Chicken Acrosin: Extraction and Purification1

Chicken Acrosin: Extraction and Purification 1 DAVID P. FROMAN Department of Poultry Science, Oregon State University, Corvallis, Oregon 97331 (Received for publication July 25, 1989)

1990 Poultry Science 69:812-817 INTRODUCTION

The discovery of proteolytic activity in chicken spermatozoa (Buruiana, 1956) was complemented by the discovery that the hen oocyte's perivitelline layer was subject to proteolysis (Saeki and Morichi, 1959). Yanagimachi and Teichman (1972) demonstrated that the proteolytic activity of chicken spermatozoa was limited to the acrosome. Subsequently, the hypothesis that acrosomal proteolytic activity is essential for fertilization was tested in vitro (Howarth and Digby, 1973; Ho and Meizel, 1975) and in vivo (Palmer and Howarth, 1973). The conclusion reached was that spermatozoal proteolytic activity is required for fertilization. The biochemical nature of spermatozoal proteolytic activity in the chicken was first investigated by Ho and Meizel (1970). They used electrophoresis to resolve distinct protein bands characterized by amidolytic activity. Such activity was suppressed by trypsin inhibitors. The existence of multiple-molecular-weight species with trypsin-like activity was confirmed by Lessley and Brown (1974) via gel filtration. Mclndoe and Lake (1974) corroborated the findings reported by Yanagimachi and Teichmen (1972) by demonstrating that > 99.7% of the seminal trypsin-like activity in chickens is associated with spermatozoa. Ho and Meizel (1976) demonstrated

similarities in the pH optimum, substrate specificity, inhibition, and other characteristics between bovine trypsin and an enzyme activity extracted from chicken spermatozoa, attributing such activity to the enzyme acrosin (EC 3.4.21.10). Apart from the work of Ho (1974), wherein acrosin was purified 4.3 fold by gel filtration, no method has been published whereby chicken acrosin can be procured with greater purity. In previous work (Ho and Meizel, 1970; Langford and Howarth, 1974; Ho and Meizel, 1976), acrosin activity typically was extracted by sonication, the use of detergents, repeated freezing and thawing, and other methods. However, in the author's experience, these approaches were inefficient in terms of extracting acrosin from turkey spermatozoa; and this realization led to the use of urea as an extraction agent (Richardson et al, 1988). Chicken acrosomes are morphologically similar to turkey acrosomes (Thurston and Hess, 1987). Therefore, the objectives of the present work were to: 1) test the efficacy of urea as an agent for solubilizing chicken acrosin; and 2) describe a method whereby chicken acrosin could be purified. MATERIALS AND METHODS

Urea as an Extraction Agent Ejaculates from 24 Single Comb White 'Oregon State University, Technical Paper Number

8959.

Leghorn (SCWL) roosters were pooled in order

to provide 11 to 12 mL of semen. A 812

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ABSTRACT The objective of the present research was to identify a procedure whereby chicken acrosin could be purified. Acrosin, as evidenced by amidase activity, was extracted with urea most efficiently at a concentration of 6 M. Extraction efficiency was enhanced by spermatozoal lysis prior to admixture with 6 M urea. Lysis was induced by passage of spermatozoal suspensions through a French pressure cell. Acrosin was purified by using gel filtration, chromatofocusing, and affinity chromatography. Based on amidase activity, a 19-fold purification was obtained with a 28% recovery. Native electrophoresis resolved two major protein bands with proteolytic activity. The methods described afford the procurement of milligram amounts of chicken acrosin. {Key words: acrosin, chicken, purification, urea, spermatozoa)

