andrologia 7 (2) : 99-104
Received October 4, 1974
(1975)
Department of Obstetrics and Gynecology University of Kiel (Germany) (Director: Prof. Dr. K. SEMM) Department of Andrology University of Hamburg (Germany) (Head: Prof. Dr. C. SCHIRREN)
Determination of sperm-antibodies in sera of sterile women by affinity-chromatography (Affinity-chromatography of sperm-antibodies) T. GRADL, L. METTLER and C. SCHIRREN
Introduction Sensitization against spermatozoa and seminal plasma as a causative of male and female sterility and infertility is currently investigated in the field of reproductive immunology. So far, humoral and cellular immune responses to sperm components were described. The occurance of circulating antibodies in female and male sera against sperm components has frequently been reported (Wilson - 1954, Riimke - 1959; Masson et al. - 1970, Mettler - 1974). The applied techniques for the detection of agglutinating sperm-antibodies comprise the test according to Kibrick et al. - 1952 and the Franklin and Dukes (FD) method (1964). Sperm immobilizing antibodies can be detected by a test according to Isojima, Li and Ashitaka (1968). Further, antiseminal sensitization was demonstrated by immunofluorescent (Hjort and Hansen 1971) and cytoxicity techniques (Hamerlynck and Riimke - 1968). In the micro- and macro-spermagglutination techniques fresh and motile spermatozoa, which are agglutinated by female sera, are used. These sperm agglutinations are predominantly found in sera of sterile woman with uwxplained causes of sterility. After inactivation of the complement-system at 56" C for 30 minutes and addition of guinea pig complement, the reduction of motility in comparison to a negative control serum is measured in the micro-spermimmobilisation test. The uptake of trypan blue is used for the determination of cytotoxicity. The immunofluorescence technique demonstrates four different antibodies reacting with antigenic sites located at the front part of the acrosume, at the equatorial segment, at the postnuclear cap and the tail. All these tests detect antigen-antibody reactions but render no information on the quantity of the antibodies. It is the aim of this paper to describe a method for the direct quantitation of sperm antibodies. In this procedure affinity-chromatography is employed and intact spermatozoa serve as antigen carriers. Antibodies, specifically eluted, are expressed in terms of pg protein/ml.
* Supported by Deutsche Forschungsgemeinschaft, SFB 111, FRG. Key words: Sperm antibody - sterile women
-
affinity chromatography
-
frozen semen
T. GRADL,L. METTLERand C. SCHIRREN
100
affinity -chromatography I I m l fractions 1
A II
I
I
0 Fig. 1:
5
10
15
20
25
cluat ( m l )
Elution diagram of a sperm-antibody containing serum sample in comparison to a blank serum sample Column: 1 ml Sephadex G 75 1 ml washed spermatozoa (8.10' spermatozoa) Sera: -= containing antibodies - - - _ _=blank without antibodies The arrow indicates the changing of the elution medium (lStelution: Phosphate buffer pH 7,2-2nd elution tris-HC1 buffer pH 8,9).
Material and methods Fresh or deep frozen semen (8. lo' spermatozoa per ml) is washed 3 times with phosphate-buffered saline (I, 155 m) and suspended in 0,066 m Sdrensen buffer at pH 7,2 adjusting the original cell count. Columns (8 cm long, 0,5 cm inner diameter) are filled with 0,5 ml swollen SephadexmG 75 and left to sediment. 1 ml of the spermatozoa suspension is added. There after the columns are overlayered each with 1 ml of the serum samples or serum dilutions. 4 serum samples which had repeatedly been positiv in the FD-test and 2 blanks were used. The agglutinating serum activity in percent was determined in 5 parallels at 1:4 serum dilations with the FD-test. Only 1-10 agglutinated motile spermatozoa were considered as positive reactions. Then these columns are washed with 5 ml of Sbrensen buffer for 5 minutes till no more proteins can be detected in the effluents. With this procedure all serum proteins are washed out while antibodies attached to spermatozoa remain within the columns. Subsequently the protein fractions, resisting the first washing, are dissoziated with 8 ml of Tris-HC1 buffer pH 8,9 (0,05 m). In the effluent, containing the antibodies against spermatozoa quantitation is performed by determination of total proteins with the Folin-Ciocalteu-method (1927). For the determination of unspecific proteins, serum andrologia 7, Heft 2 (1975)
Determination of sperm-antibodies in sera of sterile women
101
protein content of anti body - f r a c t ion
( v g protein)
adsorption - isotherme 50 0
LOO
/
/*
100
u n s p e c i f i c adsorption
