Plasminogen activator activity, plasminogen activator inhibition and plasmin inhibition in spermatozoa and seminal plasma of man and various animal species—Effect of plasmin on sperm motility

F,hr,noiy.,r~ (lYX7) I. 253 257 c? 1987 Longman Group UK Ltd

Plasminogen Activator Activity, Plasminogen Activator Inhibition and Plasmin Inhibition in Spermatozoa and Seminal Plasma of Man and Various Animal Species-Effect

A. Smokovitis,

N. Kokolis,

of Plasmin on Sperm Motility

C. Alexopoulos,

E. Alexaki, E. Eleftheriou

SU MMAR Y. Plasminogen activator activity (PAA), plasminogen activator inhibition (PAI) and plasmin inhibition (PI) were demonstrated in spermatozoa and seminal plasma of man, bull, ram, buck, boar and stallion. There was a great species variation in the PAA, PAI and PI. In spermatozoa and seminal plasma both types of plasminogen activator, namely of the tissue type (t-PA) and urokinase type (u-PA), were detected immunologically. The effect of plasmin on sperm motility was studied in the semen of bull. The addition of plasmin to the semen induced in certain concentrations a marked increase in sperm motility. The effect of plasmin on sperm motility seems to be direct. KE YWORDS. Plasminogen activator. Spermatozoa. Seminal plasma.

Plasmin.

Plasminogen

The presence of plasminogen activators’p5 as well as hbrinolytic inhibitors2.6 in human seminal plasma has been shown. Fibrinolytic activity has been also found in human, rat and rabbit spermatozoa,’ but the intracellular localisation of the plasminogen activator has not been proved because of the applied methodology. Therefore, the present study was undertaken to investigate the presence of PAA, PA1 and PI in spermatozoa and seminal plasma of various species (man, bull, ram, buck, boar and stallion) and also to identify immunologically the type of plasminogen activator, since in tissues and body fluids there exist two immunologically distinct types of plasminogen activator, called the tissue-type (t-PA) and the urokinase-type (u-PA). Furthermore, the effect of plasmin on sperm motility was studied in the semen of bull.

MATERIALS

AND METHODS

Semen samples were taken from 14 men and throughout the year from 5 bulls (104 samples), 5 rams (139), 5 bucks (129) 5 boars (129) and 3 stallions (84). Immediately after collection the semen was centrifuged (3000xg for 20-45 min). Seminal plasma was used as it was. Spermatozoa (man 5.10’ cells; stallion, A. Smokovitis, N. Kokolis, E. Alexaki, Department of Physiology. Faculty of Veterinary Medicine, Aristotelian University of Thessaloniki, C. Alexopoulos, E. Eleftheriou, Centre of Artificial Insemination and Pathology of Reproduction, Thessaloniki, Greece.

activator

inhibition.

Plasmin

inhibition.

Semen.

boar, bull 1.10” cells; ram, buck 1.lO’ cells) were washed three times in normal saline and extracted with 1 ml of a mixture of 0.1 M Tris-HCl buffer (4 ~01s) and 0.5M potassium thiocyanate (1 vol.), pH 7.4, or with 1 ml of Tris-HCl buffer, pH 7.4. The supernatants were collected. The PAA was determined both in spermatozoa and seminal plasma by a spectrophotometric method,’ using the chromogenic substrate S-2251 (Kabi, Sweden). A total volume of 0.5 ml mixture which consisted of 2OOul of the substrate S-2251 (final concentration 0.73 mM), 1OOul of the plasminogen(s) solution (final concentration 0.5 C.U./ml), lOOu1 of the Tris-HCl buffer and 100 ul of the seminal plasma or spermatozoa extract was incubated for 30 min at 37’C. The reaction was terminated by adding 1.Oml of acetic acid SOS:‘,.The procedure for the blank tube was carried out in the absence of seminal plasma or spermatozoa extract which was added to the mixture after terminating the reaction. The absorbance was read at 405 nm and the difference between the two tubes was adopted as the net optical density. The PAA in t-PA or u-PA IU was determined by referring the net optical density to standard curves, which were drawn using t-PA (Biopool, Sweden) or u-PA (Leo, Denmark), l-50 IU or 0.01-0.5 IU, respectively. The activity of plasminogen activator was expressed in terms of t-PA or u-PA IU per 1 ml of seminal plasma or per 1.1 O9 spermatozoa. The plasminogens used were isolated from each spe-

