EUROP. J. OBSTET. GYNEC. REPROD. BIOL., 1977,7/l, @ Elsevier/North-Holland Biomedical Press
5-11
Specificity of human sperm antigens solubilized by A/-cetylpyridinium chloride* L. Mettler and T. Grad1 Department of Obstetrics and Gynecology, and Midwifery School, Universityof Kiel, Kiel, Federal Republic of Germany
~_
__-
METTLER, L. and GRADL, T. (1977): Specificity of human sperm antigens solubilized by N-cetylpyridinium chloride. Europ. J. Obstet. Gynec. reprod. Biol., 7/l, 5-11. Washed human spermatozoa were treated with N-cetylpyridinium chloride following ultrasonation. The resulting extract was run against human sperm-agglutinating and sperm-immobilizing as well as rabbit anti-human spermatozoa sera. Applying spermagglutinating sera 8, applying sperm-immobilizing sera 9, and with rabbit anti-human spermatozoa sera 11, immunoprecipitation lines resulted. Only one of the determined antigen fractions designated as Gi specifically reacted with one type of the applied test sera, the sperm-immobilizing sera. It revealed a human sperma specificity with the exception that it cross-reacted with bull spermatozoa. Sera preabsorbed with intact human spermatozoa and preabsorbed with the described solubilized sperm extracts resulted in the disappearance of the Gi precipitation. Therefore, this antigenic fraction is highly suggestive to contain or to be identical with the so called sperm-immobilizing antigen. ultrasonation;
sperm agglutinization; sperm immobilization;
cross-reaction; bull spermatozoa
Introduction
A multitude of antigens has been detected in fractions gained from human spermatozoa. The majority of them have been found to be shared by blood or other organs (Li and Behrmann, 1970). A significant number of antigens fractionated from human spermatozoa occur in epididymal tissue and in seminal plasma (Rao, Sadri and Sheth, 1961). In addition to being tissue nonspecific, some sperm antigens have even proved to be species nonspecific (Mudd and Mudd, 1929). In primates, cross-reactivity was found between testicular tissue antigens of human, chimpanzee, rhesus monkey and baboon (Menge and Fuller, 1975). Human sera with sperm-agglutinating and sperm-immobilizing activities could be partially * Supported by the Deutsche Forschungsgemeinschaft 111-A-V.
SFB
absorbed by washed tree shrew spermatozoa indicating partial antigenic relationship (Mettler, Grad1 and Shirwani, 1976). Kolk, Samuel and Rtimke (1974) detected a human sperm-specific nuclear antigen which can be separated out of swollen sperm heads. Mettler and Gradl (1975) described an SDS-DTI solubilized antigen fraction of human spermatozoa which reacted specifically with human sperm-agglutinating antibodies. The extraction and solubilization of antigenic fractions from spermatozoa proved to be difficult. Physical methods of cell disruption such as sonitication, pressure cells or other mechanical homogenization techniques were found to be insufficient (Henle, Henle and Chambers, 1938; Bell and McLaren, 1970). Better results were achieved by a combination of detergents and sonifications (Zaneveld, Wagner, Schlumberger and Schumacher, 1974). A decondensation of sperm heads using guanidium chloride and
6 mercaptoethanol in chelating basic buffer proved to be the most effective (Coelingh, Rozijn and Montfort, 1969; Garner, Salisbury and Craves, 1971). It is the aim of the present study to test the specificity of sperm fractions solubilized by N-cetylpyridinium chloride,
Material and methods
Solubilized sperm fractions were tested by twodimensional immunoelectrophoresis (Laurell, 1966; Axelsen, K&l and Weeke, 1973) against sera with sperm-agglutinating and sperm-immobilizing activities. The resulting precipitations were tested for specificity by pre-absorptions of the test sera prior to electrophoresis in parallel runs. Solubilization of sperm antigens
300. lo6 human spermatozoa were washed 3 times with phosphate-buffered saline (PBS) and resuspended in 5 ml of the solubilization solution containing 11.83 sodium tetraborate; 10 HiO; 5.17 g KH,PO,; 0.37 g disodium-EDTA; 3.58 g N-cetylpyridinium chloride X Hz0 in 1000 ml HzO. The mixture was ultrasonated for 5 min in ice water (8 /.J wave. length) and centrifuged for 20 min at 300 X g. The supernatant was used for specificity tests. lIvodimensiona1 immunoelectrophoresis
A 5 mm wide strip of 1% agarose gel (approx. 1 ml) pH 9.4 (buffer solution: 9.2 tris-(hydroxymethyl)-aminomethane; 1.05 citric acid Hz0 in 1000 ml aqua dest.) was cut on a 10 X 10 cm measuring glass plate. After the gel had solidified, a well was cut and then filled with 5 /.d of the serum. The electrophoresis was run at 5 V/cm for 2 h. The rest of the plate was then covered with 4 ml of agarose containing 0.5 ml solubilized sperm fractions. The plate was turned by 90” and the electrophoresis performed in a vertical position at 1.5 V/cm for 16 h. The plates were pressed, washed and stained with coomassie blue. Parallel gels were treated with specific histochemical
