Speract. Purification and characterization of a peptide associated with eggs that activates spermatozoa.

A low molecular weight peptide (speract) associated with sea urchin eggs has been purified to apparent homogeneity by charcoal adsorption, DEAE-Sephacel chromatography, Bio-Gel P-2 filtration, and Dowex AG 50W-X4 chromatography. Gametes from 5000 female sea urchins were required for the isolation of approximately 9 mg of the peptide. The isolated peptide is homogenous based on [3H]acetic anhydride labeling, gel filtration, and reverse phase high pressure liquid chromatography. Speract is composed entirely of neutral and acidic amino acids with glycine as the major component, and it appears to have a blocked NH2 terminus based on its insensitivity to leucine aminopeptidase, its failure to react with dansyl chloride, and its chromatographic behavior on strong cation exchange resins. Speract is a potent stimulator of sea urchin sperm oxygen consumption, causing significant increases of sperm respiration rates at concentrations as low as 10(-12) M and producing 20-fold increases of oxygen consumption at maximal concentrations of 10(-8) M. Sperm cyclic GMP and cyclic AMP concentrations are also increased by speract, but concentrations of at least 10(-10) M and 10(-9) M are required for half-maximal elevations, respectively. The peptide, purified from Strongylocentrotus purpuratus eggs, also cross-reacts with spermatozoa from Lytechnis pictus sea urchins, suggesting that speract does not show species specificity. These results represent the first report of the purification of a peptide associated with eggs that may affect spermatozoa under natural conditions.

A low molecular weight peptide (speract) associated with sea urchin eggs has been purified to apparent homogeneity by charcoal adsorption, DEAE-Sephacel chromatography, Bio-Gel P-2 filtration, and Dowex AG 50W-X4 chromatography. Gametes from 5000 female sea urchins were required for the isolation of approximately 9 mg of the peptide. The isolated peptide is homogenous based on [3H]acetic anhydride labeling, gel filtration, and reverse phase high pressure liquid chromatography. Speract is composed entirely of neutral and acidic amino acids with glycine as the major component, and it appears to have a blocked NH2 terminus based on its insensitivity to leucine aminopeptidase, its failure to react with dansyl chloride, and its chromatographic behavior on strong cation exchange resins. Speract is a potent stimulator of sea urchin sperm oxygen consumption, causing significant increases of sperm respiration rates at concentrations as low as 10"' M and producing 20-fold increases of oxygen consumption at maximal concentrations of M.
Sperm cyclic GMP and cyclic AMP concentrations are also increased by speract, but concentrations of at least M and M are required for half-maximal elevations, respectively. The peptide, purified from Strongylocentrotus purpuratus eggs, also cross-reacts with spermatozoa from Lytechnis pictus sea urchins, suggesting that speract does not show species specificity. These results represent the first report of the purification of a peptide associated with eggs that may affect spermatozoa under natural conditions.
Since 1928 when Gray fist demonstrated a stimulation of sea urchin sperm respiration rates by factors associated with the sea urchin egg (l), various studies have described the activating properties of "egg water" (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). The component or components responsible for these effects have not been successfully purified or characterized, although a number of studies have characterized the sperm respiratory stimulating component of egg water as being dialyzable, alcohol-soluble, and moderately heat-stable (6-9). At one time the stimulating factor was reported to be echinochrome A, the pigment from ripe eggs, but a number of subsequent studies have disputed these reports (10)(11)(12).
Here, we report the purification to apparent homogeneity of the sperm respiratory stimulating component associated with the eggs of the sea urchin Strongylocentrotus purpura-* This study was supported by National Institutes of Health Grants HD 10254, GM 07628, and GM 00058. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
tus. The component is a low molecular weight peptide which we have named speract, because of its general sperm-activating properties. It is composed of acidic and neutral amino acids and appears to contain a blocked or modified NH2terminal amino acid. Speract is a potent stimulator of sea urchin sperm respiration with half-maximal stimulations at 10"' M concentrations; it also produces rapid elevations of sperm cyclic AMP and cyclic GMP concentrations.
Dansyl' C1 and dansyl amino acid standards were purchased from Pierce Chemical Co. and polyamide thin layer plates were obtained from Brinkman. All enzymes were purchased from Sigma or Calbiochem-Behring. All other compounds were acquired from either Sigma or Fisher.

