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Changes
in soluble interleukin-2 receptor level in serum and Na+-K+-
exchanging ATPase activity in semen of infertile men caused by antisperm
antibody
Jiang
NI, Qing-Lei LI, Wei ZHANG, Jian-Song XIE, Shu-Ling BIAN
Department
of Physiology, Harbin Medical University, Harbin 150086, China
Asian J Androl 2000 Jun; 2: 151-153
Keywords:
male
infertility; antisperm antibodies; soluble interleukin-2
receptor; Na+-K+-
exchanging ATPase
Abstract
Aim: To explore the possible mechanisms of male infertility caused by antisperm antibody (AsAb). Methods: The soluble interleukin-2 receptor (sIL-2R) level in serum was analyzed by ELISA and Na+-K+-exchanging ATPase activity in semen by phosphorus (Pi) assay. Results: The sIL-2R level in serum was significantly higher and the Na+-K+- exchanging ATPase activity in semen significantly lower in AsAb positive infertile men when compared with the controls. Conclusion: The AsAb titer varies with the sIL-2R level in serum. A decrease in Na+-K+-exchanging ATPase activity in semen may play a role in male infertility caused by AsAb.1 Introduction
It
is commonly accepted
that suspected immunological factors accounted for 3% of male infertility[1].
Antisperm antibodies (AsAb) appearing in serum or seminal plasma
have been associated with infertility in men[2,3]. But little
is known about how
autoimmune response to sperm may vary according to the circumstances under
which sperm are exposed to the immune system[4]. Recent study
has explored that cell-mediated immunity (CMI) may impair sperm function[5].
The level of the soluble form of the interleukin-2 receptor (sIL-2R) in
serum may reflect the immune cell activity, so we try to investigate the
relationship between the sIL-2R level in serum and the AsAb titer in infertile
males. Since the Na+-K+-exchanging ATPase (Na+-K+-
pump) plays an essential role in many aspects of cell biology[6],
the present study is also designed to analyze the Na+-K+-exchanging
ATPase activity in semen to explore a possible role it may play in male
infertility caused by AsAb.
2 Materials and methods
2.1
Chemicals and reagents
The
sIL-2R analyzing kit was obtained from the Department of Immunology of
Norman Bethune Medical University. The Na+-K+-exchanging
ATPase analyzing kit was purchased from Nanjing Jiancheng Bioengineering
Institute and the bovine serum albumin (BSA),
from the Sigma Chemical Co. Commassie blue G-250 was purchased
from Fluka.
2.2
Volunteers
Volunteers,
aged 28-42 years, both infertile males caused by AsAb and normal contols,
were recruited at the Second Affiliated Hospital, Harbin Medical University.
They all signed informed consent in accordance with the approved human
subject protocols. Semen was collected by masturbation and parameters
including liquefaction time, sperm concentration, sperm count, sperm viability
and white blood cell (WBC) count were evaluated. Blood was collected and
placed in 4 for clotting and serum was prepared by centrifuged at 1500g
for 10 min at 4.
2.3
sIL-2R level analysis by ELISA
The
sIL-2R level in serum was analyzed by ELISA according to the procedure
provided by the manufacturer. The ELISA plates were wrapped in monoclonal
antibody of IR-2R
(5 g/mL) and were stored at 4 overnight. Then blocking buffer containing
1% BSA in PBS was placed in each well to minimize nonspecific reaction.
After 2 h of incubation at 37, the plates were washed 3 times with PBS
containing 0.01% Tween-20. Test sera were assayed at serial dilutions
in PBS and 0.25%BSA. After incubation at 37 for 2 h and 3 times washes,
rabbit anti-IL-2R antibody
was added to each well and incubated at 37. After washing the plates,
horse radish peroxidase conjugated antibody (donkey anti-rabbit IgG) was
added to the plates after
dilution and incubated for 2 h at 37, followed by 3 times washes.
The 3,3'-diaminobenzidine tetra-hydrochloride (DAB) substrate 1
mg/mL in citrate phosphate buffer with 0.1% hydrogen peroxide was added
to each well. Results of the reaction were read at 490 nm using a microplate
reader.