ACROSIN PURIFICATION

Acrosin Extraction In each of five replicate trials, approximately 50 mL of pooled semen from 125 SCWL roosters was centrifuged at 1,200 times g for 20 min at 4 C. The supernatant was discarded, and the spermatozoa were resuspended to volume in ice-cold, .85% (wt/vol) of sodium chloride. Such centrifugation and resuspension were performed a total of four times. However, after the final centrifugation, the spermatozoa were resuspended in a total volume of 40 mL. During these manipulations, the spermatozoal suspensions were either refrigerated or were kept on ice. Following the determination of spermatozoal concentration and viability (Bilgili and Renden, 1984), the spermatozoal suspension was loaded into a prechilled, French pressure cell3 . The suspension was extruded at 24,000 pounds per square inch (1,687,392 g per cm2). The lysate was kept on ice prior to centrifugation at 1,200 times g for 30 min at 4 C. The supernatant was discarded, and the contents were returned to volume with 6 M urea. Immediately thereafter, the spermatozoal debris was resuspended thoroughly. Then the cell suspension was centrifuged at 1,200 times g for 30 min at 4 C. The

''Sigma Chemical Co., St. Louis, MO. 'American Instrument Co., Silver Spring, MD. 4 Sigma Chemical Co., St. Louis, MO. Bio-Rad Laboratories, Richmond, CA. 6 A-3912, Sigma Chemical Co., St. Louis, MO. An anion exchanger, Sigma Chemical Co., St. Louis, MO. 8 Sigma Chemical Co., St. Louis, MO.

supernatant was placed within a siliconized Erlenmeyer flask, which was kept on ice. Acrosin Purification In each of five replicate trials, approximately 35 mL of extract was applied to a column (2.5 by 19.5 cm) of Sephadex G-25 Medium4 that had been equilibrated previously with .025 M imidazole-acetic acid, pH 7.4, containing 4 M urea. Gel filtration and subsequent chromatography were performed at 5 C. Once the entire volume of extract had permeated the Sephadex, 35 mL of imidazole buffer were applied to the column; and 35 mL of eluate were collected into a siliconized Erlenmeyer flask. The amidase activity was determined as specified previously. The protein concentration was determined by using the Bio-Rad protein assay,5 with bovine serum albumin6 (BSA) as the standard. The Sephadex eluate was applied at a flow rate of 160 mLh -1 to a column (1.5 by 26 cm) of Polybuffer Exchanger 947 (PE 94) previously equilibrated with .025 M imidazole-acetic acid, pH 7.4. Once the sample application was complete, eight column volumes of imidazole buffer were passed through the anion exchanger at the same rate of flow. The amidase activity was eluted by a pH gradient, generated by titrating the anion exchanger (PE 94) with a 1: 12.5 dilution of Polybuffer 96,8 (PE 96) adjusted to pH 5.5 with glacial acetic acid. The diluted PE 96 was passed through the anion exchanger at a 48-mLh_1 flow rate. The eluate was collected into siliconized test tubes in 7.5-mL fractions. The pH and amidase activity for each fraction were determined. Fractions containing >40% of the maximal activity were pooled in a siliconized beaker, which was kept on ice. The amidase activity and protein concentration were determined for this mixture. Sodium chloride and BSA were dissolved in the chromatofocusing eluate in order to yield concentrations of .1 M and . 1 % (wt/vol), respectively. Sodium chloride was added to increase the ionic strength, whereas BSA was used to protect the amidase from autolysis prior to affinity chromatography. The pH of this solution was adjusted to 8.0 by adding NaOH prior to loading on a column (1.6 by 14 cm) of paminobenzamidine-agarose,8 equilibrated previously with .025 M triethanolamine-HCl, pH 8.0, containing .1 M NaCl. Once the sample application was complete, six column volumes of the triethanolamine buffer were passed through the

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1-mL subsample of pooled semen was placed into each of 10 microcentrifuge tubes, each one holding 1.5 mL. These tubes were centrifuged at 12,800 times g for 3 minutes. Seminal plasma supematants were discarded, and an equivalent volume of distilled water or distilled water containing 1, 2, 3, 4, 5, 6, 7, or 8 M urea was added to each tube. Following spermatozoal resuspension, the tubes were centrifuged as just described. Amidase activity within each supernatant was determined according to Richardson et al. (1988) using benzoyl arginine p-nitroanilide (BAPNA)2 as the substrate. Each activity was expressed as a percentage of the maximum activity observed. Percentages from three replicate trials were plotted against the concentration of urea by iterative least squares.