254

Effect of Plasmin

on Sperm Motility

ties plasma by affinity chromatography on LysineSepharose 4B (Sigma, USA) as described by Deutsch and Mertz.’ The PAI was determined by the following procedure:“, ’l t-PA (5 IU) or u-PA (0.05 IU) was added to 1OOul of seminal plasma or spermatozoa extract and incubated for 5min at 37°C. The sample was acidified with 100 ul of 0.16 M HCI and incubated for 10 min at room temperature to efficiently destroy plasmin inhibitors. The pH was adjusted by the addition of 1OOul of 0.16 M NaOH. Then 100 pl of Tris-HCI buffer, 100 ul of plasminogen(s) solution (0.5 CU/ml) and 200 ul of S-2251 (0.73 mM) were added. The tube was incubated at 37°C for 30 min and the reaction was terminated by adding l.Oml acetic acid 50%. In reference tube (PAA tube) t-PA or u-PA was replaced by an equal volume of buffer. In the blank tube spermatozoa extract or seminal plasma was added after the termination of the reaction, The difference in absorbance at 405 nm, due to the presence of inhibitors, was expressed in t-PA or u-PA TU per ml of seminal plasma or 1.1O9 spermatozoa using standard curves. The PI was determined as follows:” in 1OOul of seminal plasma or spermatozoa extract 100 ul(1 .O IU/ ml) of human plasmin (gift from Prof. Binder, Vienna, Austria) were added followed by 300~1 of Tris-HCI buffer; the mixture was incubated at 37°C for 2 min. To determine the amount of plasmin activity that remained after the incubation of exogenous plasmin with the seminal plasma or spermatozoa extract, 100 ul of S-2251 (final concentration 0.30mM) were added and the mixture was incubated at 37°C for 5 min. The reaction was terminated by adding 200 ul of acetic acid 50%. In blanks the seminal plasma or spermatozoa extract was added after terminating the reaction. In controls the seminal plasma or spermatozoa extract was replaced by an equal volume of buffer. The optical density was measured at 40.5 nm. Plasmin inhibition was determined from the difference in optical density of the control and the sample and expressed in plasmin IU by using a standard curve. For the immunological identification of the plasminogen activators (t-PA, u-PA) polyclonal antibodies anti (human uterus) t-PA immunoglobulin and anti (human LMW) u-PA immunoglobulin were used (Biopool, Sweden). The antibodies were diluted in phosphate buffered saline (PBS) pH 7.3. The effective concentrations were O.llO.2 mg/ml for the anti-t-PA and 0.1-0.3 mg/ml for the anti-u-PA. Each tube contained IOOul of seminal plasma or spermatozoa extract (man 5.10’ cells/ml; stallion, boar, bull 1.109 cells/ml; ram, buck 1.10’ cells/ml) and 100 111of antibody solution; blank and control tubes contained lOOu1 of PBS instead of antibody. The tubes were incubated for 90min at room remperature. Then 100 ul of Tris-HCI buffer, 100 ul of plasminogen(s) solution (0.5 CU/ml) and 2OOul S-2251 (0.73 mM) were added. In blanks S-2251 was added after terminating the reaction. The tubes were incubated at 37°C for 30 min and the reaction was terminated by adding