L. Mertlerand T. Gradl:Humanspermantigenspecificiry reagents for lipids, polynucleotides and carbohydrates. For the detection of lipids, plates were fixed in 50% ethanol for 3 min, postfixed in a mixture of 70% ethanol and acetone (1 : 1 v/v) containing 0.3% scarlet red for 5 min, washed in 50% ethanol for 3 min, and rinsed in tap-water for 2 min. The lipids stained red in this solution. For the identification of polynvcleotides, plates were incubated in a mixture of aqua dest. 2% pyronine GS and 2% methyl green (60 : 25 : 15 v/v). DNA stain blue and RNA red. Polysaccharides were demonstrated with periodic acid Schiff’s reaction. Detection of antigen specificity by absorption studies In order to detect the specificity of the solubilized sperm antigens two-dimensional immunoelectrophoresis was performed using three types of sera with anti-human sperm activity: 1.3 sera with sperm-agglutinating activity (in the micro-sperm agglutination test according to Friberg, 1974); 2.2 sera with sperm-immobilizing activity (microsperm immobilization test according to Husted and Hjort, 1974); 3.3 rabbit anti-human sperm sera; the latter was prepared as follows: 8 * lo6 human spermatozoa, washed 3 times in PBS, were mixed with Freund’s incomplete adjuvant and homogenized for 10 min in ice water using an ultraturrax. 3 rabbits were subcutaneously injected with 10 ~1 of the mixture. Two injections were given in the first week and booster doses followed after 2, 4 and 5 wk. 50 days after the first injection, 20 ml of the blood was gained by heart puncture. The sera were run against the solubilized sperm fractions as described above. Electrophoresis with each serum sample was repeated following absorption of the sera by different tissue samples. The disappearance of the immunoprecipitation bands after absorption of the sera with different tissue samples was regarded as proof of antigenicity shared by the tissues and spermatozoa used. Absorption of 100 Mg of 1 : 4 serum dilution with PBS was performed in 3 parallels for 30 min at 37’C followed by 2 h at 4°C with leukocyte concentrates, erythrocytes, human and animal spermatozoa, and human and animal organs as specified in Table I.
7
L. Mettler and T. Grad!: Human sperm antigen specificity TABLE I
Survey on tissue specimens used to absorb sera with antisperm activity
Specimen
Quantity
Treatment
Leukocytes (huffy coat) a: sperm donors b: other individuals
5x 104
washed and suspended in Hank’s solution
Sephadex G-75
100 &ll
suspended in PBS
Erythrocytes
1 x 105
suspended in Alsever solution
Spermatozoa: ejaculates human bull goose Epididymal flushings cat frog
1 x 105
washed and suspended in PBS
0.5 mg lyophilized
trypsinized, washed in Hank’s solution and lyophilized
mouse Organs, human and animal: liver spleen kidneys testes brain lungs adrenals
Results Specificity carbamylation
of human sperm antigens solubilized by and N-cetylpyridinium chloride using:
Human sperm agglutinating sera. 3 sperm-agglutinating (a) sera, of which 5 aliquot samples were run against the solubilized antigen preparation, demonstrated a constant precipitation pattern (A-H) as schematically indicated in Figure 1. 8 more or less distinct precipitation lines were discernable of which all but 1 showed a migration towards the anode. However, after thorough preabsorption with specimens indicated in Table I, some precipitation lines did not appear. This phenomenon was taken as indicative of cross-reactivity between our solubilized antigen preparation and the applied tissue specimen. Sperm fractions Aa and A’a revealed no cross-reactivity with any of the applied specimens.
Fig. 1. Precipitation pattern in two-dimensional immunoelectrophoresis of sperm-agglutinating sera vs solubilized sperm antigen fractions. 1st dimension: 5 ~1 of human sperm agglutinating serum in tris-citrate buffer (pH 9.4 at 5 V/cm for 2 h). 2nd dimension: serum vs solubilized human sperm antigen fractions at 1.5 V/cm for 16 h.
L. Mettler and T. Gradl: Human sperm antigen specificity
8 TABLE II
Survey of cross-reactivity (-) between human solubilized sperm-antigens and human and animal tissue specimens tested in two-dimensional immunoelectrophoresis (sperm-agglutinating (a) sera against N-cetylpyridinium chloride solubilized human sperm antigens (A-H))
Specimens tested for cross-reactivity
Precipitation lines following absorption Aa
A’a
Ba
Ca
Da
Ea
Fa
Ha
Leukocytes sperm donors other individuals
+ + +
+ + +
+ + +
+ + +
f + +
_ _
_ _
+ + +
Sephadex G-75
f
+
+
+
+
+
+
+
Erythrocytes
+
+
+
+
+
+
+
+
+ + +
+ + +
_ + +
_ + +
_ + _
+ + +
-
-
+ + +
+ + +
+ + f
+ + +
+ +
+ + +
f _ -
-
f +
+ +
+ +
+ +
+ +
-
-
-
-
Spermatozoa: ejaculates human bull goose Epididymal flushings cat frog mouse Organs human animal
+ = Occurrence of precipitation indicative of no cross-reactivity. - = No precipitation indicating cross-reactivity.