Methods
Gamete Collection and Incubation-Spermatozoa or eggs were obtained after the injection of sea urchins with 0.5 M KC1, and were washed as previously described (13). Spermatozoa were stored at 0-2°C as a suspension (100 to 150 mg weight/ml). Eggs were diluted approximately 1 volume of eggs to 4 volumes of buffer in acidified sea water (pH 5.0) containing 10 mM 2-(N-morpholino)ethanesulfonic acid to a final concentration of 20 to 60 mg wet weight eggs/ml. These conditions facilitated rapid removal of the egg jelly coat and resulted in a high yield of the low molecular weight factor capable of stimulating sperm respiration rates (12). After 20 min, the eggs were removed by gentle centrifugation (2000 X g for 20 min) and the supernatant fluid was stored for purification of speract. In all experiments, the cells were incubated in artificial sea water buffered to pH Determination of Respiration Rates-Respiration rates were determined using a Gilson K-IC Table Top Oxygraph equipped with' a 1.25-ml capacity temperature control chamber fitted with a Clarktype electrode. One hundred microliters of the sperm suspensions were added to 1.15 ml of sea water at 15"C, and after basal respiration has remained constant for 3 min, various agents were added and the new respiration rates were determined over the next 3 min.
Determinhtion of Cyclic Nucleotide Concentrations-The incubation mixture for the determination of cyclic nucleotide concentrations consisted of 4 to 6 mg (wet weight) sperm, 20 mM W(2-aceta-' The abbreviation used is: dansyl, Dns, 5-dimethylaminonaphtha-lene-1-sulfonyl. 1447 mido)iminodiacetic acid or 20 n m N-(2-acetamido)-2-aminoethanesulfonic acid in artificial sea water, 100 p~ 1-methyl-3-isobutylxanthine and the indicated concentration of speract in a final incubation volume of 0.5 ml . Incubations were started by the addition of sperm and were terminated by the addition of 1 ml of 0.5 N HClO, containing tracer amounts of cyclic [3H]AMP or cyclic r3H]GMP for estimation of cyclic nucleotide recoveries. Zero time cyclic nucleotide concentrations were estimated by adding sperm directly to the incubation mixture containing 0.5 N HClOA. The samples were then frozen and thawed five times and the cyclic nucleotides were subsequently purified on Dowex AG 50W-X8 (H' form) columns (0.7 X 25 cm) using the procedure of Schultz et al. (14).
The column fractions containing cyclic AMP or cyclic GMP were lyophilized, and then dissolved in 1.0 ml of deionized water. Cyclic AMP and cyclic GMP were determined by radioimmunoassay (15) with modifications by Harper and Brooker (16). Enzymatic Sensitivity of Speract-In order to determine the sensitivity of speract to various proteolytic enzymes, aliquots of solutions containing speract were tested for ability to stimulate sperm respiration after incubation with various enzymes. Incubations were carried out for 90 min at 37°C in sealed tubes with 100 pl of speract in a final incubation volume of 130 to 170 pl. The incubation mixture contained 50 pg of the respective enzymes, and the final concentrations of the following reagents: 80 mM Tris (pH 7.9), 8 mM CaC12 for trypsin, carboxypeptidase A, pronase, and subtilisin; 80 mM Tris (pH 7.9) for thermolysin; and 75 mM sodium acetate (pH 4.75), 3 mM dithiothreitol, and 1 mM EDTA for papain. Incubations with leucine aminopeptidase were carried out in 80 mM Tris (pH 7.9), 5 mM MgC1,. The reactions were generally stopped by placing the tubes in a boiling water bath for either 15 or 30 min. EDTA (11 mM) and N-ethylmaleimide (7.6 mM) were added to stop the thermolysin and papain reactions, respectively.
Dansyl Chloride Analysis-Derivatization of samples of isolated speract was carried out by the procedure of Gray (17). Samples were hydrolyzed for 12 to 18 h at 110°C in 6 N HC1 under vacuum either before or after derivatization. The dansyl derivatives were separated by two-dimensional thin layer chromatography on polyamide plat.es (18) and identification was made by comparison with standards.
Amino Acid Analysis-Amino acid analysis was carried out using a Beckman 121 amino acid analyzer equipped with a Durrum DC-6A ion exchange column (6 X 320 mm). Lyophilized samples were resuspended in 1.0 ml of 6 N HCI and hydrolyzed at 110°C for 24 h under a vacuum prior to analysis.   6 X 29 cm). The column was pre-equilibrated with 10 mM triethanolamine at pH 7.6, and after application of the sample (8.4 X IO7 units), it was washed with 200 ml of equilibration buffer. Greater than 95% of the respiratory stimulating activity was retained on the column. Speract was eluted from the column with a linear gradient of 0 to 300 mM NaCI. Respiratory stimulating activity was tested using 10 pl of a 1000-fold dilution of each fraction. buffer and 0.3-ml fractions were collected. Each fraction was tested for respiratory stimulating activity and for absorbance a t 206 nm. Peaks of absorbing material were subjected to acid hydrolysis and subsequent amino acid analysis.