1.O ml of acetic acid 50%. As before, the optical density was measured at 405nm. The degree of inhibition caused by the antibodies was determined from the difference between the optical density of the control mixture and the mixture containing the antibody and expressed as inhibition percentage. Sperm motility was determined by a modification of a sperm penetration test.i3 Plasmin was added in various concentrations (0.5, 5, 50, 500 ng/ml and 5 ug/ml) to bovine semen or to seminal plasma or to a usual diluent (containing Tris, citric acid, fructose and eggyolk). All samples were incubated at 37°C for 30min. Thereafter. seminal plasma or diluent of the semen was drawn up into capillary tubes (i.d. 1.7 mm, length 133 mm). The tubes were then positioned vertically with their lower end immersed in a sample of semen (with no plasmin) contained in a small beaker and placed in a water bath (37°C). The diluent of the semen or the seminal plasma drawn up into the capillary tubes contained no plasmin when plasmin was added to the semen. The upper end of the tube was sealed with Vaseline. In every case control samples contained instead of plasmin an equal volume of the buffer. Fifteen and 30min later the tubes were examined under a binocular dissecting microscope to measure the distance travelled by the leading spermatozoa. In separate experiments the effect of various concentrations (15, 1.5, 0.15 and 0.015mM) of EACA (Sigma, USA) or a mixture of plasmin and EACA (5/ 15 ug/mM; reduction of plasmin activity by 50%) on sperm motility was studied. A total number of 900 tubes was studied (3600 determinations of migration rate). Kinematic viscosity of the seminal plasma was measured with a Schott KPG Ubbelohde capillary viscometer. Statistical analysis was performed by F-Distribution and Dunckan test; P < 0.05 was considered significant.

RESULTS PAA, PA1 and PI in spermatozoa and seminal plasma are shown in tables, 1, 2 and 3. In ram and buck the PAA in the semen (spermatozoa and seminal plasma) was much higher than in the semen of man, bull, boar and stallion (Table 1). The species variation of PA1 and PI in the semen (spermatozoa and seminal plasma) was less marked (Tables 2 and 3). In the supernatant of washed spermatozoa affected by potassium thiocyanate the PAA was approximately 80% higher than in the supernatant of washed spermatozoa affected by Tris-HCI buffer, indicating the intracellular localization of the plasminogen activator. In spermatozoa and seminal plasma both types of plasminogen activator (t-PA and u-PA) were detected (Table 4). Plasmin added (a) to semen, (b) to seminal plasma

Journal

Table 1 Plasminogen

Activator

Activity

(PAA) in t-PA and u-PA IU of Spermatozoa

of Fibrinolysis

255

(I.109)* and Seminal Plasma (I ml). The Values

are

Mean i SD

Man Stallion Boar BUII Ram Buck

Spermatozoa PAA in t-PA IU

PAA in u-PA IU

Seminal plasma PAA in t-PA IU

PAA in u-PA IU

356.00 113.11 125.00 137.50 45 950.00 34000.49

I.77 0.50 0.55 0.48 1 127.08 922.50

379.38 108.38 91.58 17.96 1048.80 I 805.60

I.83 0.47 0.37 0.03 28.65 42.60

+ 119.32 * 52.05 i 42.87 * 60.43 k 9 850.67 + 16900.83

* extraction with potassium thiocynate Comparisons Spermatozoa PAA in t-PA IU: Spermatozoa

PAA in u-PA IU:

Seminal plasma Seminal plasma

PAA in t-PA IU: PAA in u-PA IU:

* 0.60 k 0.26 * 0.30 * 0.28 + 362.71 k 37 I .92

) i + k & i_

Activator Inhibition (PAI) in t-PA and u-PA IU of Spermatozoa (I ml). The Values are Mean f SD

Man Stallion Boar Bull Ram Buck Comparisons Spermatozoa

PA1 in u-PA IU

Seminal plasma PA1 in t-PA IU

PA1 in u-PA IU

46.15 13.50 33.50 42.90 3527.78 3200.00

0.88 0.46 0.37 0.12 225.00 135.00

13.00 15.00 38.38 46.80 I I .06 37.56

0.19 0.41 0.38 0.12 0.40 I .56

_t 2.76 I .08 i f I .22 f I.98 * 480.31 f 669.X9

* 0.01 + 0.03 * 0.02 If: 0.01 k 29.72 k 46.72

PA1 in u-PA IC:

Table 3 Plasmin

Inhibition (PI) in Plasmin IU of Spermatozoa (1.10’) and seminal plasma (I ml). The Values arc Mean I SD

Spermatozoa

Seminal plasma

0.1 I4 0.036 0.025 0.086 0.061 0.050

0.244 0.142 0.383 0.775 0.650 0.381

+ + i + + *

0.052 0.020 0.015 0.050 0.017 0.015

f i_ + i & i

3.83 I.22 4.85 1.36 6.22 9.10

f 0.03 _+ 0.08 * 0.02 & 0.02 & 0.15 & 0.23

P < 0.001 in all cases except ram/buck. where the difference was not significant (P > 0.05). P < 0.001 in all casts. P < 0.001 in all cases except manstallion, man/ram. stallion; man, boar/buck. where the difference was not significant (P > 0.05). P < 0.01 in all cases except stallion/boar. stallion~ram. boar; ram. where the difference was not significant (P > 0.05).