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FROMAN

Two milligrams of lyophilized protein were dissolved in .5 mL of distilled water. Proteolytic activity was determined with a 15-u.L subsample of this solution, using the Bio-Rad protease assay.9 Native electrophoresis was performed according to Richardson et al. (1988), using 125 |j.g of protein per lane. The proteolytic activity of the protein bands so resolved was determined by overlaying the polyacrylamide gel with a 1% (wt/vol) agar gel containing casein.9 RESULTS AND DISCUSSION

Urea was tried as an agent for extracting chicken acrosin for the following reasons. First, Richardson et al. (1988) found 4 M urea to be very effective in solubilizing turkey acrosin. Second, once solubilized, turkey acrosin could be partially purified by chromatofocusing, which requires the use of buffers with a low ionic strength. The presence of urea had no effect on the acrosin adsorption to or the elution from the anion exchanger. As shown in Figure 1, 6 M urea was most effective in solubilizing the amidase activity from chicken spermatozoa. The decreased amidase activities of the extracts prepared with 7 and 8 M urea were attributed to the denaturing action of the concentrated urea solutions. Based on preliminary work, there was no need to freeze the spermatozoa suspended in urea; nor was there any benefit in sonicating

^Bio-Rad Laboratories, Richmond, CA. 1( Tharmacia Fine Chemicals, Uppsala, Sweden.

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FIGURE 1. Solubilized spermatozoal amidase activity (O) as a function of the urea concentration. The data points shown are from three replicate trials.

such suspensions after thawing, as was done with turkey spermatozoa (Richardson et al., 1988). However, approximately 20% more amidase activity was recovered when the spermatozoa were lysed by passage through a French pressure cell prior to extraction. The recovery of lysate was 97% (CV equals 1.4%). Consequently, subsequent extractions with 6 M urea were preceded by spermatozoal lysis. These procedures resulted in the solubilization of 90% of the spermatozoal amidase activity. Repeated washing of the spermatozoa reduced the concentration of extracellular protein from approximately 4 mg per mL to 10 u.g per mL. As evidenced by ethidium bromide exclusion, the final suspensions of spermatozoa contained an average of 93% viable spermatozoa (CV equals 5.3%). Therefore, the washing procedure selectively removed seminal plasma proteins, which include a proteinase inhibitor (Lessley and Brown, 1978). This may explain why Mclndoe and Lake (1974) extracted approximately 20% more amidase activity from washed than from unwashed chicken spermatozoa. In contrast with the method of Richardson et al. (1988), gel filtration was performed prior to chromatofocusing. According to literature published by Pharmacia Fine Chemicals,10 sample composition is critical if the proteins are to be separated successfully by chromato-

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Confirmation of Proteolytic Activity and the Electrophoretic Assessment of Purity

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agarose. The amidase activity was eluted with .25 M acetate-acetic acid, pH 4. Both the sample application and elution were performed at a 75-mLh-1 flow rate. The eluate was collected as 7.5-mL fractions into 10, siliconized test tubes. The pH and amidase activity for each fraction were determined. The two to three fractions containing the preponderance of amidase activity were pooled. The amidase activity and protein concentration were determined for this mixture. Prior to lyophilization, the amidaseenriched solution was dialyzed (molecular weight cutoff = 8,000 daltons) overnight against 1 mM HC1.

815

ACROSIN PURIFICATION

TABLE 1. Recovery of spermatozoal amidase activity and specific activity, according to the purification step1

Purification step

Recovery of amidase activity (%)

