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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
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

Data were expressed as means. Statistical analysis was performed using the Student's t-test and the significance of differences was set at P<0.05.

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)



Sperm conc. (107/mL)



Sperm count (109)



Sperm viability (%)



WBC count (106/mL)



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).

Figure  2. Change of Na+-K+-exchanging ATPase activity in semen of infertile males caused by AsAb.  Control (n=12), AsAb(+) (n=7). means. bP<0.05, compared with the control.

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 

We thank Prof Zhi-Ping CHENG for his beneficial advice on the work.


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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.
Received 2000-01-23     Accepted 2000-05-16