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Biology of Reproduction 63, 42-48 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Trypsin/Acrosin Inhibitor Activity of Rat and Guinea Pig Caltrin Proteins. Structural and Functional Studies1

Daniel E. Winnicaa,b, Maria L. Novellaa, Andrea Dematteisa, and Carlos E. Coronel2,a,b

a Cátedras de Química Biológica, Facultades de Ciencias Médicas and b Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Córdoba, Argentina

ABSTRACT

Dramatic inhibition of trypsin activity by rat caltrin and guinea pig caltrin I was spectrophotometrically demonstrated using the artificial substrate benzoylarginyl ethyl ester. Approximately 6% and 21% of residual proteolytic activity was recorded after preincubating the enzyme with 0.22 and 0.27 µM rat caltrin and guinea pig caltrin I, respectively. Reduction and carboxymethylation of the cysteine residues abolished the inhibitor activity of both caltrin proteins. Rat caltrin and guinea pig caltrin I show structural homology with secretory trypsin/acrosin inhibitor proteins isolated from boar and human seminal plasma and mouse seminal vesicle secretion and share a fragment of 13 amino acids of almost identical sequence (DPVCGTDGH/K/ITYG/AN), which is also present in the structure of Kazal-type trypsin inhibitor proteins from different mammalian tissues. Bovine, mouse, and guinea pig caltrin II, three caltrin proteins that have no structural homology with rat caltrin or guinea pig caltrin I, lack trypsin inhibitor activity. Rat caltrin, guinea pig caltrin I, and the mouse seminal vesicle trypsin inhibitor protein P12, which also inhibits Ca2+ uptake into epididymal spermatozoa (mouse caltrin I), bound specifically to the sperm head, on the acrosomal region, as detected by indirect immunofluorescence. They also inhibited the acrosin activity in the gelatin film assay. Caltrin I may play an important role in the control of sperm functions such as Ca2+ influx in the acrosome reaction and activation of acrosin and other serine-proteases at the proper site and proper time to ensure successful fertilization.

calcium, male reproductive tract, seminal vesicles, sperm capacitation/acrosome reaction

INTRODUCTION

The presence of serine-protease inhibitors in the seminal plasma and in prostate and seminal vesicle secretions has been reported in several species of mammals [1–4]. Although functions for these inhibitors in fertilization have been proposed [5, 6], their actual role in reproduction is still unclear. It is well known that natural trypsin inhibitors (soybean trypsin inhibitor, lima bean trypsin inhibitor, and ovomucoid trypsin inhibitor) and synthetic trypsin inhibitors (p-aminobenzamidine, benzamidine) inhibit in vitro fertilization by blockage of mouse sperm binding to the zona pellucida (ZP) [7] and human sperm penetration through the ZP [8, 9]. It has also been reported that the sperm trypsin-like protease, acrosin, regulates Ca2+ influx and sperm acrosome reaction by progesterone in humans [10].

There is evidence that serine-protease inhibitors are associated with other physiological events that occur in areas other than the sperm head. In the boar, for example, a seminal plasma trypsin inhibitor protein inactivates the membrane-bound trypsin-like proteinase in the flagella of epididymal spermatozoa, which regulates the activity of adenylate cyclase [11]. This proteinase inhibitor seems to be involved in the regulation of sperm motility through the bicarbonate-sensitive adenylate cyclase system.

Most of the trypsin-like proteinase inhibitors isolated from mammals are small (5–15 kDa) and basic (pI > 8.0). A protein with inhibitory activity on extracellular Ca2+ uptake by epididymal spermatozoa was purified from bovine seminal plasma [12]. Its properties were characterized [13–15], and it was designated caltrin (calcium transport inhibitor) [16]. Two different molecular forms of caltrin (I and II) were later identified and sequenced in the guinea pig [17], rat [18, 19], and mouse [18, 20]. In the guinea pig, caltrin I binds to the acrosomal region of the sperm head and inhibits acrosomal exocytosis, whereas caltrin II binds to the principal segment of the tail and prevents the onset of sperm hyperactivated motility [21].