Gel filtration 95 ± 1 Chromatofocusing 54 ± 4 Affinity chromatography 28 ± 3 1

6

11

16

21

26

31

36

41

46

51

56

61

FRACTION NUMBER

focusing. In particular, the ion strength should be <.05, and the sample pH should be equivalent to that used to equilibrate the polybuffer exchanger. Richardson et al. (1988) applied sample volumes equivalent to 45% of column bed volume; in the present work, though, sample volumes equivalent to 75% of bed volume were used. Therefore, gel filtration was performed in order to desalt die extract and to transfer the extracted proteins into the imidazole buffer. As shown in Table 1, 95% of extracted amidase activity was recovered following gel filtration. Chromatofocusing resolved distinct amidase activities (Figure 2), which represent proteins with different isoelectric points. The primary peak of amidase activity was eluted at a pH of 6.23 (CV = .56%). Thus, the pi of chicken acrosin seems to be 6.2; this value is similar to the pi of 6.4 observed following the chromatofocusing of turkey acrosin by Richardson et al. (1988). As shown in Table 1, 54% of the extracted amidase activity was recovered following chromatofocusing. Affinity chromatography utilizing the acrosin inhibitor p-aminobenzamidine as the immobilized ligand afforded a further purification of the spermatozoal amidase activity (Figure 3). As shown in Table 1, the amidase activity was purified 19-fold by chromatofocusing and by affinity chromatography, with a 28% recovery. While the purification factor is comparable to that reported by Richardson et al. (1988), the recovery of chicken acrosin was 18.4 percentage units greater than that obtained

428 ± 77 6,895 ± 669 8,171 ± 961.

'Each value represents a x ± SEM from five replicate trials.

for turkey acrosin. This difference in the present work is attributed to the utilization of a more-favorable ratio between the binding capacity and die amount of enzyme applied. As shown in Figure 4, the protein eluted from p-aminobenzamidine agarose was proteolytic. Based on the hydrolysis of BAPNA at a pH of 8.1, the inhibition of amidase activity with the acrosin inhibitor tosyl lysine chloromethyl ketone (Schleuning and Fritz, 1976), protein binding to p-aminobenzamidine, and the hydrolysis of casein, the author concluded that the purification of amidase activity (Table 1) indicated the purification of acrosin. Two major and several minor protein bands were resolved by native electrophoresis of protein obtained by affinity chromatography (Figure 5). Based on the proteolysis within an agar gel containing casein (data not shown),

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FIGURE 3. Elution of amidase activity (O) from paminobenzamidine agarose. The dissociation of protein from the immobilized ligand was induced by a reduction in the pH (A).

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FIGURE 2. Fractionation of spermatozoal amidase activity (O) by chromatofocusing. The amidase activity was eluted by the development of a pH gradient (A). Peaks of activity denote proteins with distinct isoelectric points. In this particular case, Fractions 38 to 55 were pooled prior to further purification by affinity chromatography.

Specific activity (IU per mg of protein)

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FROMAN

which was overlaid on a replicate of the acrylamide gel shown in Figure 5, proteolytic activity was associated with the major bands alone. The specific activity for turkey acrosin similarly purified (Richardson et al., 1988) was 1.86 times greater than the 8,171 IU per mg shown for chicken acrosin in Table 1. Therefore, the proteins within minor bands may represent contaminants. However, and more likely, these proteins probably represent inactive fragments of acrosin formed by proteolysis (Mueller-Esterl and Fritz, 1981). The resolution of amidase activities by chromatofocusing and the demonstration of distinct forms of acrosin following native electrophoresis corroborate the observations of Ho and Meizel (1970) as well as those of Lessley and Brown (1974). Therefore, following the conversion of chicken proacrosin into acrosin, as suggested by Brown and Hartree (1976), the outcome of the present study establishes the fact that chicken acrosin exists in multiple-molecular-weight forms similar to mammalian acrosin (Parrish and Polakoski, 1979). In summary, a simple technique has been described for the extraction of chicken acrosin. Furthermore, a purification scheme developed for the purification of turkey acrosin by Richardson et al. (1988) was modified for the large-scale purification of chicken acrosin. Milligram amounts of chicken acrosin may be procured by the method described herein.

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FIGURE 5. Resolution of proteins recovered from affinity chromatography via polyacrylamide gel electrophoresis. The protein bands were stained with Coomassie Blue R-250. The replicate bands denoted by arrows were characterized by proteolytic activity when a second polyacrylamide gel was overlaid with a 1% (wt/vol) agar gel containing casein.

