Properties of A Human Seminal Plasma Inhibitor for Trypsin*

Vol. 22. No.6, June 1971

FERTILITY AND STERILITY

Copyright

©

Printed in U.S.A.

1971 by The Williams & Wilkins Co.

PROPERTIES OF A HUMAN SEMINAL PLASMA INHmITOR FOR TRYPSIN* CLAUDIUS HIRSCHHAUSER, DR.

MED.,

t

AND

MANFRED KIONKE

Dermatological Clinic and Polyclinic, Philipps-University, Marburg/Lahn, Western Germany

Human seminal plasma inhibits the fibrinolytic activity of trypsin, as described by Rasmussen, and Albrechtsen. 1 Haendle, Fritz, Trautschold, and Werle; 2 and Ingrisch, Haendle, and Werle, 3 measured the amount of the inhibitor for trypsin in human seminal plasma. They suggested that the inhibitor is formed under hormonal control in seminal vesicles of mice. Rabbit spermatozoa contain a trypsin-like enzyme, which is important for the penetration ofthe spermatozoa into the zona pellucida. 4-6 This part of the ovopenetration is suppressed by soybean trypsin inhibitor 6 and also by rabbit sperm inhibitor for the trypsin-like enzyme. 7 The previous studies on the human seminal plasma inhibitor have been made with crude seminal plasma. To find out more about function, importance, and properties of this human seminal plasma inhibitor, we thought it necessary to purify the inhibitor and to specify it more closely.

hydrochloride (TRA) buffer, pH 7.3, and then homogenized with an "Ultra-Turrax" (Jahnke and Kunkel) in the same buffer. The spermatozoa concentration was about 100 million/ml. The spermatozoa were counted in a Thoma's chamber after staining with fuchsin. One gram of human seminal vesicles (2 hr. after the operation) was homogenized in 9 ml. of 0.25 M sucrose-TRA buffer, pH 7.3, with an "Vltra-Turrax", and then centrifuged for 15 min. at 30,000 X g and -4° C. The supernatant was taken for examination. The fructose content was determined according to the Seliwanoff method (resorcin/ HCI), as described by Tonutti, Weller, Schuchhard, and Heinke. 8 The protein content was determined according to Lowry, Rosebrough, Farr, and Randall. 9 Fibrin agar electrophoresis and fibrin plate diffusion were made according to Heimburger, and Schwick. 1o

Definition of Inhibitor Units

MATERIAL AND METHODS

We examined seme:p. obtained by masturbation from the Andrological Department. The liquified semen was centrifuged for 15 min. at 3000 r.p.m. The supernatant was frozen at - 21 ° C. When required for examination, the seminal plasma was brought to room temperature and then centrifuged again. The clear supernatant was used. The spermatozoa sediment was washed twice with 0.25 M sucrose-triethylamine * Presented in part at a annual meeting of the German Society for the Study of Fertility, Hamburg, 1970. t Present address: 4 Dusseldorf, Universitats-HautkIinik, Moorenstrall,e 5, Germany.

According to Trautschold (cited by Vogel, Trautschold, and Werle l l), one inhibitor unit is that inhibitor content, which, under standard conditions, is able to inhibit one enzyme unit completely (1 LV. = 1000 m. LV.).

Enzyme Solutions The following enzyme solutions were used in our studies. Trypsin Trypsin, 10 mg. (Boehringer 15330 Etab) was diluted in 100 ml. of 10 -3 M hydrochloric acid and kept at - 21 ° C.

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HUMAN SEMINAL PLASMA INHIBITOR

TABLE 1. Volume

mi.

Seminal plasma Supernatant after treatment with "Sephazyme TRY" Washing water 1 Washing water 2 Washing water 3 Inhibitor fraction 1 Inhibitor fraction 2

Protein

Protein

100

11

14

73

5

0.2

0.125

100

,

42

5.3 1.9 0.6 0.48

Inhibitor amount

c·

mg./ml.

5

5 5 5 8

LV/m!.

10 4.5 1.4 1.7 0.46

Purifying factor

Specific activity of inhibitor

';

0.03

36

0.046

36

3 m.I.U./mg. protein

21

65 m.I.U'/mg. protein 230 m.I.U./mg. protein

77

m lUI test

3,0

• seminal plasma limit of mesuremenl

o""~~ 0 (Inh,b,l,on continually Increases)

/

2,0

to

o /

blood serum

l/ 11 0/ I"

.