They could not even be detected on the surface membranes of intact human spermatozoa. Fractions Ba and Ca were found only on the surface membranes of human spermatozoa. All 4 fractions seem so far to be species and sperm-specific, the first 2 occurring inside and the last 2 on the surface membrane of human spermatozoa. Antigen Da showed sperm specificity, but no clear-cut species specificity could be detected on frog, goose or human spermatozoa. Antigens Ea, Fa and Ha revealed neither sperm nor species specificities. The details of these absorption studies are given in Table II. Human sperm immobilizing
sera
Two human sperm-immobilizing (i) sera run in 5 aliquot samples against the solubilized antigen preparation constantly presented the precipitation (A-H) pattern show in Figure 2. 9 precipitation lines could be discerned of which 2 showed a migration towards the cathode.
Fig. 2. Precipitation pattern in two-dimensional immunoelectrophoresis of sperm-immobilizing serum vs solubillled sperm fractions. 1st dimensions: 5 ~1 of sperm-immobilizing serum: triscitrate buffer, pH 9.4 at 5 V/cm for 2 h. 2nd dimension: serum vs solubilized human sperm fractions at 1.5 V/cm for 16 h.
L. Mettler and T. Gradl: Human sperm antigen specificity
9
only be withdrawn after preabsorption with intact human but not with bull spermatozoa. A similar absorption pattern as for Ha was found for Hi (Table III).
Sperm antigen fraction Ai showd cross-reactivity with cat epididymal flushing. It could also be detected on the surface membrane of intact human spermatozoa. The fraction Ai proved to be sperm and species specific as far as tested. This fraction seems to be identical with Aa as to its electrophoretic migration and cross-reactivity. Fraction Bi was found to be sperm specific but not species specific. With the exception of being species nonspecific it seems to be similar to Ba. Fraction Ci could only be detected on the surface membrane of intact human spermatozoa as was fraction Ca. Di proved to be sperm but not species specific. Sperm fractions Ei and Fi were found to be ubiquitous similar to Ea and Fa. Sperm fraction Gi produced precipitations only when sperm-immobilizing sera were used and showed cross-reactivity only with bull spermatozoa. In accordance with these results the precipitate also disappeared after preabsorption with intact human spermatozoa. However, the sperm-immobilizing activity of female sera could
TABLE III
Rabbit antiserum to human spermatozoa 3 rabbit anti-human spermatozoa (r) sera run in 5 aliquot samples against the solubulized antigen preparation constantly demonstrated the precipitation pattern shown in Figure 3. 11 precipitation lines could be distinguished; 3 showed migration towards the cathode. Sperm fractions Ar and Br revealed sperm specificities, demonstrating cross-reactivity with intact human, bull and goose spermatozoa. Sperm fraction Cr showd cross-reactivity with human and animal organs. This was clearly detectable on the surface membranes of human intact spermatozoa. Fraction Fr, Gr and Hr revealed no sperm or species spe-
Survey of cross-reactivity (-) between human solubilized sperm antigens and human and animal tissue specimens tested in two-dimensional irnmunoelectrophoresis (sperm-immobilizing (i) sera against IV-cetylpyridinium chloride solubilized human sperm antigens (A-H))
Specimens tested for cross-reactivity
Precipitation lines following absorption ____Ai
A’i
Bi ______
Ci
Di
Ei
Fi
Gi
Hi
Leukocytes sperm donors other individuals
+ +
+ +
+ +
+ +
+ +
_ _
_ -
+ +
_ -
Sephadex G-15
+
+
+
+
+
+
+
+
+
Erythrocytes
+
+
+
+
+
_
-
+
+
Spermatozoa: ejaculates human bull goose Epididymal flushings cat frog mouse
_ + +
+ + +
_ + _
_ + +
+
_ _ _
_ -
_ _ +
_ -
+ +
+ + +
_ + +
+ + +
-
_ _
_ -
+ + +
_ + _
Organs human animal
+ +
+ +
+ + + + -__+ = Occurrence of precipitation indicative of no cross-reactivity. - = No precipitation indicating cross-reactivity.
+ +
_ _
_
+ +
_
L. Mettler and T. Gradl: Human sperm antigen specificity
10
cificities, 3 fractions showing migration towards the cathode (Ir, Lr and Kr); Ir and Lr did not reveal sperm nor species specificities. Kr was only detectable on the surface membrane of intact human and mouse spermatozoa. 2,dimension 1
Histochemistry The applied histochemical methods suggest a considerable lipid content of A, B, D, F, I and L antigen fractions. Fraction C was only visible in protein stains. Fraction E gave a weak positivity to lipid stains and was found to be PAS positive, thus indicating its lipopolysaccharid moiety. No positive results were obtained with polynucleotide stains.