2.4
Na+-K+-exchanging ATPase activity assay
The
Na+-K+-exchanging ATPase activity of semen was analyzed
according to the procedure provided by the manufacturer. Protein quantification
was done using the Commassie blue staining method. The Na+-K+-exchanging
ATPase activity was expressed in the phosphor production from the decomposition
per mg protein per h (MPi per mg protein per h).
2.5
Statistical analysis
3
Results
3.1
Semen Parameters
In
infertile men caused by AsAb, the sperm concentration and count were significantly
lower (P<0.05 and P<0.01, respectively) as compared
with the controls, but between the two groups, there were no significant
differences in liquefacation time, sperm viability and WBC count (P>0.05)
(Table 1).
Table
1. AsAb-caused changes of semen parameters in infertile males.
bP<0.05, cP<0.01 vs
control.
|
Semen
parameters |
AsAb
positive (n=24) |
Control
(n=40) |
|
Liquefaction
time (min) |
32.50.5 |
31.50.3 |
|
Sperm
conc. (107/mL) |
4.82.5b |
6.38.2 |
|
Sperm
count (109) |
1.00.4c |
1.40.8 |
|
Sperm
viability (%) |
74.46.1 |
76.44.8 |
|
WBC
count (106/mL) |
2.11.8 |
1.81.4 |
3.2
sIL-2R level
ELISA
showed that the sIL-2R level in serum of infertile males caused by AsAb
was 43572 U/mL, which was significantly higher than that of the control
(28057 U/mL) (P<0.01,
Figure 1).
Figure 1. Change of sIL-2R level in serun of infertile males caused by AsAb. Control (n=10), AsAb(+) (n=7). means. cP<0.01, compared with the control.
3.3
Na+-K+-exchanging ATPase activity
The
Na+-K+-exchanging ATPase activity in semen of AsAb
positive patient was 25.35.2 MPi/(mg proteinh), which was significantly
lower than that of the control 34.612.4 MPi/(mg proteinh) (P<0.05,
Figure 2).
4
Conclusions
Studies
in animals and human beings have demonstrated conclusively that antisperm
antibodies can interfere with fertilization. In the male, autoantibodies
to sperm can be detected both in the serum and seminal plasma[7].
So AsAb detection is one of
the most important steps in the evaluation of male infertility today.
But the mechanism of infertility caused by AsAb has not been clearly illustrated
yet. Prior infection, vasectomy,
vas reversal were commonly associated with the presence of antisperm antibodies[7,8].
Gubin[7] reported that antisperm antibodies did not
significantly decrease the sperm motility and seminal volume. We found
that in infertile men caused
by AsAb, the sperm concentration and sperm count were decreased significantly,
but not the liquefaction time, sperm viability and WBC count.
In
B cell chronic lymphocytic leukemia (B-CLL) patients the level of sIL-2R
was higher as compared with age-matched normal controls[9].
In the present study, it was found that the serum level of sIL-2R was
increased in the infertile males and the increase was ralated to AsAb
in the serum. The result can be explained as follows: when sperm antigens
began to be exposed to the immune
system under some pathogenic conditions, T cells were activated and IL-2
released, expressing IL-2R, so that the level of sIL-2R in serum was raised.
B cells were activated afterwards by the action of T cells, causing the
production of AsAb. Thus the
AsAb titer varied with the sIL-2R level in the serum.
Potassium
homeostasis is a determinant factor in the maintenance of many vital functions.
Na+-K+-exchanging ATPase is involved in potassium
transfer between the extracellular and intracellular compartments, enabling
the creation of an appropriate trans-membrane K gradient[10,11].
The present study showed that the Na+-K+-exchanging
ATPase activity in semen of AsAb positive patients was decreased significantly
compared with that of normal control (P<0.05). It is suggested
that a decrease in the Na+-K+-exchanging ATPase
activity may damage sperm function and may be a possible cause of AsAb
caused infertility.
5
Acknowledgments
References
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[10] Jaisser F. Molecular and functional diversity of Na,K-ATPase
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[11] Blanco G, Mercer RW. Isozymes of the Na+-K+-ATPase:
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Correspondence
to: Prof
Jiang NI, Department of Physiology, Harbin Medical University,
Harbin 150086, CHINA.
Tel +86-451-6674538
e-mail: liql@ems.hrbmu.edu.cn
Received
2000-01-23 Accepted 2000-05-16