P-4 Gel
13H]Acetic Anhydride Labeling-Acetic anhydride labeling of speract was accomplished with the following procedure: An aliquot of purified speract (74 nmol of glycine) was lyophilized to dryness in a 3.0-ml conical vial and redissolved in 100 pl of pyridine. Two hundred microliters of pyridine containing 1 mCi of [JH]acetic anhydride (500 mCi/mol) was added, and the vial was sealed and blended on a Vortex mixer. After 14 h at room temperature, 500 p1 of deionized water was added and the sample was incubated 6 h more. The sample was then frozen a t -70°C and lyophilized using a liquid nitrogen cold trap to collect the volatile radioactivity. Portions of the lyophilized sample were chromatographed on a Bio-Gel P-2 column (1.5 X 86 cm).
High Pressure Liquid Chromatography Analysis-High pressure liquid chromatography analysis of purified speract was carried out using either a Waters Associates system consisting of two 6MH)A solvent delivery units, an "660 solvent programmer, and a U6K injector or a Beckman model 322 chromatograph system. All separations were performed on a Cx Ultrasil column (4.6 mm X 25 cm) from Beckman and the column effluent was monitored for absorbance a t 206 nm using a Beckman model 155 variable wavelength detector with a 20-111 flow cell. In general, separations were carried out using the following program: with a constant flow rate of 1.0 ml/min, the column was first eluted with a mixture of 10% Solvent B (90% Solvent A) for 2 min, then a linear gradient from 10 to 60% Solvent B was developed over the next 25 min to 1 h. Solvent A consisted of 10 mM KHsPO, in water and Solvent B was acetonitrile.

RESULTS
Purification of S p e r a c t By using the ability of speract to stimulate sea urchin sperm respiration as an assay, the purification of speract was accomplished as outlined below.
Charcoal Column Chromatography-The supernatant fluid obtained from the egg treatment was applied to a column of activated charcoal (2.6 X 32 cm) which had been preequilibrated with water. The respiratory stimulating activity completely adsorbed to the charcoal, and after extensive washing with H20 and 50% ethanol, speract was eluted with 70% ethanol containing 0.2% NH,OH (Fig. 1). The fractions containing speract were pooled, flash-evaporated at 30"C, and the remaining aqueous solution was frozen and lyophilized.
DEAE-Sephacel Chromatography-The dried residue from the pooled charcoal column fractions was reconstituted in 10 mM triethanolamine buffer at pH 7.6 to a dilution of less than 1 mg of residue/ml, and the pH of the solution was readjusted to 7.6 with 0.5 N NaOH. This solution was then applied to a DEAE-Sephacel column (1.6 X 29.0 cm) which had been equilibrated with 10 m~ triethanolamine buffer at pH 7.6. The column was washed with 200 ml of the equilibration buffer and the respiratory stimulating activity was eluted from the column using a linear gradient from 0 to 300 mM NaCl in 10 mM triethanolamine at pH 7.6. Speract eluted from the column at approximately 100 mM NaCl (Fig. 2). P-2 Gel Filtration-The fractions from the DEAE-Sephacel chromatography which contained speract were desalted on a Bio-Gel P-2 column (5.0 X 23 cm) equilibrated with 0.2 M pyridine acetate at pH 5.0 and the fractions containing speract were then pooled and lyophilized.
Dowex 50 Chromatography-The lyophilized fractions containing speract were dissolved in 4.0 ml of 100 mM HCl. The acidified sample was then applied to a column (0.7 X 15 cm) of Dowex AG 50W-X4 (H') which had equilibrated with 100 mM HCl. Speract did not bind to the resin and was eluted with 100 mM HCl (Fig. 3). The fractions containing speract