In the present study, we analyzed the structural homology among guinea pig, rat, mouse, and bovine caltrin. Inhibitory effects of caltrins on trypsin and acrosin activity have been tested by direct and indirect procedures. A molecular model for the structure of rat and mouse caltrin I and a functional role for these proteins in fertilization is proposed.

MATERIALS AND METHODS

Animals

Animals were housed in air-conditioned rooms under a photoperiod of 14L:10D. Food and water were available ad libitum, and the animals were killed with carbon dioxide gas. All experimental protocols were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Protein Purification

Caltrin Rat and guinea pig caltrins were purified from seminal vesicle contents as previously reported [17, 18]; bovine caltrin, obtained from seminal plasma, was a gift from Dr. Henry Lardy (Institute for Enzyme Research, The University of Wisconsin-Madison).

Mouse seminal vesicle trypsin inhibitor protein P12 Seminal vesicle secretion from approximately 50 mice was extracted in 50 ml of 5% acetic acid at 4°C [22]. After 30 min, the preparation was centrifuged at 17 000 x g at 2°C for 30 min. The supernatant was brought to 36% saturation with solid ammonium sulfate (the preparation was adjusted to pH 2.0 with 6 N HCl), stirred for 30 min, and allowed to stand at 0°C for 1 h. The sample was centrifuged again, and the supernatant was dialyzed overnight against several volumes of 0.5% acetic acid and then lyophilized.

The lyophilized sample was dissolved in a minimum volume of 25 mM Tris-HCl pH 7.6 and loaded onto a column (2.6 x 50 cm) of Sephadex G-50 superfine (Pharmacia LKB Biotechnology, Uppsala, Sweden) equilibrated with the same buffer. The elution was performed with the equilibration buffer at a rate of 4.0 cm/h at room temperature. The fractions containing trypsin inhibitory activity, which was detected by enzymatic assay, were pooled and loaded immediately onto a column (1.0 x 16 cm) of carboxymethyl-cellulose (CM-32, Whatman, Maidstone, UK) equilibrated with 25 mM Tris-HCl, pH 7.6. The elution was carried out applying a stepwise NaCl gradient from 0.1 to 0.4 M in equilibration buffer. Fractions with trypsin inhibitor activity were pooled, and the purity of the protein was analyzed.

The purity of all isolated proteins was assessed by SDS-PAGE according to the method of Schägger and von Jagow [23] or by HPLC using a Beckman (Fullerton, CA) gradient system with a dual pump, a 165 dual channel variable wavelength detector, and an IBM PC-based data system/controller (Software Beckman System Gold). A Beckman Ultrapore C8 (4.6 x 250 mm) column was used. Eluant A was trifluoracetic acid (TFA), and eluant B was 0.01% TFA in acetonitrile. The column was equilibrated with a mixture of 80% A/20% B for 1 h at a flow rate of 1 ml/min. After sample injection, a gradient from 20% B to 100% B for 1 h was applied. Proteins were detected by recording the absorbance at 280 nm.

Trypsin Inhibitory Activity

Trypsin activity was assessed spectrophotometrically at 253 nm using the synthetic substrate benzoylarginyl ethyl ester (BAEE) in 50 mM phosphate buffer, pH 7.8, at 25°C [24]. Trypsin inhibitory activity was determined by measuring the reduction of trypsin-catalyzed hydrolysis of BAEE after preincubating the enzyme with purified caltrin proteins. Each 3-ml assay contained 10 µg trypsin, 0.15 mM BAEE, and various amounts of buffered pure caltrin proteins. One enzyme unit hydrolyzes 1 µmole of substrate per minute under assay conditions. One inhibitor unit causes the reduction of the enzyme activity by one enzyme unit.

