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Determination of sperm acrosin activity for evaluation of male fertility

Yun-He CUI; Rui-Lan ZHAO1; Qiang WANG; Zi-Ying ZHANG

Jining Medical College, Jining 272013, China
1Mother and Child Care Hospital, Central District, Jining, China

Asian J Androl  2000 Sep; 2: 229-232

Keywords: sperm; acrosin assay; male fertility
Abstract
Aim: To investigate a simple method for assaying acrosin activity for the evaluation of male fertility. Methods: The acrosin activity of 7.5106  sperm without  seminal  plasma and acrosin activity inhibitors was assayed using N--benzoyl-DL-arginine-p-nitroanilide (BAPNA) and detergent (Triton X-100) as substrate. Results: The acrosin activity of 60 normal fertile men (3510 IU/106 sperm ) was higher than that of 168 infertile men (168 IU/106 sperm) (P0.01). It was indicated that there was a significant positive correlation between the acrosin activity and the sperm motility (r0.6534, P0.01)and a significant negative correlation between the sperm malformed rate and the WBC number (rܣ0.5426, P0.01).  The temperature and time of incubation and the sperm concentration could influence the assay results. Conclusion: Acrosin activity is an important index for the evaluation of male fertility. The approach developed by the authors is a simple method for the determination of acrosin activity.

1 Introduction

Sperm acrosin is a trypsin-like serine proteinase existing in acrosomes. It is an important proteolytic enzyme that is capable of hydrolyzing the zona pellucida (ZP) in oocyte, and plays a vital role in the process of fertilization. Traditionally, several assay methods for acrosin activity have been used in the laboratories, including radioimmunoassay (RIA), fluorometric enzyme method (FEM)[1] and Gelatinolytic technique[2,3]. But these methods are complicated and time-consuming. Later, the methods were improved by Kennedy et al[4], Blackwell et al[5] and Wang et al[6].This investigation was aimed at the exploration of a concise and practical method for determining acrosin activity, and clarification of the clinical significance of sperm acrosin determination .
2 Materials and methods

2.1 Subjects

One hundred and sixty-eight infertile males, aged 27-39 years (mean 29.3) and 60 normal controls, aged 25-28 years (mean 28.2) were recruited. The infertile patients were all married and lived together with their spouses for two or more years without bearing children; the gynecological examination of their spouses was normal. The controls had fathered one or more children and had their routine semen analysis within the normal range. Semen were obtained by masturbation after 5-7 days of abstinence. Semen samples were liquefied in a 37 water bath before analysis.

2.2 Instruments and reagents

Spectrophotometer 722 (or 721) , centrifuge, constant temperature culture-box, and three-use mini water tank.  N--benzoyl-DL-arginine-p-nitroanilide  (BAPNA); benzamidine ; Ficoll 400 ; N-2-hydroxylpiperazine-N'-2-ethane sulfonic acid (HEPES); dimethyl sulfoxide (DMSO) (all purchased from the Sigma Chemical Co, St. Louis, MO, USA).

2.3 Preparation of solutions

Solution A (g/100 mL deionized water): NaCl  0.70,  HEPES 0.60,  Ficoll 11.0 (pH 7.4).

Solution B (g/100 mL deionized water): HEPES 1.31,  NaCl 0.32,  1% TritonX-100 primary solution 1.0 mL (primary solution: Triton 1.0 mL, deionized water 99 mL, pH8.0).

Solution C: benzamidine 8.73 (g/100 mL deionized water). Solution A, B, and C were then stored at 4  for no more than 3 months.

Solution D: BAPNA 5 mgDMSO 0.5 mL. The solution should be freshly prepared and the  solid completely dissolved.

Solution E: Thoroughly mixed up one part of Solution B and three parts of Solution D.

2.4 Assay method

The required amount of semen containing 7.5106 sperm was placed into a plastic tube. Added 0.5 mL Solution A and centrifuged at 1500g for 10 min.

The supernatant was removed with 100 L remaining. To the control tube added 100 L of Solution C. Then added 1.0 mL of Solution E to both tubes, mixed well and incubated at 24 for 3 hours; shaking well every hour.

After incubation, added 100 L of Solution C to end the reaction, shook well and centrifuged at 1500g for 10 min.

Added 0.5 mL of the supernatant from both the experimental and control tubes to different 0.5 mL cells and a similar amount of Solution E to another cell, which served as the zero control. The absorbance at 400 nm was determined. One IU of acrosin activity was defined as the substrate amount that hydrolyzed 1.0 mol/min of BAPNA at 24. Formula: Acrosin activity (IU)/7.5106 sperm=(experimental OD valuecontrol OD value)106 /14857.5[7]. In this case the amidase activity had been determined, but it could be inhibited by benzamidine, indicating that the activity was primarily or entirely due to acrosin. Therefore, the term acrosin activity rather than amidase activity is used[4,5].