~
Received October 4, 1974
(1975)
Department of Obstetrics and Gynecology University of Kiel (Germany) (Director: Prof. Dr. K. SEMM) Department of Andrology University of Hamburg (Germany) (Head: Prof. Dr. C. SCHIRREN)
Determination of sperm-antibodies in sera of sterile women by affinity-chromatography (Affinity-chromatography of sperm-antibodies) T. GRADL, L. METTLER and C. SCHIRREN
Introduction Sensitization against spermatozoa and seminal plasma as a causative of male and female sterility and infertility is currently investigated in the field of reproductive immunology. So far, humoral and cellular immune responses to sperm components were described. The occurance of circulating antibodies in female and male sera against sperm components has frequently been reported (Wilson - 1954, Riimke - 1959; Masson et al. - 1970, Mettler - 1974). The applied techniques for the detection of agglutinating sperm-antibodies comprise the test according to Kibrick et al. - 1952 and the Franklin and Dukes (FD) method (1964). Sperm immobilizing antibodies can be detected by a test according to Isojima, Li and Ashitaka (1968). Further, antiseminal sensitization was demonstrated by immunofluorescent (Hjort and Hansen 1971) and cytoxicity techniques (Hamerlynck and Riimke - 1968). In the micro- and macro-spermagglutination techniques fresh and motile spermatozoa, which are agglutinated by female sera, are used. These sperm agglutinations are predominantly found in sera of sterile woman with uwxplained causes of sterility. After inactivation of the complement-system at 56" C for 30 minutes and addition of guinea pig complement, the reduction of motility in comparison to a negative control serum is measured in the micro-spermimmobilisation test. The uptake of trypan blue is used for the determination of cytotoxicity. The immunofluorescence technique demonstrates four different antibodies reacting with antigenic sites located at the front part of the acrosume, at the equatorial segment, at the postnuclear cap and the tail. All these tests detect antigen-antibody reactions but render no information on the quantity of the antibodies. It is the aim of this paper to describe a method for the direct quantitation of sperm antibodies. In this procedure affinity-chromatography is employed and intact spermatozoa serve as antigen carriers. Antibodies, specifically eluted, are expressed in terms of pg protein/ml.
* Supported by Deutsche Forschungsgemeinschaft, SFB 111, FRG. Key words: Sperm antibody - sterile women
-
affinity chromatography
-
frozen semen
T. GRADL,L. METTLERand C. SCHIRREN
100
affinity -chromatography I I m l fractions 1
A II
I
I
0 Fig. 1:
5
10
15
20
25
cluat ( m l )
Elution diagram of a sperm-antibody containing serum sample in comparison to a blank serum sample Column: 1 ml Sephadex G 75 1 ml washed spermatozoa (8.10' spermatozoa) Sera: -= containing antibodies - - - _ _=blank without antibodies The arrow indicates the changing of the elution medium (lStelution: Phosphate buffer pH 7,2-2nd elution tris-HC1 buffer pH 8,9).
Material and methods Fresh or deep frozen semen (8. lo' spermatozoa per ml) is washed 3 times with phosphate-buffered saline (I, 155 m) and suspended in 0,066 m Sdrensen buffer at pH 7,2 adjusting the original cell count. Columns (8 cm long, 0,5 cm inner diameter) are filled with 0,5 ml swollen SephadexmG 75 and left to sediment. 1 ml of the spermatozoa suspension is added. There after the columns are overlayered each with 1 ml of the serum samples or serum dilutions. 4 serum samples which had repeatedly been positiv in the FD-test and 2 blanks were used. The agglutinating serum activity in percent was determined in 5 parallels at 1:4 serum dilations with the FD-test. Only 1-10 agglutinated motile spermatozoa were considered as positive reactions. Then these columns are washed with 5 ml of Sbrensen buffer for 5 minutes till no more proteins can be detected in the effluents. With this procedure all serum proteins are washed out while antibodies attached to spermatozoa remain within the columns. Subsequently the protein fractions, resisting the first washing, are dissoziated with 8 ml of Tris-HC1 buffer pH 8,9 (0,05 m). In the effluent, containing the antibodies against spermatozoa quantitation is performed by determination of total proteins with the Folin-Ciocalteu-method (1927). For the determination of unspecific proteins, serum andrologia 7, Heft 2 (1975)
Determination of sperm-antibodies in sera of sterile women
101
protein content of anti body - f r a c t ion
( v g protein)
adsorption - isotherme 50 0
LOO
/
/*
100
u n s p e c i f i c adsorption
~