10

20

30

50

FIG. 1. Relationship between the inhibition and the amount (Ill.) of seminal plasma and blood serum in the test.

Chymotrypsin

Urokinase

Chymotrypsin, 6.66 mg., (Serva 17160) was diluted in 10 ml. of 0.05 M Tris buffer, pH 7.5, and 0.0066 M CaCl 2 and kept at -210 C.

Urokinase 5000 Ploug units, (Nutritional Biochemicals Corp.) was diluted in 0.5 ml of twice distilled water and kept at - 21 0 C.

Thrombin Test thrombin, 60 NIH units, (Behring Werke) was diluted in 0.5 ml. of twice distilled water and used immediately.

Plasmin Plasmin, 2 Novo units, (Plasmin Novo stabilized with {-lysin; a gift of the Novo-Industrie GmbH, Mainz) was suspended III 0.1 ml. of twice distilled water.

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FIG. 2. Fibrin agar electrophoresis of blood serum (A)-and human seminal plasma (B) inhibitor.

Benzoyl- L-argininethylester (BAEE) BAEE was used for trypsin, thrombin, and plasmin. The test was made according to Trautschold and Werle. 2 For the 1-ml. test composition, 0.01 ml. oftrypsin solution, respectively; 0.010 ml. of plasmin solution, respectively; and 0.012 ml. thrombin solution were used. FIG. 3. Fibrin plate diffusion of plasmin against homogenate of guinea pig seminal vesicles, human seminal plasma inhibitor (HSI) and blood serum (from left to right).

Substrates The following substrates were used.

Benzoylarginine-p-nitranilid (BAPNA) BAPNA was used for trypsin, thrombin, and plasmin. Test Composition. The composition was 0.1 ml. of 0.01 M BAPNA solution (test composition, Boehringer) with 0.05 ml. of trypsin solution (5 J,lg.) , respectively, 0.25 ml. of thrombin solution (30 NIH units), respectively, and 0.025 ml. of plasmin solution. The inhibitor solution to be tested was diluted to 1 ml. with 0.2 M Tris buffer, pH 7.8, and 0.025 M CaC1 2 for trypsin, respectively; with 0.2 M TRA buffer, pH 7.8, and 0.05 M l-lysin for plasmin. For the thrombin determination diethylbarbituric acid buffer, pH 8.2 was used, as described by Heimburger and Schwick. 10 Enzyme activity was measured at 405 nm.

Carboxypropionyl-phenylalanine-p-nitranilid (CPPNA) CPPNA was used for chymotrypsin. Test Composition. The composition was 0.035 ml. of 0.13 M CPPNA solution (test composition, Boehringer, Mannheim) with 0.065 ml. of chymotrypsin solution. The inhibitor solution to be tested was diluted to 1.1 ml. with 0.5 M Tris buffer, pH 7.5, and 0.066 M CaC1 2 • All analyses were carried out at a temperature of 25° C. and 405 nm.

Isolation of the Human Seminal Plasma Inhibitor Seminal plasma, 5 ml., was mixed with 5 ml. of 0.2 M Tris buffer, pH 7.8, and 0.025 M CaC1 2 and centrifuged for 15 min. at 3000 r.p.m. The seminal plasma was stirred overnight at +4° C. with previously sedimented Sephazyme Trypsin (TRy) (Pharmacia Fine Chemicals, Uppsala; Lot L 5861); on the following day the Sephazyme TRY was centrifuged and washed three times with 5 ml. of the same Tris buffer at +4° C. (Table 1, Washing water 1-3).

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The inhibitor was then eluted, according to Fritz, Gebhard, Fink, Schramm, and Werle,I3 with 10 ml. of 0.2 M KCI for an hour at pH 2.0 (Inhibitor fraction 1, Table 1). The sediment was washed again with 5 ml. of 0.2 M KCl and centrifuged (Inhibitor fraction 2, Table 1). The inhibitor fractions were adjusted to pH 4-5. RESULTS

Qualities of the Crude Human Seminal Plasma Inhibitor

~

~

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HUMAN SEMINAL PLASMA INHIBITOR

The average inhibitor content is about 1.3 1. U./total sperm. The activity per milliliter is 0.242 1.U'/ml. (S.D. 0.048); the minimum activity found was 0.13 1.U'/ml., the maximum 0.32 1.U'/ml. This inhibitor activity is heat resistant to 80 0 C. and cannot be precipitated by trichloroacetic acid (TCA 3.35~) and perchloric acid (PCA 1 M). Chloroform and acetone (40 vol. 5~) have no influence on the activity. After ultracentrifuging at 140,000 X g for 1 hr. a remarkable decrease in the inhibitor activity in the supernatant was observed. The amount of lost activity in three different seminal plasmas was about 25, 35, and 54%, respectively.