.o
0
t.dimension Fig. 3. Precipitation pattern in two-dimensional immunoelectrophoresis of rabbit anti-human spermatozoa serum vs solubilized human sperm fractions. 1st dimension: 5 crl rabbit anti-human spermatozoa serum in tris-citrate buffer, pH 9.4 at 5 V/cm for 2 h. 2nd dimension: serum vs solubilized human sperm fractions at 1.5 V/cm for 16 h.
TABLE IV
Discussion
Using N-cetylpyridinium chloride, a medium polar cationic detergent, 8 antigenic sperm fractions could
Survey on cross-reactivity (-) between human solubilized spermantigens and human and animal tissue specimens tested in two-dimensional immunoelectrophoresis (rabbit anti-human spermatozoa serum against N-cetylpyridinium chloride solubilized human sperm-antigens (A-K)).
Specimens tested for cross-reactivity
Precipitation lines following absorption
Ar
Br
Cr
Dr
Er
Fr
Gr
Hr
Ir
Kr
Lr
Leukocytes sperm donors other individuals
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
Sephadex G-75
+
+
+
+
+
+
+
+
+
+
+
Erythrocytes
+
+
+
+
+
+
+
+
+
+
+ _ -
Spermatozoa: ejaculates human bull goose Epididymal flushings cat frog mouse
-
_ -
_ + +
+ + +
+ + +
+ + +
+ -
-
+
-
+
+ +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+
_ + +
+ + +
+ + -
-. +
Organs human animals
+ +
+ +
-
+ +
+ +
+ -
_ _
-
+ -
+ +
-
+ = Occurrence of precipitation indicative of no cross-reactivity. - = No precipitation indicating cross-reactivity.
L. Mettler and T. Gradl: Human sperm antigen specificity
be extracted from ultrasonated spermatozoa run against human sperm-agglutinating sera in two-dimensional immunoelectrophoresis. In runs against spermimmobilizing sera, 9 sperm fractions and in those against rabbit anti-human spermatozoa sera 11 fractions could be visualized. Most of the antigenic fractions which were separated showed cross-reactivity with allogenic or xenogenic tissues. Cross-reactivity with human and animal organs was observed as follows: a. fractions Ea, Fa and Ha in runs vs sperm-agglutinating sera (Table 11); b. fractions Ei, Fi and Hi in runs vs sperm-immobilizing sera (Table III), and c. fractions Cr, Fr, Gr, Hr, Ir and Lr in runs against rabbit anti-human spermatozoa sera (Table IV). Sperm as well as species specificities were found in fractions Aa and A’a as well as in A’i, Dr and Er. However, none of these fractions could be detected on the surface membranes of intact human spermatozoa. Fractions Ba, Ca, Da, Ai, Bi, Ci, Di, Gi and Ar, Br and Er were found to be sperm specific and species independent. Evaluation of the obtained precipitation patterns showed evidence that only Ba, Ca and Ci reacted with the surface membrane of human spermatozoa. They proved to be human and sperm specific. Thus, the number as well as the character of the antigens solubilized by N-cetylpyridinium chloride compares well with the antigenic patterns described by other authors (Li and Behrmann, 1970; Rao et al., 1961). Applying this sperm-solubilization method, specific reactivity against a sperm-immobilizing serum (Gi) was detected, but no specific reactivity against a sperm-agglutinating serum could be found. Gi is considered as the ‘immobilizing’ antigen. Its chemical components and its stability remain to be determined. The experiments allow no conclusions as to the origin of the antigens detectable on the surface membrane of intact spermatozoa. The possibility has not been excluded that these antigens originally derive from sperm plasma being attached to the surface membrane during sperm passage through the male genital tract. Considering the fact that the antigenic fractions participating in the immune precipitation have not been studied further in respect to their heterogeneity, the possibility exists that we are dealing with various antigens.
11 Acknowledgement
We gratefully acknowledge the technical assistance of Ulla Bachmann-Diekmann.