Sensitivity of speract to various enzyme treatments
The respiration-stimulating activity was determined in samples of speract which had been incubated with 50 pg of the respective enzyme as described under "Experimental Procedures." The results are expressed as the percentage of respiratory stimulating activity remaining as compared to samples incubated with 50pg of denatured enzyme. were pooled, lyophilized, reconstituted in H20, and stored at -35°C.

Recovery
Based on half-maximal stimulation of sperm respiration,

The Purification
of Speract the recovery of speract through this purification scheme was approximately 14% (Table I).

Characterization of Speract
In order initially to establish the nature of the isolated respiratory stimulating material, the sensitivity of speract (based on ability to stimulate respiration) to various enzymatic treatments was examined (Table 11). After a 90-min incubation with subtilisin, thermolysin, pronase, and papain, the majority of the respiratory stimulating activity was destroyed while trypsin and leucine aminopeptidase failed to inactivate the factor. Based on these data it appeared that the respiratory stimulating component might be a peptide where Lys or Arg residues either were not present or did not donate a carbonyl function to a peptidyl linkage. Insensitivity to leucine aminopeptidase suggested the presence of a blocked or modified NHs-terminal amino acid. The chromatographic behavior of the respiratory stimulating activity on the strong cation exchanger Dowex 50 at low pH (Fig. 3) also suggested the absence of any primary amino groups in the presumed peptide (19). Furthermore, NHs-terminal analysis by the dansyl chloride method failed to identify any dansyl amino acids unless the isolated material was hydrolyzed prior to dansyl chloride derivatization (Fig. 4).

Assessment of Homogeneity
The isolated respiratory stimulating material appears homogeneous based on several criteria.

P-4 Gel Filtration
The isolated material migrated as a single peak of protein on Bio-Gel P-4 columns. Monitoring absorbance of the column effluent at 206 nm indicated one major peak of UV-absorbing material which co-migrated with the respiratory stimulating activity. The amino acid composition of this peak also appeared identical with the original material. A minor peak of 206 nm absorbing material appeared slightly beyond the total volume of the column. This peak varied in size in various runs and no detectable amino acids were observed before or after acid hydrolysis of this peak.

r3H]Acetic Anhydride Labeling
Incubation of the isolated speract with ["HJacetic anhydride yielded a single nonvolatile radioactive peak on Bio-Gel P-2 gel filtration (1.5 X 86 cm column) which eluted at the same position as unlabeled speract (Fig. 5).

High Pressure Liquid Chromatography Analysis
Further analysis of the isolated speract yielded one major peak of 206 nm absorbing material which co-chromatographed with the respiratory stimulating activity and corresponded to the only detectable peak of amino acid-containing material detected after acid hydrolysis of the various fractions (Fig. 6). This step again failed to alter the amino acid composition from that of the original material.

Amino Acid Composition of Speract
The amino acid composition of purified speract is consistent with the previously observed properties of the molecule (Table  111). The lack of Lys or Arg residues explains the resistance of speract to trypsin inactivation and the presence of Leu and Val explains its sensitivity to thermolysin treatment. The amino acid composition from three different preparations was identical and each preparation had the same relative biological specific activity of 6 X lo3 units/nmol of glycine. One unit of activity is defined as the amount of speract which produces a half-maximal increase in sperm respiration under standard conditions (1.25 ml final volume, pH 6.6 sea water, 15"C, and a final sperm concentration of 8 to 16 mg wet weight/ml).