Acrosin Proteolytic Activity

Proteolytic activity of individual spermatozoa was determined using a gelatin substrate film assay following the procedure described by Liu and Baker [8]. Gelatin substrate film slides were prepared on one side of glass slides with a 5% gelatin solution. Epididymal spermatozoa treated with or without rat caltrin protein or mouse P12 for 1 h were spread on the surface of gelatin-coated slides and incubated at 37°C to allow proteolysis of the gelatin by acrosin. At intervals, the slides were observed by phase contrast microscopy. Formation of a clear halo around the sperm head indicated acrosin proteolytic activity.

Antibodies Production and Purification

Monospecific polyclonal antibodies were prepared by injecting purified proteins into adult male rabbits as described previously [25]. Approximately 0.2–0.3 mg of caltrin or mouse P12 protein in Freund's complete adjuvant (1:1 ratio) were injected s.c. into 2.5-kg male adult rabbits. Four weeks later, a second injection of 0.2–0.3 mg of caltrin or mouse P12 in Freund's incomplete adjuvant was administrated. The antisera titers reached satisfactory levels 2–4 wk after the second injection, and blood was collected from the rabbits at that time. Approximately 15 ml of serum was recovered from each rabbit. The antisera were decomplemented by heating at 60°C for 30 min and then stored frozen in aliquots at -20°C. The specificity and titers of the antisera were tested by Western blotting using 1–5 µg of pure caltrin.

Rat caltrin antiserum was purified by affinity chromatography in a protein A-Sepharose (Pharmacia LKB Biotechnology) column equilibrated with PBS containing 150 mM NaCl and 10 mM phosphate buffer at pH 7.4. The antiserum was eluted with 0.1 M glycine-HCl buffer at pH 2.0 [26]. Collected IgG fractions were immediately neutralized with 2 M Tris-HCl buffer (pH 7.5), and BSA was added to the samples up to 1% (v/v) final concentration. Purified antibodies were stored in small aliquots at -20°C until used.

Indirect Immunofluoresence

The experiments were performed according to the method of Irwing et al. [27] as described previously for guinea pig spermatozoa [21] using monospecific polyclonal antibodies against rat caltrin and mouse trypsin inhibitor P12 protein prepared in rabbits as described above and fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG. Washed rat and mouse epididymal spermatozoa were fixed with 0.5% formaldehyde in PBS at pH 7.4. After washing twice with PBS, the cells were incubated with rat caltrin or mouse trypsin inhibitor P12 protein, respectively, for 1 h and washed with PBS, and the cell suspensions were spread on acetone-cleaned glass slides to dry. Dried smears were fixed in absolute ethanol for 20 min and then treated with 3% normal goat serum for 1 h. The slides were washed with PBS and then covered with rat caltrin antiserum or mouse P12 antiserum, respectively. After 1 h, the slides were washed with PBS and then treated for 30 min with goat anti-rabbit IgG labeled with FITC diluted 1:80 in PBS. The preparations were washed with PBS for 1 h and then covered with a drop of FluorSave Reagent (Calbiochem, La Jolla, CA), and a coverslip was fixed in place. The preparations were observed in a fluorescence microscope (MicroStar IV, Leica, Buffalo, NY). Photographs were taken using AGFA PAN 400 film.

SDS-PAGE and Western Blotting

Electrophoresis was performed using the Mini-Protean II system (Bio-Rad Laboratories, Richmond, CA). Using the procedures reported by Laemmli [28], the stacking and running gels were 7.5% and 15.0% acrylamide, respectively. Following the procedures of Schägger and von Jagow [23], the gels were prepared with the following acrylamide/bisacrylamide mixtures: 4% T/3% C stacking gel, 10% T/3% C spacer gel, and 16.6% T/6% C separating gel. The electrode buffers were 0.2 M Tris-HCl at pH 8.9 for the anode and 0.1% SDS, 0.1 M Tris/0.1 M tricine at pH 8.25 for the cathode. Under these conditions, peptides and proteins in the range of 1 to 100 kDa were separated.

Proteins separated by SDS-PAGE were transferred to nitrocellulose membranes by electrotransference using a Trans Blot Cell (Bio-Rad) according to the method of Towbin [29] as previously described [18]. Membranes used for immunodetection of caltrin proteins were incubated with the corresponding antiserum. A goat anti-rabbit IgG conjugated with alkaline phosphatase (AP) or horseradish peroxidase (HRP) was used as the secondary antibody.