2.5 Statistical analysis

Data were expressed as s wherever applicable. Significance of differences were analyzed by the Student's t-test and P<0.05 was considered significant.

3 Results

3.1 Effect of incubation temperature and time on acrosin activity

Semen samples were collected from one normal control and one infertile man. Each sample was assayed  six times: the effect of incubation temperature on acrosin activity was observed in 3 assays, and the effect of incubation time on acrosin activity in other 3. It was showed that the optimal temperature was 20-30 (Table 1) and the optimal incubation time, 4-5 hours (Table 2).

Table 1. Effect of incubation temperature on acrosin activity (IU/106sperm). cP<0.01, compared with 15, 20, and 37. fP<0.01, compared with 15.

Incubation temp.

15

20

25

30

37

Normal sperm

24.11.0

30.21.2

35.40.8c

37.20.9

25.81.8

Infertile sperm

9.40.7

12.01.0

16.10.6f

18.20.8

13.71.1

Table 2. Effect of incubation time on acrosin activity (IU/106 sperm, means). cP<0.01, compared with 1 and 2 h and fP<0.01, compared with 1, 2 and 3 h in normal sperm group.  iP<0.01, compared with 1 h and lP<0.01, compared with  1, 2 and 3 h in infertile sperm group.

Incubation time

1 h

2 h

3 h

4 h

5 h

Normal sperm

23.21.2

27.41.3

37.85.6c

52.45.6f

60.54.4f

Infertile sperm

12.40.9

17.51.0

20.31.0i

30.11.2l

35.22.3l

3.2 Effect of sperm concentration on acrosin activity

Acrosin activity of ten normal semen samples were examined with 5 different sperm concentrations ranging from 1.0106 to 10106  sperm/mL. Table 3 showed that the acrosin activity increased with increasing sperm concentration. There was a significant positive correlation between the sperm acrosin activity and the sperm concentration (r =0.964, P<0.01).

Table 3. Relationship between acrosin activity and sperm concentration.

Sperm concentration

Acrosin activity (IU/106sperm)

10.0106/mL

169

2.5106/mL

2110

5.0106/mL

2810

7.5106/mL

3711

10.0106/mL

5011

12.5106/mL

6110

3.3 Effect of Triton X-100 concentration on acrosin activity

Acrosin activity of ten normal semen samples was examined with 6 different Triton concentrations (from 0 to 0.10%). Maximal acrosin activity occurred at the concentration of 0.01%, and there was no significant difference between the acrosin activity values obtained at Triton concentrations of 0.03% to 0.10% (Table 4).

Table 4. Effect of Triton concentration on acrosin activity. bP<0.05, compared with 0 concentration.

Triton concentration (%)

Acrosin activity (IU/106sperm)

0

146

0.001

187

0.01

3611b

0.03

2410

0.05

218

0.10

208

3.4 Acrosin activity in fertile and infertile men

The acrosin activity in the normal fertile men was significantly higher than that in the infertile men (3510 and 168 IU/106 sperm, respectively, P0.01).

3.5 Relationship between acrosin activity and sperm motility

Semen samples with the sperm motility above 50% were classified as High Motility Group, and below 50% as Low Motility Group. Semen samples with the sperm forward progression of Grade a and b were classified as Good Progression Group, and Grade c and d, Poor Progression Group. The different grades of sperm forward progression were classified according to the WHO manual[7]. Table 5 shows that there is a significant positive correlation between the acrosin activiy and the movement characteristics of the sperm.

Table 5. Relationship between acrosin activity (IU/106 sperm) and sperm motility.

Group

n

Acrosin activity

r

P

Good Progression

138

349

 

 

Poor Progression

90

156

0.6534

0.01

High Motilioty

182

339

 

 

Low Motility

46

148

0.7321

0.01

3.6 Correlation between acrosin activity and malformed sperm rate

From Table 6 it can be seen that the acrosin activity was higher in semen samples with the malformed rate 20% than in those with the malformed rate higher than 20%. There was a significant negative correlation between the acrosin activity and the malformed rate.

Table 6. Correlation between acrosin activity (IU/106sperm) and sperm malformed rate.

Malformed rate

n

Acrosin activity

r

P

20%

180

3812

 

 

>20%

48

217

0.5426

<0.01

3.7 Correlation between acrosin activity and leukocyte number

As shown in Table 7 there was a significant negative correlation between the acrosin activity and the leukocyte number in semen.

Table 7. Correlation between acrosin activity (IU/106sperm) and leukocyte number.