Comparison of the Trypsin Inhibition by Seminal Plasma and Blood Serum In order to determine more about the function of the inhibitor in the in vitro test and to exclude an interference of other substances of the seminal plasma, increasing amounts of seminal plasma and, for comparison, blood serum were examined with regard to their inhibitory effect (Fig. 1). Remarkable differences were found. Increasing amounts of seminal plasma show increasing inhibition, whereas the inhibition of the blood serum for trypsin only rises to a maximum and then slowly decreases. In the fibrin agar electrophoresis the human seminal plasma inhibitor shows a different electrophoretical movement from that of the blood serum inhibitors (Fig 2).

These experiments prove that there is a different inhibitor system in human seminal plasma from that in blood serum.

Isolation of the Plasma Inhibitor

Human

Seminal

Table 1 shows the results of an isolation of human seminal plasma inhibitor from a sperm pool by Sephazyme TRY. We were able to purify the inhibitor up to Factor 70, based on the protein values (Table 1). The disc electrophoresis, according to Davis, 14 and electrophoresis on cellulose acetate strips gave no colorable bands. We assume that these results are due to the peptide character of the inhibitor.2 The fibrin agar electrophoresis showed a uniform picture for human seminal plasma inhibitor. Additional investigations will be carried out to improve the yield.

Characterization of the Purified Human Seminal Plasma Inhibitor The purified human seminal plasma inhibitor is a specific inhibitor for trypsin. It has neither an influence on the esterase and amidase activity of thrombin, nor on the plasminogen activation by urokinase. The inhibitor does not significantly influence the plasminifibrinolysis (Fig. 3). Furthermore, it does not inhibit the plasmin activity measured with the substrates BAPNA and BAEE. It is impossible to demonstrate plasmin inhibition for the substrate BAEE in seminal plasma, as seminal plasma contains esterase activity.15 The amidase activity of chymotrypsin is neither influenced by seminal plasma nor by the purified human seminal plasma inhibitor. These results are in disagreement with the findings of Syner, and Moghissi. 16

Comparison of the Trypsin Inhibition of Purified Human Seminal Plasma Inhibitor with Trasylol When comparing the same inhibitor amounts of Trasylol (Bayer AG) and puri-

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HIRSCHHAUSER AND KIONKE

5'Jbstrafp, BAPNA enzyme: Trypsin

0.250

HSI

0.125

0-0-'-=

TRASYLOL

®

+ - + - - = human seminal plasma inhibitor = HSI

5

10

15

20 minutes

FIG. 4. Comparison of human seminal plasma inhibitor (HSD and the polyvalent protease inhibitor Trasylol.

fied human seminal plasma inhibitor it was the inhibitor content and the number of found that the inhibition equilibrium for spermatozoa. Comparing the inhibitor achuman seminal plasma inhibitor is formed tivity with the fructose content in the semiinstantaneously, whereas Trasylol needs nal plasma of the same patient, we found no under the same conditions about 10 min. relationship either (Table 3). Six sperm to reach the inhibition maximum (Fig. 4). plasmas were selected with regard to their The inhibition of the esterase activity of fructose content, from 0 to very high, and trypsin by human seminal plasma inhibi- were analyzed for their inhibitor activity tor, tested with BAEE, is, in comparison (Table 4). There was no relationship to be with that of amidase, much slower; possibly found. According to our results the diagdue to the influence of the alkaline pH 8.7. nostic usefulness of the inhibitor content in According to Haendle and co-workers, 2 pathologic seminal plasma, as described by we found an inhibitor content of 0.6 LU.! Ingrisch and co-workers,3 seems somewhat gm. human seminal vesicles. doubtful. We examined five different spermatozoa DISCUSSION homogenates with a sperm count of about The human seminal plasma inhibitor, as 100 million/ml. These homogenates showed no trypsin inhibitor activity when they were described above, is very specific for trypsin. We could not find the plasmin inhibition in measured with our methods. human seminal plasma, as described by Relationship of the Inhibitory Activity to Haendle and co-workers,2 but we did find some Fertility Parameters in Man it in extracts of guinea pig seminal vesicles (Fig. 3). The extract of guinea pig seminal The inhibitor content was determined in 25 patients with diverse diagnoses. The re- vesicles shows different electrophoretical sults were related to the sperm count (Table movement compared with human seminal 2). There is no evident relationship between plasma inhibitor in fibrin agar electrophore-

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June 1971

TABLE 2. Sperm Count/Inhibitor Spennatozoa/ml.