References
Axelsen, N.H., Kr#l, J. and Weeke, B. (1973): Quantitative immunoelectrophoresis. &and. J. Immunol., 2, Suppl. 1. Bell, E.B. and McLaren, A. (1970): Reduction of fertility in female mice iso-immunized with a sub-cellular sperm fraction. J. Reprod. Fertil., 22, 345. Coelingh, J.P., Rozijn, R.H. and Montfort, C.H. (1969): Isolation and partial characterization of a basic protein from bovine sperm heads. Biochim. biophys. Acta, 188, 353. Friberg, J. (1974): A simple and sensitive micro method for demonstration of sperm agglutinating activity in serum from infertile men and women. Acta obstet. gynec. stand., Suppl. 36, 21-29. Garner, D.L., Salisbury, G.W. and Craves, C.N. (1971): Electrophoretic fraction of bovine acrosomal proteins and proteinase. Biol. Reprod., 4, 93. Henle, W., Henle, G. and Chambers, L.A. (1938): Studies on the antigenic structure of some mammalian spermatozoa. J. exp. Med., 68, 335. Husted, S. and Hjort, T. (1974): Microtechnique for detection of immobilization and cytotoxicity. Presented at WHO-workshop, July 15-19, Aarhus, 1974. Kolk, A.H.J., Samuel, T. and Rtimke, P. (1974): Autoantigens of human spermatozoa. I. Solubilization of a new auto-antigen detected on swollen spermheads. J. clin. exp. Immunol., 16, 63. Laurell, C.B. (1966): Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Analyt. Biochem., IS, 45. Li, T.S. and Behrmann, S.J. (1970): The sperm and seminal plasma specific antigens of human. Fertil. and Steril. 21. 565. Menge, A.C. and Fuller, B. (1975): Testis antigens of man and some other primates. Fertil. and Steril., in preparation. Mettler, L. and Gradl, T. (1975): Solubilization of human spermatozoa and antigen isolation responsible for agglutiMettler, L., Gradl, T. and Shirwani, D. (1976): Tissue and species specificity of spermantibodies. In press. Mudd, S. and Mudd, E.B.H. (1929): The specificity of mammalian spermatozoa with special reference to electrophoresis as a means of serological differentiation. J. Immunoi., 17, 39. Rao, S.S., Sadri, K.K. and Sheth, A.R. (1961): The antigenicity of human spermatozoa and its significance. In: Proc. Symp. Proteins 1960, C.F.T.R.I. Mysore. Zaneveld, L.J.D., Wagner, L., Schlumberger, H.D. and Schumacher, G.F.B. (1974): Immunological and biochemical studies on fractionated bull spermatozoa. J. Reprod. Fertil., 38, 411.
5-11
Specificity of human sperm antigens solubilized by A/-cetylpyridinium chloride* L. Mettler and T. Grad1 Department of Obstetrics and Gynecology, and Midwifery School, Universityof Kiel, Kiel, Federal Republic of Germany
~_
__-
METTLER, L. and GRADL, T. (1977): Specificity of human sperm antigens solubilized by N-cetylpyridinium chloride. Europ. J. Obstet. Gynec. reprod. Biol., 7/l, 5-11. Washed human spermatozoa were treated with N-cetylpyridinium chloride following ultrasonation. The resulting extract was run against human sperm-agglutinating and sperm-immobilizing as well as rabbit anti-human spermatozoa sera. Applying spermagglutinating sera 8, applying sperm-immobilizing sera 9, and with rabbit anti-human spermatozoa sera 11, immunoprecipitation lines resulted. Only one of the determined antigen fractions designated as Gi specifically reacted with one type of the applied test sera, the sperm-immobilizing sera. It revealed a human sperma specificity with the exception that it cross-reacted with bull spermatozoa. Sera preabsorbed with intact human spermatozoa and preabsorbed with the described solubilized sperm extracts resulted in the disappearance of the Gi precipitation. Therefore, this antigenic fraction is highly suggestive to contain or to be identical with the so called sperm-immobilizing antigen. ultrasonation;
sperm agglutinization; sperm immobilization;
cross-reaction; bull spermatozoa
Introduction
A multitude of antigens has been detected in fractions gained from human spermatozoa. The majority of them have been found to be shared by blood or other organs (Li and Behrmann, 1970). A significant number of antigens fractionated from human spermatozoa occur in epididymal tissue and in seminal plasma (Rao, Sadri and Sheth, 1961). In addition to being tissue nonspecific, some sperm antigens have even proved to be species nonspecific (Mudd and Mudd, 1929). In primates, cross-reactivity was found between testicular tissue antigens of human, chimpanzee, rhesus monkey and baboon (Menge and Fuller, 1975). Human sera with sperm-agglutinating and sperm-immobilizing activities could be partially * Supported by the Deutsche Forschungsgemeinschaft 111-A-V.