Potency of Purified Speract
In addition to the effects of speract on sperm respiration, it also is capable of producing rapid elevations of sperm cyclic AMP and cyclic GMP concentrations (Fig. 7). Speract is more potent toward the stimulation of sperm respiration with half- maximal effects occurring at 3 X lo-" M (based on a molecular weight of 1885) than toward elevating cyclic nucleotide concentrations. Half-maximal elevations of cyclic GMP are at 2 X M while half-maximal elevations of cyclic AMP concentrations required at least 2 X 10" M speract. Speract is also capable of stimulating sperm fatty acid oxidation (20) with half-maximal effects at concentrations of lo-" M (data not shown).

DISCUSSION
A peptide associated with eggs has been purified that stimulates sperm respiration rates and elevates sperm cyclic AMP and cyclic GMP concentrations. This peptide, which we have named speract because it appears to act as a general sperm activator, is probably similar or identical with the sperm respiratory stimulating component previously studied in crude extracts of sea urchin eggs (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). The isolated speract appears homogeneous based on several criteria. Gel filtration profiles on Bio-Gel P-2 columns indicate one peak of protein.
['HI-Acetic anhydride labeling of the isolated material using a procedure designed to acetylate the hydroxyl groups of threonine and serine (21) produced one radioactive band with the same chromatographic behavior as unlabeled speract. The isolated material retained a constant amino acid composition through further chromatographic steps, including reverse phase high pressure liquid chromatography (Fig. 6), a technique which is considered a high resolution analytical technique for peptide analysis (22,23). The lack of prior reports on the purification and characterization of speract may be due to both its high potency and the low amounts associated with sea urchin eggs. In our studies with S. purpuratus eggs, we find approximately 5 pmol of speract/kg wet weight of eggs, based on estimates of biological activity extracted into acidified sea water. One kilogram of eggs is the approximate amount of eggs we collect from 5000 female sea urchins. Thus, these purification procedures were designed for large scale preparations, utilizing several thousand sea urchins.
From its insensitivity t o leucine aminopeptidase, failure to produce an amino acid derivative with dansyl C1, and the chromatographic behavior on Dowex 50, it can be concluded that speract contains a modified NH1 terminus. The lack of a primary amino function also results in speract not being reactive toward any standard peptide reagents such as fluorescamine or ninhydrin.
Speract is an extremely potent activator of sea urchin sperm respiration and fatty acid oxidation, but appears somewhat less potent at elevating cyclic AMP or cyclic GMP concentrations. The apparent discrepancy between the potency of speract toward the elevation of cyclic GMP concentrations and the stimulation of sperm respiration may not support the theory that cyclic GMP serves as a second messenger for speract (12,20). Analogous discrepancies have been previously observed, however, between physiological responses to a hormone and elevations of cyclic AMP. For example, although adrenocorticotropin appears to elevate cyclic AMP concentrations a t higher concentrations than required for the stimulation of steroidogenesis ( 2 4 ) , it appears that changes in intracellular receptor bound cyclic AMP actually occur a t much lower adrenocorticotropin concentrations (25,26). The changes in receptor bound cyclic AMP appear more closely related to the stimulation of steroidogenesis. If cyclic GMP is actually involved as a second messenger for speract, closer analysis of the compartmentalization of cyclic GMP may reveal a similar situation.
The occurrence of factors associated with either the female reproductive tract or the egg itself which can stimulate the metabolism, motility, or cyclic nucleotide metabolism of spermatozoa appears to be widespread in nature (27,28). It is now clear that in at least one animal, the sea urchin, the factor is a peptide. It is also clear that the peptide has extremely high biological potency. Since the jelly coat of eggs is actually a female secretion, it remains to be determined whether or not the peptide is synthesized by follicular cells of the ovary and deposited as a part of the jelly coat, or whether the peptide is synthesized and secreted by the egg itself.