I.u./ml.

I.U'/sperma

o o

0.225 0.235 0.274 0.268 0.235 0.23 0.183 0.23 0.24 0.162 0.263 0.288 0.129 0.225 0.17 0.215 0.3 0.321 0.236 0.478 0.299 0.31 0.273 0.26 0.225

1.125

8 13 14 15 17 19 25 27 27 28 28 38 41 41 58 64 66 91 110 112 154 158 216

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HUMAN SEMINAL PLASMA INHIBITOR

1.230 1.340 1.060

1.680 0.970 1.450 1.090 0.970 1.800 0.595 1.125 1.200 1.775 0.590 1.435 1.950 1.240 1.230 2.040 1.130

TABLE 3. Fructose/Inhibitor Fructose, mg./ml.

Fructose,

mg./ml.

I.U./ml.

3475 4060 2670 2800 3850 5000

165 390 515 1665 2220 2430 2560 2600 2670 2800 3115 3155 3195 3475 3705 3735 3850 4060 4150 4205 4585 4960 5000 5200

0.478 0.225 0.236 0.170 0.162 0.263 0.299 0.129 0.274 0.268 0.321 0.215 0.310 0.225 0.230 0.240 0.235 0.235 0.273 0.225 0.288 0.26 0.23 0.30

3705 3735 2220 2430 4585 2600 4205 1665 3155 5200 3115 515 165 2560 3195 4150 4960 390

I.U'/spenna

1.435 1.13 0.59 0.59 0.97 1.45 1.95 0.97 1.23 1.34 1.775 1.125 1.24 1.125 1.68 1.06 1.23 1.8 1.09 2.04 1.2

TABLE 4.

.,

sis (Fig. 5). We would, therefore, like to assume that the human seminal plasma inhibitor is different from the inhibitor of guinea pig seminal vesicles. Zaneveld, Srivastava, and Williams 7 demonstrated m rabbit spermatozoa a change m trypsin-like enzyme activity. Their results show that the trypsin-like enzyme activity is nearly the same in epididymal and capacitated spermatozoa. They found almost no activity in crude ejaculates. Gaddum, and Blandau 17 demonstrated a protease activity in the acrosomal part of human spermatozoa. It seems probable that the human seminal plasma inhibitor is responsible for the inhibition of this spermatozoa protease activity. Therefore, the biologic importance of the human seminal plasma inhibitor can be seen in the fact that the inhibitor, which possesses great affinity to trypsin, is bound to the

Fructose, mg./ml.

o 730

985 1655 5125 5455

I.U'/ml.

0.244 0.218 0.268 0.240 0.244 0.260

sperm head and thus prevents the penetration of spermatozoa into human cells other than the ovum. Inhibitors for trypsin are found in the whole female genital tract. With the fibrin agar electrophoresis we were able to demonstrate the presence of trypsin inhibitors in cervical mucus, previously demonstrated by Schumacher, and Peare 18 and Haendle, Ingrisch, and Werle, 19 uterine endometrium, uterine mucus, fallopian tube epithelium, and fallopian tube mucus. 20 The re-

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HIRSCHHAUSER AND KIONKE

. •

-~

~ ~ ~.:.....;::;".;y~~

.' .

FIG. 5. Fibrin agar electrophoresis of homogenate of guinea pig seminal vesicles.

moval of the inhibitor from the acrosomal spermatozoa protease can be regarded as part of the polyvalent actions, which precede penetration of spermatozoa into the ovum. SUMMARY

Human seminal plasma inhibitor is a specific inhibitor for trypsin. Its mean value in seminal plasma is 0.242 LU'/ml. The inhibitor was purified by absorption on Sephazyme TRY, and its properties were then described. There is no correlation between spermatozoa count, fructose value, and inhibitor content. The possible function of human seminal plasma inhibitor is discussed.