SFB
absorbed by washed tree shrew spermatozoa indicating partial antigenic relationship (Mettler, Grad1 and Shirwani, 1976). Kolk, Samuel and Rtimke (1974) detected a human sperm-specific nuclear antigen which can be separated out of swollen sperm heads. Mettler and Gradl (1975) described an SDS-DTI solubilized antigen fraction of human spermatozoa which reacted specifically with human sperm-agglutinating antibodies. The extraction and solubilization of antigenic fractions from spermatozoa proved to be difficult. Physical methods of cell disruption such as sonitication, pressure cells or other mechanical homogenization techniques were found to be insufficient (Henle, Henle and Chambers, 1938; Bell and McLaren, 1970). Better results were achieved by a combination of detergents and sonifications (Zaneveld, Wagner, Schlumberger and Schumacher, 1974). A decondensation of sperm heads using guanidium chloride and
6 mercaptoethanol in chelating basic buffer proved to be the most effective (Coelingh, Rozijn and Montfort, 1969; Garner, Salisbury and Craves, 1971). It is the aim of the present study to test the specificity of sperm fractions solubilized by N-cetylpyridinium chloride,
Material and methods
Solubilized sperm fractions were tested by twodimensional immunoelectrophoresis (Laurell, 1966; Axelsen, K&l and Weeke, 1973) against sera with sperm-agglutinating and sperm-immobilizing activities. The resulting precipitations were tested for specificity by pre-absorptions of the test sera prior to electrophoresis in parallel runs. Solubilization of sperm antigens
300. lo6 human spermatozoa were washed 3 times with phosphate-buffered saline (PBS) and resuspended in 5 ml of the solubilization solution containing 11.83 sodium tetraborate; 10 HiO; 5.17 g KH,PO,; 0.37 g disodium-EDTA; 3.58 g N-cetylpyridinium chloride X Hz0 in 1000 ml HzO. The mixture was ultrasonated for 5 min in ice water (8 /.J wave. length) and centrifuged for 20 min at 300 X g. The supernatant was used for specificity tests. lIvodimensiona1 immunoelectrophoresis
A 5 mm wide strip of 1% agarose gel (approx. 1 ml) pH 9.4 (buffer solution: 9.2 tris-(hydroxymethyl)-aminomethane; 1.05 citric acid Hz0 in 1000 ml aqua dest.) was cut on a 10 X 10 cm measuring glass plate. After the gel had solidified, a well was cut and then filled with 5 /.d of the serum. The electrophoresis was run at 5 V/cm for 2 h. The rest of the plate was then covered with 4 ml of agarose containing 0.5 ml solubilized sperm fractions. The plate was turned by 90” and the electrophoresis performed in a vertical position at 1.5 V/cm for 16 h. The plates were pressed, washed and stained with coomassie blue. Parallel gels were treated with specific histochemical
L. Mertlerand T. Gradl:Humanspermantigenspecificiry reagents for lipids, polynucleotides and carbohydrates. For the detection of lipids, plates were fixed in 50% ethanol for 3 min, postfixed in a mixture of 70% ethanol and acetone (1 : 1 v/v) containing 0.3% scarlet red for 5 min, washed in 50% ethanol for 3 min, and rinsed in tap-water for 2 min. The lipids stained red in this solution. For the identification of polynvcleotides, plates were incubated in a mixture of aqua dest. 2% pyronine GS and 2% methyl green (60 : 25 : 15 v/v). DNA stain blue and RNA red. Polysaccharides were demonstrated with periodic acid Schiff’s reaction. Detection of antigen specificity by absorption studies In order to detect the specificity of the solubilized sperm antigens two-dimensional immunoelectrophoresis was performed using three types of sera with anti-human sperm activity: 1.3 sera with sperm-agglutinating activity (in the micro-sperm agglutination test according to Friberg, 1974); 2.2 sera with sperm-immobilizing activity (microsperm immobilization test according to Husted and Hjort, 1974); 3.3 rabbit anti-human sperm sera; the latter was prepared as follows: 8 * lo6 human spermatozoa, washed 3 times in PBS, were mixed with Freund’s incomplete adjuvant and homogenized for 10 min in ice water using an ultraturrax. 3 rabbits were subcutaneously injected with 10 ~1 of the mixture. Two injections were given in the first week and booster doses followed after 2, 4 and 5 wk. 50 days after the first injection, 20 ml of the blood was gained by heart puncture. The sera were run against the solubilized sperm fractions as described above. Electrophoresis with each serum sample was repeated following absorption of the sera by different tissue samples. The disappearance of the immunoprecipitation bands after absorption of the sera with different tissue samples was regarded as proof of antigenicity shared by the tissues and spermatozoa used. Absorption of 100 Mg of 1 : 4 serum dilution with PBS was performed in 3 parallels for 30 min at 37’C followed by 2 h at 4°C with leukocyte concentrates, erythrocytes, human and animal spermatozoa, and human and animal organs as specified in Table I.
7
L. Mettler and T. Grad!: Human sperm antigen specificity TABLE I
Survey on tissue specimens used to absorb sera with antisperm activity
Specimen
Quantity
Treatment
Leukocytes (huffy coat) a: sperm donors b: other individuals
5x 104
washed and suspended in Hank’s solution
Sephadex G-75
100 &ll
suspended in PBS
Erythrocytes
1 x 105
suspended in Alsever solution
Spermatozoa: ejaculates human bull goose Epididymal flushings cat frog
1 x 105
washed and suspended in PBS
0.5 mg lyophilized
trypsinized, washed in Hank’s solution and lyophilized
mouse Organs, human and animal: liver spleen kidneys testes brain lungs adrenals
Results Specificity carbamylation
of human sperm antigens solubilized by and N-cetylpyridinium chloride using:
Human sperm agglutinating sera. 3 sperm-agglutinating (a) sera, of which 5 aliquot samples were run against the solubilized antigen preparation, demonstrated a constant precipitation pattern (A-H) as schematically indicated in Figure 1. 8 more or less distinct precipitation lines were discernable of which all but 1 showed a migration towards the anode. However, after thorough preabsorption with specimens indicated in Table I, some precipitation lines did not appear. This phenomenon was taken as indicative of cross-reactivity between our solubilized antigen preparation and the applied tissue specimen. Sperm fractions Aa and A’a revealed no cross-reactivity with any of the applied specimens.