Bound conjugates were visualized by staining for enzymatic activity with 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt and nitro blue tetrazolium (NBT) for AP or with 4-chloro-1-napthol and hydrogen peroxide for HRP.

Reduction and Carboxymethylation

Reduction and alkylation of cysteine residues of rat and guinea pig caltrin were carried out with dithiothreitol and iodoacetic acid following the procedure of Allen [30], as described previously [17, 18]. Dried caltrins (1–2 mg) were dissolved in 1 ml of 6 M guanidine hydrochloride, 1 mM EDTA, and 0.1 M Tris-HCl (pH 8.3) saturated with nitrogen. Dithiothreitol was added (~3 mM), and the preparation was left at room temperature for 1 h. Iodoacetic acid neutralized with NaOH was added (~5 mM), and the reaction was allowed to proceed under nitrogen in the dark at 37°C for 1 h. A new cycle was initiated under similar conditions. At the end, 2-mercaptoethanol (1%) was added, and the mixture was dialyzed against 50 mM NH4HCO3/0.01% thiodiglycol. The protein samples were freeze-dried.

Sequencing

Proteins purified by cation exchange chromatography in CM-cellulose (native form) and reduced and alkylated (modified form) were sequenced at the University of Wisconsin Biotechnology Center (Madison, WI) as previously described [17, 18]. Both the gas phase and pulse liquid phase sequencers with on-line HPLC for phenylthiohydantoin analysis and the data system were from Applied Biosystems (Foster City, CA). Standard cycles and procedures were those described by Hewick et al. [31], and procedures and reagents were provided by the manufacturer.

Sequence Comparisons

Protein sequences were compared with those obtained by computer searches of the GenBank database using a BLASTP program (NCBI, Bethesda, MD).

Protein Assay

Protein concentration of native and chemically modified proteins was estimated with bicinchoninic acid following the procedure described by Smith [32]. Bovine serum albumin fraction V was used as the standard protein.

Chemicals

Bovine serum albumin, FITC-labeled goat anti-rabbit IgG, trypsin, bicinchoninic acid, and BAEE were from Sigma Chemical Co. (St. Louis, MO). All other reagents were of the highest quality commercially available and from the sources described previously [18, 33].

RESULTS

Inhibition of Trypsin by Rat Caltrin and Guinea Pig Caltrin I

Figure 1 shows the trypsin-inhibiting effects of native and reduced and carboxymethylated rat caltrin (A) and guinea pig caltrin I (B) proteins. Almost complete inhibition of the enzyme (95%) was registered with 0.22 µM of rat caltrin, whereas no inhibition was detected when the assay was carried out with 0.42 µM and 0.85 µM of reduced and alkylated protein (Fig. 1A). A similar inhibitor effect was determined with native guinea pig caltrin I, but more protein was required to obtain the highest inhibition (Fig. 1B). Thus, 78% and 96% inhibition of trypsin activity was recorded with 0.27 and 2.97 µM of guinea pig caltrin I, respectively. Reduced and carboxymethylated protein did not inhibit the enzyme at the concentrations used (1.54 and 3.08 µM).



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  		FIG. 1. Trypsin inhibition by rat caltrin (A) and guinea pig caltrin I (B). Trypsin (10 µg) was preincubated with various concentrations of native caltrin proteins (0.22, 0.44, and 0.89 µM rat caltrin [•], and 0.165, 0.275, and 2.97 µM guinea pig caltrin I [{blacksquare}]) or chemically modified caltrin proteins (0.42 and 0.85 µM rat caltrin [{circ}], and 1.54 and 3.09 µM guinea pig caltrin I [{square}]) in 50 mM phosphate buffer pH 7.8 at 25°C for 5 min. Residual trypsin activity was assessed with 0.15 mM BAEE as substrate. Data are average of results of triplicate experiments