Spermatozoa PA1 in u-PA IU: Seminal plasma PA1 in t-PA IU:

Man Stallion Boar Bull Ram Buck

(1.10’) and Seminal

Spermatozoa PA1 in t-PA IU

PA1 in t-PA IU:

Semmal plasma

+ 0.59 * 0.10 + 0.15 * 0.01 k 9.37 5 I I .33

P < 0.001 in all cases except stallioq’boar, boar/bull, ram/buck. where the difference was not significant (P > 0.05). P < 0.001 in all cases except stallion/boar, stallion~bull, boarjbull, ram/buck, where the difference was not significant (P > 0.05). P < 0.001 in all cases. P i 0.001 in all cases except stallion/boar, where the difference was not significant (P > 0.05).

Table 2 Plasminogen Plasma

132.94 15.24 31.50 3.62 206.72 401 .X2

_+ 0.041 i 0.083 & 0.085 _+ 0.156 i 0.143 * 0.073

and 5 ugjml) the increase of sperm motility was inversely proportionate to the concentration of plasmin (P < 0.001). The increase in sperm motility was reduced by EACA (P < 0.001). while EACA itself had no effect on sperm motility. Plasmin did not affect significantly the viscosity of the seminal plasma.

DISCUSSION Comparisons Spermatozoa

PI:

Seminal plasma

PI:

P < 0.001 in all casts except man,!bull, stallion/boar, where the difference was not significant (P > 0.05). P < 0.001 in all cases except boar/buck, where the difference was not significant (P > 0.05).

and (c) to a diluent of the semen induced an increase in sperm motility (Table 5). The highest increase of motility was induced by a concentration of plasmin 5 ng/ml (P < 0.001). Lower concentrations had no effect, while in higher concentrations (50, 500ngiml

PAA was demonstrated in spermatozoa and seminal plasma of various species. The PAA in spermatozoa and seminal plasma was much higher in ram and buck than in man, bull, boar and stallion. After the applied procedure the intracellular localisation of the plasminogen activator in spermatozoa has been shown. The activator is mostly membrane bound (unpublished data). In human spermatozoa the application of antibodies raised against human t-PA and u-PA showed that 68:J, of their PAA was due to t-PA and 14q,, to u-PA. In human seminal plasma the 72J’:, and 269; of

256

Effect of Plasmin

Table 4 Immunological Inhibition Percentages

on Sperm Motility

Identification

of t-PA and u-PA

in Spermatozoa*

Spermatozoa anti t-PA IgG anti u-PA IgG 1mg/ml PBS 2 mg/ml PBS 1 mg/ml PBS 3 mg/ml PBS

Man Stallion Boar BUll Ram Buck

*

**

68% 27% 23% 6% 6% 5% man: stallion: boar: bull: ram: buck: non tested

NT** 47% 25% 7% 7% 7%

14% 0% 0% 0% 0% 0%

NT**

and Seminal

5% 6% 5%

(0.1 ml). The Values

are Expressed

as

Seminal plasma anti t-PA IgG anti u-PA IgG 1 mg/ml PBS 2 mg/ml PBS 1mg/ml PBS 3 mg/ml PBS NT**

72% 17% 19% 13% 14% 5O’ /”