ACKNOWLEDGMENTS

The author thanks Jim Soodsma, Oregon State University Chemistry Department, for advice regarding the use of the French pressure cell and John D. Kirby for the preparation of the line graphs. REFERENCES Bilgili, S. F., and J. A. Renden, 1984. Fluorometric determination of avian sperm viability and concentration. Poultry Sci. 63:2275-2277. Brown, C. R„ and E. F. Hartree, 1976. Comparison of

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FIGURE 4. Agar gel containing casein used to assess the proteolytic activity of protein recovered from affinity chromatography. The well on the left received 15 uL of distilled water. The well on the right received 15 UL of water containing .8 |ig of protein.

ACROSIN PURIFICATION

properties of a proteinase inhibitor from chicken seminal plasma. Biol. Reprod. 19:223-234. Mclndoe, W. M., and P. E. Lake, 1974. The distribution of some hydrolytic enzymes in the semen of the domestic fowl, Galius domesticus. J. Reprod. Fertil. 40: 359-365. Mueller-Esterl, W., and H. Fritz, 1981. Sperm acrosin. Methods Enzymol. 80:621-632. Palmer, M. B., and B. Howarth, Jr., 1973. The requirement of a trypsin-like acrosomal enzyme for fertilization in the domestic fowl. J. Reprod. Fertil. 35:7-11. Parrish, R. F., and K. L. Palakoski, 1979. Mammalian sperm proacrosin-acrosin system. Int. J. Biochem. 10: 391-395. Richardson, M. E., A. B. Bodine, D. P. Froman, and R. J. Thurston, 1988. Turkey acrosin. I. Isolation, purification, and partial characterization. Biol. Reprod. 38: 645-651. Saeki, Y., and T. Morichi, 1959. Action of semen inducing rupture of yolk membrane in chicken egg. Proc. See. Exp. Biol. Med. 101:648-650. Schleuning, W. D., and H. Fritz, 1976. Sperm acrosin. Methods. Enzymol. 65:330-342. Thurston, R. J., and R. A. Hess, 1987. Ultrastructure of spermatozoa from domesticated birds: Comparative study of turkey, chicken, and guinea fowl. Scanning Microsc. 1:1829-1837. Yanagimachi, R., and R. J. Teichman, 1972. Cytochemical demonstration of acrosomal proteinase and avian spermatozoa by a silver proteinate method. Biol. Reprod. 6:87-97.

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neutral proteinase activities in cock and ram spermatozoa and observations on a proacrosin in cock spermatozoa. J. Reprod. Fertil. 46:155-164. Buruiana, L. M., 1956. Sur l'activite hyaluronidosique et trypsinique du sperme. Naturwissenschaften 43:523. Ho, J.J.L., 1974. Page 21 in: Biochemistry and physiology of acrosin from the spermatozoa of the domestic fowl, Galius domesticus. Ph.D. diss., University of California, Davis, CA. Ho, J.J.L., and S. Meizel, 1970. Electrophoretic detection of multiple forms of trypsin-like activity in spermatozoa of the domestic fowl. J. Reprod. Fertil. 23:177-179. Ho, J.J.L., and S. Meizel, 1975. Hydrolysis of the hen egg vitelline membrane by cock sperm acrosin and other enzymes. J. Exp. Zool. 194:429-437. Ho, J.J.L., and S. Meizel, 1976. Biochemical characterization of an avian spermatozoan acrosin and comparison of its properties to those of bovine trypsin and mammalian acrosins. Comp. Biochem. Physiol. 54B:213-218. Howarth, B., Jr., and S. T. Digby, 1973. Evidence for the penetration of the vitelline membrane of the hen's ovum by a trypsin-like acrosomal enzyme. J. Reprod. Fertil. 33:123-125. Langford, B. B., and B. Howarth, Jr., 1974. A trypsin-like enzyme in acrosomal extracts of chicken, turkey, and quail spermatozoa. Poultry Sci. 53:834-837. Lessley, B. A., and K. I. Brown, 1974. Release of multiple molecular weight forms of trypsin-like enzyme from frozen-thawed chicken spermatozoa. Poultry Sci. 53: 52. (Abstr.) Lessley, B. A., and K. I. Brown, 1978. Purification and

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