8. TONUTTI, E., WELLER, 0., SCHUCHHARD, E., AND HEINKE, E. Die miinnliche Keimdriise. Ferd. Enke Verlag, Stuttgart, Germany, 1960. 9. LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. Protein measurement with the folin phenol reagent. J Bioi Chem 193:265, 1951. 10. HEIMBURGER, N., AND SCHWICK, G. Die Fibrinagarelektrophorese. Thromb Diath Haemorrh 7:432, 1962. 11. ThAUTSCHOLD, 1. Cited by VOGEL, R., TRAUTSCHOLD, 1., AND WERLE, E. Naturliche Proteasen Inhibitoren. Georg Thieme Verlag, Stuttgart, Germany, 1966. 12. TRAUTSCHOLD, 1., AND WERLE, E. In Hoppe-Seyler/

13.

REFERENCES 1. RASMUSSEN, J., AND ALBRECHTSEN, O. K Fibrinolytic activity in human seminal plasma. Fertil 2.

3.

4.

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6.

7.

SteriI11:264, 1960. HAENDLE, H., FRITZ, H., TRAUTSCHOLD, 1., AND WERLE, E. Uber einen hormonabhiingigen Inhibitor fiir proteolytische Enzyme in miinnlichen accessorischen Geschlechtsdriisen und im Sperma. Hoppe-Seylers Z Physiol Chem 343:185, 1965. lNGRISCH, H., HAENDLE, H., <\ND WERLE, E. Uber die Konzentration des Trypsin-Inhibitors im Sperma von Gesunden und andrologisch Kranken und iiber ihre Beziehung zu anderen Parametem des Spermas. Andrologie 2:103, 1970. STAMBAUGH, R., AND BUCKLEY, J. ZONA pellucida dissolution enzymes of the rabbit sperm head. Science (New York) 161:585, 1968. STAMBAUGH, R., AND BUCKLEY, J. Identification and subcellular localization of the enzymes effecting penetration of the Zona pellucida by rabbit spermatozoa. J Reprod Fertil19:423, 1969. STAMBAUGH, R., BRACKETT, B. G., AND MASTROIANNI, L. Inhibition of in vitro fertilization of rabbit ova by trypsin inhibitors. Bioi Reprod 1:223, 1969. ZANEVELD, L. J. D., SRIVASTAVA, P. N., AND WILLIAMS, W. L. Relationship of a trypsin-like enzyme in rabbit spermatozoa to capacitation. J Reprod FertiI20:337, 1969.

14.

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Thierfelder: Handbuch der physiologisch-chemischen Analyse. Enzyme (Bd. 6, Teil C) Springer Verlag, New York, 1966, p. 745. FRITZ, H., GEBHARD, M., FINK, E., SCHRAMM, W., AND WERLE, E. Verwendung wasserunloslicher Enzymharze mit polyanionischer und polyamphoterer Harzmatrix zur Isolieurung von Proteinaseinhibitoren. Hoppe-Seylers Z Physiol Chem 350: 129, 1969. DAVIS, B. J. Disc electrophoresis. II. Method and application to human serum protein. Ann NY Acad Sci 121:404, 1964. LUNDQUIST, F., THORSTEINSSON, TH., AND Buus, O. Purification and properties of some enzymes in human seminal plasma. Biochem J 59:69, 1955. SYNER, F. N., AND MOGHISSI, K. S. Properties of proteolytic enzymes isolated from human seminal plasma and spermatozoa. Abstract of papers of Society (or the Study of Reproduction, 1969. GADDUM, P., AND BLANDAU, R. J. Proteolytic activity of mammalian spermatozoa. Annual Conference of Society for the Study of Fertility, Liverpool, 1970. SCHUMACHER, G. B. F., AND PEARE, M. J. Alpha, Antitrypsin in cervical mucous Fertil SteriI19:91, 1968. HAENDLE, H., lNGRISCH, H., AND WERLE, E. Uber einen neuen Trypsin-Chymotrypsin-Inhibitor im Cervixselret der Frau. Hoppe-Seylers Z Physiol Chem 341:545, 1970. HIRSCHHAUSER, C., AND KIONKE, M. Untersuchungen zur trypsininhibierenden Wirkung des Spermaplasmas. Vortrag d. Deutschen Gesellschaftzum Studium der Fertilitlit und Sterilitiit Hamburg, 1970.

t