluids may be depleted of positive modulatory activity by CaM affinity chromatography and this activity can be partially restored by a synthetic caltrin. Rigorous demonstration of this identity requires complete sequencing of PMF and accumulation of the necessary quantities of material are in progress.
It is worth noting that caltrin accounts for some, although not all, of the PMF activity in seminal secretions (see Table  I11 in particular). Differences may reflect complex and poorly understood requirements for caltrin stability and function (San Agustin and Lardy, 1990) or contributions of other peptides to this activity. In this regard, it has been reported that heparin affinity chromatography of seminal fluids results in several retained peaks of PMF activity (Miller et al., 1990). It is plausible that additional factors (other than caltrin) are functional and this must be addressed in future studies.
Thus, caltrin has two effects on sperm Ca2+ homeostasis. Previously, Lardy and co-workers have shown that caltrin inhibited 45Ca2+ sequestration in sperm (Babcock et al., 1979;San Agustin et al., 1987), a process that largely reflects mitochondrial function. Yet, studies with plasma membrane preparations (Rufo et al., 1982(Rufo et al., , 1984 and with permeabilized cells (Breitbart et al.,199Ob) indicate that the peptide probably acts primarily by inhibiting plasma membrane ion transport, thereby effecting mitochondrial accumulation indirectly. It has been suggested that a Na'-Ca2+ antiporter, present in mammalian sperm (Bradley and Forrester, 1980), is the caltrin-sensitive transport pathway (Rufo et aL, 1984;Breitbart et al., 1990a) and that inhibition of this pathway contributed to the inhibition of spontaneous acrosomal exocytosis (Lardy and San Agustin, 1989). Further studies are required in order to determine the mechanism and functional significance of the caltrin-mediated inhibition of sperm Ca2+ transport, as well as the relationship (if any) between effects on 45Ca2' sequestration and on agonist-induced exocytosis.
Here, we report cross-talk between caltrin and ZP-agonist signaling pathways. Agonist signal transmission in sperm includes obligate elevation of internal ionic mediators, including Ca2+i (Florman et al., 1989(Florman et al., , 1992Storey et al., 1992). Caltrin modulates this pathway at an early stage, coupling external stimulus to the processes that regulate Ca2+i (Fig. 4). In mammalian sperm, these processes include a high voltagethreshold, dihydropyridine-sensitive (L-type) calcium channel (Babcock and Pfeiffer, 1987;Cox and Peterson, 1989;Cox et al., 1991;Florman et al., 1992) that is activated by ZP agonist and mediates an essential component of the Ca2+ influx required for the acrosome reaction (Florman et al., 1992). It is reasonable to propose that the L-type channel of sperm is functionally regulated by caltrin, and as a consequence may then be activated by agonist. A similar situation may pertain in muscle and neurons where the function of Ltype Ca" channels (Kazazogolou et al., 1983;Renaud et al., 1984Renaud et al., , 1989, as well as Na+ channels (Renaud et al., 1981;Zhang et al., 1992), nicotinic (Berg et al., 1989), and muscarinic receptors (Renaud et al., 1980) is modulated during embryogenesis. The mechanism of activation is not well understood, although conductance through somatic L-type channels is controlled by several protein kinases and by direct G protein interaction (Brown, 1990(Brown, ,1991Hille, 1991). Studies to determine whether similar regulatory pathways are present in sperm and are modulated by caltrin are in progress.
These experiments also provide some indication of the functional significance of caltrin during fertilization. Addition of caltrin to CESp enhances fertilizing ability (Fig. 6). Although bovine CESp will fertilize eggs in vitro (Ball et al., 1983;Parrish et al., 1988), relatively high sperm concentrations are required. A plausible mechanism that accounts for fertilization under these circumstances is based on the occurrence in mammalian sperm of spontaneous (ZP agonist-independent) exocytosis, in addition to the agonist dependent process. It may be that sperm, if present at the ZP surface and undergoing a spontaneous acrosome reaction, can proceed through the zona. Given previous estimates of the rate of spontaneous exocytosis in bovine CESp (=l%/h) (Florman and First, 1988b), it would be predicted that fertilization by this mechanism would require high concentrations of cells. Caltrin can account for the difference in fertility of CESp and EjSp that is produced by seminal fluids, thereby associating the development of the ZP3 signal transduction pathway with increased fertility of sperm populations.
The development of zona agonist signal transduction occurs gradually following treatment with caltrin, requiring approximately 1 h for completion. In contrast, a more protracted incubation is needed before coupling is apparent following application of unfractionated seminal fluids (=5 h). These data are consistent with the notion that seminal fluids contain both positive and negative modulators of fertility (Florman and First, 1988b;Florman and Babcock, 1990). The presence of negative regulators has been reported previously (Chang, 1957) and is indicated here by the delay in the development of signal transduction in sperm treated with unfractionated seminal fluids as compared to sperm treated with purified caltrin. The mechanism of positive modulation by caltrin and of negative modulation by other constituents must be defined.
In conclusion, a seminal fluid peptide with biological and biochemical properties similar to those of caltrin has been shown to regulate ZP agonist signal transduction pathways in bovine sperm. The most reasonable site of action is on the high voltage-sensitive, L-type Ca2' channel that is present in mammalian sperm (Babcock and Pfeiffer, 1987;Cox and Peterson, 1989;Florman et al., 1992) and that mediates zonapromoted elevations of Ca2+i (Florman et al., 1989).