Amino Acid Sequences

Protein databases were searched for homologies to caltrin proteins. Rat caltrin and guinea pig caltrin I sequences had significant amino acid homologies with several Kazal-type trypsin inhibitors from different tissues and types of seminal plasma. The 56-residue sequence of rat caltrin was identical (100% homology) to that of rat pancreatic secretory trypsin inhibitor II (PSTI-II) [34] and to the trypsin inhibitor protein isolated from rat liver [35]. Significant homology was also found with the sequences of human, bovine, porcine, ovine, and canine PSTIs, mainly in a central domain of 29 amino acids (residues 15–43 in rat caltrin, Fig. 2). The spatial position of six cysteine residues was well conserved in rat caltrin and the Kazal-type trypsin inhibitors.



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  		FIG. 2. Amino acid sequences of rat caltrin and PSTI proteins. Rat caltrin (1) is compared with those of human (2), porcine (3), bovine (4), ovine (5), and canine (6) PSTIs. Amino acids identical in all seven structures are in gray. Rat caltrin is identical in all residues to rat PSTI-II

Primary structure of rat caltrin overlapped a fragment of 24 amino acids (Leu13–Ile36) with that of guinea pig caltrin I (Leu21–Ile44), with 58.5% homology (Fig. 3, sequences 2 and 5). The greatest degree of overall homology between rat caltrin and guinea pig caltrin I was found in the fragment of 13 amino acids, DPVCGTDGK/HTYA/GN (residues 21–33 in rat caltrin and 29–41 in guinea pig I), whose sequence resembles that of a highly conserved segment in PSTIs and trypsin/acrosin inhibitors from seminal secretions (Figs. 2 and 3).



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  		FIG. 3. Amino acid sequences of trypsin/acrosin inhibitor proteins from seminal secretion of mammals. Amino acid sequences of rat caltrin (2) and guinea pig caltrin I (5) are compared with those of HUSI-II (1), mouse P12 (3), and BAI (4). Identical amino acids are indicated in gray

A comparison of the amino acid sequences of rat caltrin and guinea pig caltrin I with those of boar acrosin inhibitor (BAI) [1], human acrosin-trypsin inhibitor (HUSI-II) [36], and mouse seminal vesicle trypsin inhibitor protein P12 [22] is shown in Figure 3. Higher sequential homology was detected between rat caltrin and mouse P12. These two proteins overlapped along their whole peptidic chains (56 and 57 amino acid residues, respectively) with 64% homology (Fig. 3; sequences 2 and 3). Fragments with structural homology among those proteins are summarized in Tables 1 and 2.


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  		TABLE 1. Structural homology of rat caltrin with trypsin/acrosin inhibitors

Fragments of 24 to 56 amino acids from rat caltrin overlapped the sequences of mouse P12, guinea pig caltrin I, HUSI-II, and BAI with 64%, 58.5%, 57%, and 43% homology, respectively (Table 1). Fragments from guinea pig caltrin I were shorter (18–34 residues), but they overlapped the sequences of mouse P12, HUSI-II, and BAI with 67%, 54%, and 48% homology, respectively (Table 2).


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  		TABLE 2. Structural homology of guinea pig caltrin-I (GPC-I) with trypsin/acrosin inhibitors

Purification of Mouse Seminal Vesicle Trypsin Inhibitor P12

To study functional properties of rat caltrin and guinea pig I in comparison with those of seminal vesicle protease inhibitors, a mouse trypsin inhibitor protein that inhibits Ca2+ uptake of epididymal spermatozoa [20] was purified from seminal vesicle secretion by gel permeation and cation exchange chromatography. A protein peak with trypsin inhibitory activity, eluted from a CM-cellulose column, was revealed by SDS-PAGE as a single band of ~7 kDa. The sequence of the first 20 amino acids was identical to the N-terminal of the mouse trypsin inhibitor protein P12 (Fig. 3, sequence 3) reported by Lai [22]. The purified protein was injected into adult male rabbits to generate polyclonal antibodies.