Leukocyte No.

n

Acrosin activity

r

P

1.0106/mL

190

3510

 

 

>1.0106/mL

38

208

0.4638

<0.01

3.8 Acrosin activity in infertiles with normal semen data

It is interesting to note that in 25 out of 168 infertile cases the routine semen data were within the normal range, i.e., sperm density 4.0107/L, sperm forward progression = Grade a or b, sperm motility 50%, leukocyte 1.0106/mLtheir sperm acrosin activity was still significantly lower than that of the controls (20 and 3510 IU/106 sperm, respectively, P<0.01).

4 Discussion

The temperature and time of incubation could influence acrosin activity directly, so it is very important to choose them properly. Suitable temperature is 24 and time is 3 h.

With the increase of sperm concentration, acrosin activity were also increased gradually when the sperm concentration was between 1.0106-12.5106. Therefore, it is of utmost importance to choose the proper sperm concentration[8]. The present results have shown that the sperm acrosin activity in the normal fertile men is significantly higher than that in the infertile men, and that there is a positive correlation between acrosin activity and sperm motility, suggesting that the activity may reflect the fertilizing ability of sperm. Acrosin is a delicate enzyme that can be disturbed under various conditions, e.g., sperm malformation and local inflammation with leukocyte infiltration. In these cases, infertility may result[4,9,10]. In 15 infertile men without demonstrable causes, Agarwal and Loughlin found that the acrosin activity of 10 cases were less than 14 IU/106 sperm (mean 7.8)[11].

Acrosin activity of the fertile men in the present paper is similar to those reported by Xiao et al[8] and Zhao et al[9], but lower than that indicated by Tong et al[12], which may be the result of a difference in the methodology.

With the conventional methods for acrosin determination, eg, the RIA and FEM[1], besides its complexity in methodology, the main drawback of these methods is that they only determine the acrosin itself, but not the activity. For this reason their clinical usefulness is limited. The authors put forward a modified method on the basis of the work of Kennedy et al[4], Blackwell et al[5], and Wang et al[6].  The new method reflects the acrosin activity in a fixed number (7.5106) of sperm, with the exclusion of factors inhibitory to acrosin activity. In the assay, the acrosome membrane was destroyed through resuspension of sperm in BAPNA and Triton and the acrosin was completely released and activated by pH 8.0 solution. The method is simple and easy to be extended  for clinical use.

References

[1] Moammad M, Frank NS, Kamran SM. A study of sperm acrosin in patients with unexplained  infertility. Fertil Steril 1982; 37: 223-9.
[2] El-Mulla KF, Kohn FM, Kl-Beheiry AH, Schill WB. The effect of smoking and varicocele on human sperm acrosin activity and acrosome reaction. Hum-Reprod. 1995; 10:3190-4.
[3] Henkel R, Muller C, Miska W, Schill WB, Kleinstein J, Gips H. Acrosin activity of human spermatozoa by means of a simple gelatinolytic technique: a method useful for IVF. J Androl 1995; 16: 272-7.
[4] Kennedy WP, Kaminski JM, Van Der Ven HH, Jeyendran RS,  Reid DS, Blackwell J, et al. A simple clinical assay to evaluate the acrosin activity of human spermatozoa. J Androl 1989; 10: 221-31.
[5] Blackwell J, Kaminski J, Bielfeld P, Mack SR, Zaneveld LJ. Human sperm acrosin further studies with the clinical assay and activity in a group of presumably fertile men. J Androl 1992; 13: 571-8.
[6] Wang HJ, Xu ZG, Wang CZ, Zuo WJ, Wang ZS. Analysis of influencing factor on human sperm acrosin activity in 1077 male infertiles. Chinese J Androl (in Chinese) 1999; 13: 153-5.
[7] World Health Organization. Laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 3rd ed. Cambridge: Cambridge University Press; 1992. 
[8] Xiao CH, Wang ZS, Zuo WJ, Wang CZ, Xu ZG, Li YW. Assay of sperm acrosin activity and its clinical application. Chinese J Androl (in Chinese) 1994; 8: 198-207.
[9] Zhao ZW, Xiao CH, Xu N, Shi AP. Human sperma acrosin and male infertility. Chinese J Urol (in Chinese) 1998; 19: 499-500.
[10] Wang CZ, Xu ZG, Zuo WJ, Wang HJ, Wang ZS, Xiao CH, et al. Analysis on influencing factor and activity of human sperm acrosin. J Norman Bethune Med Univ (in Chinese) 1996; 22: 583-5.
[11] Agarwal A, Loughlin KR. Acrosin activity in patients with idiopathic infertility. Arch Androl 1991; 27: 97-101.
[12] Tong JS, Hu YZ, Wang R, Qian SZ. Effect of gossypol on human acrosin activity. Prog Biochem Biophys (in Chinese) 1986; (5): 41-2.
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Correspondence to:  Dr Yun-He CUI, Jining Medical College, Jining 272013, China.
e-mail: cuiyunhe@ji-public.sd.cninfo.net
Received 2000-01-25     Accepted 2000-05-23