Fig. 1. Precipitation pattern in two-dimensional immunoelectrophoresis of sperm-agglutinating sera vs solubilized sperm antigen fractions. 1st dimension: 5 ~1 of human sperm agglutinating serum in tris-citrate buffer (pH 9.4 at 5 V/cm for 2 h). 2nd dimension: serum vs solubilized human sperm antigen fractions at 1.5 V/cm for 16 h.
L. Mettler and T. Gradl: Human sperm antigen specificity
8 TABLE II
Survey of cross-reactivity (-) between human solubilized sperm-antigens and human and animal tissue specimens tested in two-dimensional immunoelectrophoresis (sperm-agglutinating (a) sera against N-cetylpyridinium chloride solubilized human sperm antigens (A-H))
Specimens tested for cross-reactivity
Precipitation lines following absorption Aa
A’a
Ba
Ca
Da
Ea
Fa
Ha
Leukocytes sperm donors other individuals
+ + +
+ + +
+ + +
+ + +
f + +
_ _
_ _
+ + +
Sephadex G-75
f
+
+
+
+
+
+
+
Erythrocytes
+
+
+
+
+
+
+
+
+ + +
+ + +
_ + +
_ + +
_ + _
+ + +
-
-
+ + +
+ + +
+ + f
+ + +
+ +
+ + +
f _ -
-
f +
+ +
+ +
+ +
+ +
-
-
-
-
Spermatozoa: ejaculates human bull goose Epididymal flushings cat frog mouse Organs human animal
+ = Occurrence of precipitation indicative of no cross-reactivity. - = No precipitation indicating cross-reactivity.
They could not even be detected on the surface membranes of intact human spermatozoa. Fractions Ba and Ca were found only on the surface membranes of human spermatozoa. All 4 fractions seem so far to be species and sperm-specific, the first 2 occurring inside and the last 2 on the surface membrane of human spermatozoa. Antigen Da showed sperm specificity, but no clear-cut species specificity could be detected on frog, goose or human spermatozoa. Antigens Ea, Fa and Ha revealed neither sperm nor species specificities. The details of these absorption studies are given in Table II. Human sperm immobilizing
sera
Two human sperm-immobilizing (i) sera run in 5 aliquot samples against the solubilized antigen preparation constantly presented the precipitation (A-H) pattern show in Figure 2. 9 precipitation lines could be discerned of which 2 showed a migration towards the cathode.
Fig. 2. Precipitation pattern in two-dimensional immunoelectrophoresis of sperm-immobilizing serum vs solubillled sperm fractions. 1st dimensions: 5 ~1 of sperm-immobilizing serum: triscitrate buffer, pH 9.4 at 5 V/cm for 2 h. 2nd dimension: serum vs solubilized human sperm fractions at 1.5 V/cm for 16 h.
L. Mettler and T. Gradl: Human sperm antigen specificity
9
only be withdrawn after preabsorption with intact human but not with bull spermatozoa. A similar absorption pattern as for Ha was found for Hi (Table III).
Sperm antigen fraction Ai showd cross-reactivity with cat epididymal flushing. It could also be detected on the surface membrane of intact human spermatozoa. The fraction Ai proved to be sperm and species specific as far as tested. This fraction seems to be identical with Aa as to its electrophoretic migration and cross-reactivity. Fraction Bi was found to be sperm specific but not species specific. With the exception of being species nonspecific it seems to be similar to Ba. Fraction Ci could only be detected on the surface membrane of intact human spermatozoa as was fraction Ca. Di proved to be sperm but not species specific. Sperm fractions Ei and Fi were found to be ubiquitous similar to Ea and Fa. Sperm fraction Gi produced precipitations only when sperm-immobilizing sera were used and showed cross-reactivity only with bull spermatozoa. In accordance with these results the precipitate also disappeared after preabsorption with intact human spermatozoa. However, the sperm-immobilizing activity of female sera could
TABLE III
Rabbit antiserum to human spermatozoa 3 rabbit anti-human spermatozoa (r) sera run in 5 aliquot samples against the solubulized antigen preparation constantly demonstrated the precipitation pattern shown in Figure 3. 11 precipitation lines could be distinguished; 3 showed migration towards the cathode. Sperm fractions Ar and Br revealed sperm specificities, demonstrating cross-reactivity with intact human, bull and goose spermatozoa. Sperm fraction Cr showd cross-reactivity with human and animal organs. This was clearly detectable on the surface membranes of human intact spermatozoa. Fraction Fr, Gr and Hr revealed no sperm or species spe-
Survey of cross-reactivity (-) between human solubilized sperm antigens and human and animal tissue specimens tested in two-dimensional irnmunoelectrophoresis (sperm-immobilizing (i) sera against IV-cetylpyridinium chloride solubilized human sperm antigens (A-H))
Specimens tested for cross-reactivity
Precipitation lines following absorption ____Ai
A’i
Bi ______
Ci
Di
Ei
Fi
Gi
Hi
Leukocytes sperm donors other individuals
+ +
+ +
+ +
+ +
+ +
_ _
_ -
+ +
_ -
Sephadex G-15
+
+
+
+
+
+
+
+
+
Erythrocytes
+
+
+
+
+
_
-
+
+
Spermatozoa: ejaculates human bull goose Epididymal flushings cat frog mouse
_ + +
+ + +
_ + _
_ + +
+
_ _ _
_ -
_ _ +
_ -
+ +
+ + +
_ + +
+ + +
-
_ _
_ -
+ + +
_ + _
Organs human animal
+ +
+ +
+ + + + -__+ = Occurrence of precipitation indicative of no cross-reactivity. - = No precipitation indicating cross-reactivity.
