Editor-in-Chief
Prof. Yi-Fei WANG,
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 7.5×106 sperm without seminal plasma and acrosin activity inhibitors was assayed using N-α-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and detergent (Triton X-100) as substrate. Results: The acrosin activity of 60 normal fertile men (35±10 μIU/106 sperm ) was higher than that of 168 infertile men (16±8 μIU/106 sperm) (P＜0.01). It was indicated that there was a significant positive correlation between the acrosin activity and the sperm motility (r≥0.6534, P＜0.01)and a significant negative correlation between the sperm malformed rate and the WBC number (r≤－0.5426, P＜0.01). 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-α-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 mg＋DMSO 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 7.5×106 sperm was placed
into a plastic tube.
Added 0.5 mL Solution A and centrifuged at 1500×g for 10 min.
The
supernatant was removed with 100 μL remaining. To the control tube
added 100 μL 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 μL of Solution C to end the reaction, shook
well and centrifuged at 1500×g 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
μmol/min of BAPNA at 24℃. Formula: Acrosin activity (μIU)/7.5×106
sperm=(experimental OD
value－control OD value)×106 /1485×7.5[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
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 (μIU/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 |
24.1±1.0 |
30.2±1.2 |
35.4±0.8c |
37.2±0.9 |
25.8±1.8 |
| Infertile
sperm |
9.4±0.7 |
12.0±1.0 |
16.1±0.6f |
18.2±0.8 |
13.7±1.1 |
Table
2. Effect of incubation time on acrosin activity (μIU/106
sperm, mean±s). 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 |
23.2±1.2 |
27.4±1.3 |
37.8±5.6c |
52.4±5.6f |
60.5±4.4f |
| Infertile
sperm |
12.4±0.9 |
17.5±1.0 |
20.3±1.0i |
30.1±1.2l |
35.2±2.3l |
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 1.0×106 to 10×106
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 (μIU/106sperm) |
| 10.0×106/mL |
16±9 |
| 2.5×106/mL |
21±10 |
| 5.0×106/mL |
28±10 |
| 7.5×106/mL |
37±11 |
| 10.0×106/mL |
50±11 |
| 12.5×106/mL |
61±10 |
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 (μIU/106sperm) |
| 0 |
14±6 |
| 0.001 |
18±7 |
| 0.01 |
36±11b |
| 0.03 |
24±10 |
| 0.05 |
21±8 |
| 0.10 |
20±8 |
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 (35±10 and 16±8 μIU/106 sperm,
respectively, P＜0.01).
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 (μIU/106 sperm) and
sperm motility.
|
Group |
n |
Acrosin
activity |
r |
|
| Good
Progression |
138 |
34±9 |
|
|
| Poor
Progression |
90 |
15±6 |
0.6534 |
＜0.01 |
| High
Motilioty |
182 |
33±9 |
|
|
| Low
Motility |
46 |
14±8 |
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 (μIU/106sperm) and
sperm malformed rate.
|
Malformed
rate |
n |
Acrosin
activity |
r |
P |
| ≤20% |
180 |
38±12 |
|
|
| >20% |
48 |
21±7 |
－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 (μIU/106sperm) and
leukocyte number.
|
Leukocyte
No. |
n |
Acrosin
activity |
r |
P |
| ≤1.0×106/mL |
190 |
35±10 |
|
|
| >1.0×106/mL |
38 |
20±8 |
－0.4638 |
<0.01 |
3.8
Acrosin activity in infertiles with “normal” semen data
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 1.0×106-12.5×106.
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 μIU/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.
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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