Effect of Rat Caltrin and Mouse Seminal Vesicle Trypsin Inhibitor P12 on Acrosin Activity

After 15–17 h, the heads of approximately 70–80% of rat and mouse epididymal spermatozoa had protein-digested halos generated by acrosin activity on the gelatin substrate films (Fig. 4A). When rat and mouse sperm cells were preincubated with rat caltrin or mouse P12, respectively (0.1 mg/2 x 107 cells), only small refringent areas were observed in some cells after 72 h of incubation (Fig. 4B).



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  		FIG. 4. Acrosin activity of rat epididymal spermatozoa visualized by incubation on a gelatin substrate slide. Approximately 80% of the sperm digested the gelatin substrate film, producing a protein-free halo around the head at 15–17 h of incubation (A). Rat spermatozoa preincubated with rat caltrin (0.1 mg/2 x 107 cells) did not form as big or as clear protein-free halos even after 72 h (B)

Specific Binding of Rat Caltrin and Mouse Seminal Vesicle Trypsin Inhibitor P12 to the Sperm Surface

Washed and fixed rat and mouse epididymal spermatozoa were incubated with rat caltrin and mouse P12 (0.1 mg/2 x107 cells), respectively, and then treated with the corresponding rabbit-generated specific antibodies. Protein binding to the sperm surface was revealed by indirect immunofluorescence using FITC-labeled goat anti-rabbit IgG.

Rat caltrin bound on the acrosomal region of epididymal spermatozoa was visualized as a thin fluorescent zona on the convex margin of the head (Fig. 5A). No fluorescence was detected on the tail.



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  		FIG. 5. Binding of rat caltrin (A) and mouse protein P12 (C) to epididymal spermatozoa. The cells were exposed to each protein in a ratio of 0.5 mg/108 spermatozoa for 1 h. Specific binding on the cells was immunolocalized by an indirect immunofluorescence method using anti-rat caltrin and anti-mouse trypsin inhibitor protein antisera prepared in rabbits (primary antibodies) and goat anti-rabbit IgG conjugated with FITC. A and C) Immunofluorescence photomicrographs; B and D) corresponding phase contrast photomicrographs

Mouse P12 bound on the anterior region of the acrosome, as recently reported by Chen [20]. Occasionally, faint staining at the beginning of the principal tail of some cells was observed (Fig. 5C).

Spermatozoa incubated without rat caltrin or mouse P12 but submitted to antibodies treatment did not show immunofluorescence. No immunofluorescence was observed with cells preincubated with rat caltrin or mouse P12 but treated with normal rabbit serum.

DISCUSSION

The sequence of rat caltrin is identical (100% homology) to those of Kazal-type trypsin inhibitor proteins isolated from pancreas (PSTI-II) [34], and liver [35] of rats and consequently shows high homology with human, porcine, bovine, ovine, and canine PSTIs (Fig. 2). Otherwise, rat caltrin exhibits homology with guinea pig caltrin I (~60%) and other trypsin/acrosin inhibitors from the seminal vesicle secretion of boar [1], humans [36], and mice [22]. The greatest degree of homology among the sequences of these secretory proteins has been detected in a central domain of 13 amino acids, which closely resembles that of the highly conserved region in the PSTIs sequences (Figs. 2 and 3). Taking into account the amino acid homology between rat caltrin and the trypsin/acrosin inhibitors from seminal and pancreatic secretions, we propose for rat caltrin a molecular arrangement (Fig. 6) similar to that of boar seminal vesicle acrosin inhibitor reported by Tschesche [1]. This arrangement was developed using as a model the schematic diagram of the structure of bovine PSTI [37]. Rat caltrin can form three loops, A, B, and C, containing 9, 20, and 33 amino acid residues, respectively, stabilized by three disulfide bonds between six cysteine residues localized in homologous positions as in the Kazal-type trypsin inhibitor proteins (Fig. 2). The reactive site residues, Arg18–Asp19, are localized within loop B between cysteines II and III as in the structure of Kazal-type trypsin inhibitor proteins [37]. Mouse seminal vesicle protein P12, which has both a trypsin inhibitory effect and calcium transport inhibitor activity [20] in epididymal spermatozoa, may also present the same structural arrangement. We propose "caltrin I" as the most appropriate designation for these two secretory proteins from rat and mouse seminal vesicles.