30% 28%

Plasma

20% 46% 52% 24% 6%

26% 0% 0% 0% 0% 0%

NT** 13% 39% 36% 7% 5%

5.10’ spermatozoa/ml 1.10’ spermatozoa/ml 1.109 spermatozoa/ml 1.109 spermatozoa/ml 1, IO’ spermatozoa/ml 1.10’ spermatozoa/ml

the PAA were due to t-PA and u-PA, respectively. The presence of both types of PA in human spermatozoa and seminal plasma has been confirmed as well as with the use of monoclonal antibodies (gift from Prof. Binder, Vienna, Austria, unpublished data). The immunological characterisation of t-PA5 and u-PA4s5 in human seminal plasma has been also done by others.4,5 In stallion spermatozoa the corresponding percentages were only 47% (t-PA) and 30% (u-PA), while in seminal plasma these percentages were 20% (t-PA) and 13% (u-PA) and with the use of a much higher concentration of antibodies. The lowest percentages of cross-reaction were observed in spermatozoa and seminal plasma of the buck. It seems that antibodies against human t-PA and u-PA bind more firmly to human t-PA and u-PA than to t-PA and u-PA from stallion, boar, bull, ram and buck. Among the various animal species there were also great differences (Table 4). It is suggested that t-PA and u-PA in spermatozoa and seminal plasma of the species studied are not identical. The immunological identification of both types of plasminogen activator (t-PA, u-PA) in spermatozoa provides definite evidence that the acrosomal proteinase acrosin should be distinguished from the plasminogen activator.2,‘4 Spermatozoa and seminal plasma of all species studied showed also PA1 and PI. The PAI was particularly high in ram and buck spermatozoa, while the highest PI of spermatozoa was seen in man and of seminal plasma in bull and ram. The physiological significance of the PAA, PA1 and PI of the semen is still largely unclear. The seminal

plasma PAA probably maintains the patency of the pathways in areas of the male genital tract. In addition, this activity could affect spermatozoa. The present study showed that plasmin added to the semen can affect sperm motility. It is of interest that after a concentration of plasmin (5 ng/ml) which induced the highest increase in sperm motility, higher concentrations of plasmin induced an increase in sperm motility inversely proportionate to the concentration of plasmin. The effect of plasmin on sperm motility should be direct, since plasmin did not affect at least the viscosity of the seminal plasma. Furthermore, plasmin added to a diluent of the semen instead of the seminal plasma affected sperm motility to the same degree and this effect was marked even after some minutes (15 min) from the addition of plasmin (figures not given in Table 5). A marked increase in the motility was also noticed when plasmin was added to semen and 30 min later the spermatozoa were allowed to migrate into a medium (seminal plasma or diluent of the semen) containing no plasmin. The effect of plasmin on sperm motility was reduced by the inhibitor EACA, while EACA itself (at the concentrations used) had not any effect on sperm motility. As to the PAA of spermatozoa, this proteolytic activity should be of particular importance for the penetration of spermatozoa through ovum layers as shown by in vitro studies;2 there is an accumulation of evidence that the PAA can exert a tissue degradative effect. “-” The relationship between activators and inhibitors in spermatozoa may be of importance for the fertilisation process. The independent influence of

Table 5 Distance (mm) Travelled by the Leading Spermatozoa in 30min (after the addition semen diluent and incubation at 37°C for 30min). The values are mean + SD

Addition of plasmin to

Control (addition of Tris-buffer)

0.5 n&/ml

5 ng/ml

Semen Seminal plasma Semen diluent

37.31 + 1.14 37.00 + 1.71 34.37 * 0.88

38.12 f 1.02 37.56 k 0.96 35.56 k 0.89

48.56 + 0.96 46.31 f 0.87 43.62 k 0.96

of plasmin

Plasmin concentrations 50 ngjml 45.62 f 1.02 42.94 1 1.00 40.56 f 0.89

to semen or to seminal plasma

or to

500 ng/ml

5 ug/ml

42.94 I 1.06 40.87 + 1.09 38.62 f 0.81

41.25 + 1.00 39.69 + 1.40 37.06 + 1.12

Journal

activators and inhibitors by biochemical (hormonal, etc) factors could open new avenues to fertility control. The physiological meaning of the great variation in PAA, PAI and PI of the semen (spermatozoa and seminal plasma) among some of the species studied is under investigation.

9.

IO.

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orders to: Professor A. Smokovitis, Department of Physiology, Faculty of Veterinary Medicine, Aristotelian University. Thessaloniki 54006. Greece.

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