+ +
_ _
_
+ +
_
L. Mettler and T. Gradl: Human sperm antigen specificity
10
cificities, 3 fractions showing migration towards the cathode (Ir, Lr and Kr); Ir and Lr did not reveal sperm nor species specificities. Kr was only detectable on the surface membrane of intact human and mouse spermatozoa. 2,dimension 1
Histochemistry The applied histochemical methods suggest a considerable lipid content of A, B, D, F, I and L antigen fractions. Fraction C was only visible in protein stains. Fraction E gave a weak positivity to lipid stains and was found to be PAS positive, thus indicating its lipopolysaccharid moiety. No positive results were obtained with polynucleotide stains.
.o
0
t.dimension Fig. 3. Precipitation pattern in two-dimensional immunoelectrophoresis of rabbit anti-human spermatozoa serum vs solubilized human sperm fractions. 1st dimension: 5 crl rabbit anti-human spermatozoa serum in tris-citrate buffer, pH 9.4 at 5 V/cm for 2 h. 2nd dimension: serum vs solubilized human sperm fractions at 1.5 V/cm for 16 h.
TABLE IV
Discussion
Using N-cetylpyridinium chloride, a medium polar cationic detergent, 8 antigenic sperm fractions could
Survey on cross-reactivity (-) between human solubilized spermantigens and human and animal tissue specimens tested in two-dimensional immunoelectrophoresis (rabbit anti-human spermatozoa serum against N-cetylpyridinium chloride solubilized human sperm-antigens (A-K)).
Specimens tested for cross-reactivity
Precipitation lines following absorption
Ar
Br
Cr
Dr
Er
Fr
Gr
Hr
Ir
Kr
Lr
Leukocytes sperm donors other individuals
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
+ +
Sephadex G-75
+
+
+
+
+
+
+
+
+
+
+
Erythrocytes
+
+
+
+
+
+
+
+
+
+
+ _ -
Spermatozoa: ejaculates human bull goose Epididymal flushings cat frog mouse
-
_ -
_ + +
+ + +
+ + +
+ + +
+ -
-
+
-
+
+ +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+
_ + +
+ + +
+ + -
-. +
Organs human animals
+ +
+ +
-
+ +
+ +
+ -
_ _
-
+ -
+ +
-
+ = Occurrence of precipitation indicative of no cross-reactivity. - = No precipitation indicating cross-reactivity.
L. Mettler and T. Gradl: Human sperm antigen specificity
be extracted from ultrasonated spermatozoa run against human sperm-agglutinating sera in two-dimensional immunoelectrophoresis. In runs against spermimmobilizing sera, 9 sperm fractions and in those against rabbit anti-human spermatozoa sera 11 fractions could be visualized. Most of the antigenic fractions which were separated showed cross-reactivity with allogenic or xenogenic tissues. Cross-reactivity with human and animal organs was observed as follows: a. fractions Ea, Fa and Ha in runs vs sperm-agglutinating sera (Table 11); b. fractions Ei, Fi and Hi in runs vs sperm-immobilizing sera (Table III), and c. fractions Cr, Fr, Gr, Hr, Ir and Lr in runs against rabbit anti-human spermatozoa sera (Table IV). Sperm as well as species specificities were found in fractions Aa and A’a as well as in A’i, Dr and Er. However, none of these fractions could be detected on the surface membranes of intact human spermatozoa. Fractions Ba, Ca, Da, Ai, Bi, Ci, Di, Gi and Ar, Br and Er were found to be sperm specific and species independent. Evaluation of the obtained precipitation patterns showed evidence that only Ba, Ca and Ci reacted with the surface membrane of human spermatozoa. They proved to be human and sperm specific. Thus, the number as well as the character of the antigens solubilized by N-cetylpyridinium chloride compares well with the antigenic patterns described by other authors (Li and Behrmann, 1970; Rao et al., 1961). Applying this sperm-solubilization method, specific reactivity against a sperm-immobilizing serum (Gi) was detected, but no specific reactivity against a sperm-agglutinating serum could be found. Gi is considered as the ‘immobilizing’ antigen. Its chemical components and its stability remain to be determined. The experiments allow no conclusions as to the origin of the antigens detectable on the surface membrane of intact spermatozoa. The possibility has not been excluded that these antigens originally derive from sperm plasma being attached to the surface membrane during sperm passage through the male genital tract. Considering the fact that the antigenic fractions participating in the immune precipitation have not been studied further in respect to their heterogeneity, the possibility exists that we are dealing with various antigens.
11 Acknowledgement
We gratefully acknowledge the technical assistance of Ulla Bachmann-Diekmann.
References
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