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  		FIG. 6. Schematic representation of the covalent structure of rat caltrin. Filled circles indicate identical residues similarly located in the sequence of guinea pig caltrin I

Caltrin I from rats and guinea pigs showed potent inhibitor effect on trypsin activity as registered by spectrophotometric enzyme assay (Fig. 1). This inhibition was completely abolished by reduction and alkylation of cysteine residues, indicating the important role of the disulfide bridges on the biological activity of caltrin proteins as reported previously [17, 18, 21].

Rat and mouse caltrin I bound to the head of epididymal spermatozoa on the acrosomal region, as demonstrated with guinea pig caltrin I [21], and inhibited acrosin activity, as assessed by the gelatin substrate film method. Absence of gelatin digestion by epididymal spermatozoa coincubated with excess caltrin I may be the consequence of acrosin inhibition, blockage of proacrosin/acrosin activation, or inhibition of acrosin release from the acrosome.

The inhibitor effect of rat caltrin I on proacrosin/acrosin activation or on acrosin activity may have physiological significance because the enzyme [38] and its zymogen precursor have been identified as molecules involved in sperm-ZP binding and sperm penetration through the ZP [39]. Several authors have proposed that trypsin inhibitors block fertilization by preventing the primary sperm-ZP binding and/or sperm penetration of the ZP [7–9, 40]. By contrast, we determined that rat caltrin with its high trypsin-inhibiting activity promotes sperm-ZP binding [41]. The proportion of fertilized eggs is reduced by the presence of excess amount of caltrin in the medium [42]. Apparently, rat caltrin I facilitates the primary sperm-ZP3 binding, but excess caltrin inhibits ZP3-induced acrosome reaction and subsequent sperm penetration through the ZP. Experiments using monoclonal anti-acrosin antibodies revealed that these antibodies block ZP penetration by spermatozoa even though sperm attachment to and initial entry into the ZP take place [43]. These results suggest that the availability of the acrosin protease determines the ability of sperm to penetrate the ZP but not the ability to binding to the ZP. This interpretation is consistent with the proposals of Crosby [44] and Jansen [39].

Calcium-induced hyaluronidase release is inhibited by guinea pig caltrin I [21] and by rat caltrin I [45]. Blockage of acrosomal enzymes release that results from sperm Ca2+-uptake inhibition by caltrin suggests inhibition of the acrosomal exocytosis.

Rat caltrin I, guinea pig caltrin I, and mouse caltrin I or P12 protein are synthesized in the secretory epithelium of the seminal vesicles under strict androgenic control [33, 20, 46] and share structural and functional features that indicate their identical functional role. These three proteins appear to be engaged in the fertilization process, regulating not only the onset of the acrosome reaction but also sperm-ZP recognition, binding, and penetration, where the proacrosin/acrosin system seems to have a significant role.

ACKNOWLEDGMENTS

The authors thank Drs. Antonio Blanco and Ryuzo Yanagimachi for critical review of the manuscript.

FOOTNOTES

First decision: 4 November 1999.

1 This work was supported by a grant from the Agencia Nacional de Promoción Científica y Tecnológica (PICT 01225, Préstamo BID 802/OC-AR) and grants from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Consejo de Investigaciones Científicas y Tecnológicas de la Provincia de Córdoba (CONICOR), and Secretaría de Ciencia y Tecnología de la Universidad Nacional de Córdoba. C.E.C. is a Career Scientist of CONICET, and A.D. is a Fellow of CONICOR. Back

2 Correspondence: Carlos E. Coronel, Cátedra de Química Biológica, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Casilla de Correo 35, Suc. 16, 5016-Córdoba, Argentina. FAX: 54 351 433 3072; ccoronel{at}biomed.uncor.edu Back

Accepted: February 8, 2000.

Received: October 5, 1999.

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