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Antigenicity of Spermatozoa
Antigenicity of Spermatozoa SEYMOUR KATSH, PH.D., AND GRACE F. KATSH, PH.D.
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will present a brief review of our previous efforts and a report of our current attempts to elucidate the chemistry of spermatozoal antigens. The subject of this paper may have had its beginnings, by inference at least, in the Bible (Sarah able to conceive only after long continence) or in later writings such as Darwin's Descent of Man (the profligacy of women accounting for their infertility), the inference being that exposure to sperm had resulted in sterility. For us, however, the beginning must be dated from 1899 to 1900 when Landsteiner,29 Metchnikoff, and Metalnikoff first reported that injections of spermatozoa or testicular extracts into experimental animals led to antibody production. In the initial illumination of this revelation, much effort was directed toward accomplishing infertility by injections of testicular tissue or sperm; success in those ventures was elusive, however. During the intervening years, it has been amply demonstrated that spermatozoa are antigenic and that the antigenic nature of spermatozoa is remarkably species-selective and organ-selective if not species-specific and organ-specific. The following synopsis of reports on the subject is given not for the value of the findings per se, but rather for their fruitful implications. Farnum injected semen or testicular material of the dog, bull, or man into experimental animals and reported that the resultant antibodies were specific for each kind of material. There was no cross-reaction to indicate similarity of antigenicity: this was an indication of species-specificity. Pfeiffer injected IDS ARTICLE
From the University of Colorado Medical Center, Denver Colo. Presented at the Seventeenth Annual Meeting of the American Society for the ~tudy of Sterility, Apr. 21-23, 1961, Bal Harbour, Fla. It is a pleasure to acknowledge the continuing encouragement and financial support of Drs. Warren O. Nelson and Sheldon J. Segal of the Population Council, Inc. 522
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bull spenn into rabbits and reported that the antiserum reacted only with bovine testis and negligibly, if at all, with extracts of other bovine organs. This was an indication of organ-specificity. Hektoen and Manly injected human seminal fluid and spenn as well as swine, bovine, and equine seminal fluid into rabbits and reported species- and semen-specific precipitins. Pommerenke found cross-reactions between rabbit and rat spenn: the serum of rabbits injected with rabbit spenn was toxic for rat spenn and vice versa. Mudd and Mudd demonstrated that the spenn of man, guinea pig, bull, and ram induced species-specific antibodies in rabbits. However, cross-reactions between bull and ram spenn and their corresponding antisera were noted. These and other experiments led to the conclusion that mammalian spenn possessed both species- and tissue-specificity. The absoluteness of species and tissue specificity was modified by Lewis31 who disclosed that antitesticle sera reacted strongly with brain, but not with liver, lung, heart, spleen, kidney, or ovary; and that testicle and brain have a marked organ- and species-selectivity, but not absolute specificity. Henle7 supported the contention that species-specificity of spennatozoa was of a dominant rather than an absolute nature, for he found cross-reactions between spenn of different species. For example, there was strong antigenic resemblance between bull and sheep spenn, but less reactivity between bull and human spenn. Henle et al.,s using bull spenn, found: 1. heat-labile, head-specific and tail-specific antigens and a heat-stable antigen common to both heads and tails that was species-specific; 2. three different cross-reacting antigens, two of which were in the heads and one in the tails; 3. one head antigen that was not active in the native cell (discovered after rupture of the spenn). Lewis32 demonstrated that not only did antitestis sera react with brain tissue but also that antisera against alcoholic extracts of these two organs had the same antigenic properties. Moreover, both organs showed otherwise complete organ specificity except for cross-reactions with corpus luteum. Voisin et al. 4o• 41 and Freund and colleagues4 • II reported that homologous testicular tissue in adjuvant could induce aspennatogenesis in the guinea pig. The latter workers also found that testis or spenn from the bull, rabbit, hamster, or sheep failed to elicit testicular damage in the guinea pig. Freund and co-workers recorded experiments in which aspennatogenesis, anaphylaxiS, and cutaneous sensitization were induced in the guinea pig by homologous testicular extract. II We have also reported a species-specificity among the spenn of several animal fonns and a speCies-selectivity among the spenn of other fonns, using the isolated ileum or uterus of the sensitized guinea pig and aspennatogenesis as test systems. lO
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At this point it is well to call to mind our reason for setting forth the information regarding the antigenic specmcity of sperm. What is the signmcance to be extracted from the above studies? Elicitation of antibody production (in a competent biological system) is a function of antigenic stimulation. The reaction between antigen and its corresponding antibody is so specific as to constitute one of the most discriminating analytical tools available to biologists. Indeed, antibodies can be employed, for example, to distinguish between the a and f3 glucosides of glucose1 and the 3 stereoisomers (d-, 1- and meso-) of tartaric acid. 30 There are many and noteworthy instances of the remarkable ability of antibodies to detect quite minor differences between antigens. But the points we wish to establish are simply that antigen-antibody interactions can be exquisitely and faithfully specmc; that cross-reactions denote a degree of antigenic similarity; and, that nonreactivity implies antigenic dissimilarity. Now, an antigen is nothing more than a chemical moiety capable of eliciting antibody production to that moiety. Applying this background to sperm, the clear and simple conclusion is that the degree of reactivity and the lack of reactivity of different spermatozoa are referrable to the degree of similarity and dissimilarity, respectively, of the chemical moieties of the sperm cells that are responsible for the antibody produced. This reasoning has directed our experimental investigations, for it was clear that a major advance in an understanding of immunologically-based clinical cases of infertility in human males and females awaits a firm knowledge of the chemical identity of the antigens involved. Equally, it was recognized that any concerted attempt to control fertility (as in population problems) by immune methods would be suspended until the proposed antigenic materials could be identified. MATERIALS AND METHODS
Sensitizing Procedures While many procedures and a great variety of laboratory animals have been employed, for the purpose of this discussion a brief description of a few approaches will suffice. Induction of Aspermatogenesis. This technic has been described in detail elsewhere. 4 , 5,11,12,17 Briefly, the testis homogenate (in 0.9% NaCI), the epididymal spermatozoal suspension (in saline), or the purified material (in saline) is emulsmed with an equal volume of the Freund-type adjuvant so as to form a stiff water-in-oil emulsion. The emulsion is injected intracutaneously in several sites in the nuchal region of male guinea pigs. At intervals
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from one month following injection, the animals are sacrificed, the blood is taken for subsequent serological examination, the ileum is removed and placed in Locke's solution for later anaphylactic tests, and the testes are removed, weighed, sectioned and stained for histological study. The degree of testicular damage is rated from 0 to 4 depending upon the degree of depletion of spermatogenic tissue. Obtaining Sperm Antisera. Male and female guinea pigs and rabbits are injected intradermally with testicular homogenates, washed epididymal sperm, or purified extracts thereof in adjuvant or in saline. Cardiac blood is drawn at intervals and the sera tested for antibody, using spermatozoa or purified extracts as antigens.
Procedures for Detecting Antisperm Antibodies A battery of tests is employed to detect antibody to sperm and extracted sperm antigens. Some of these are briefly as follows. Sperm-immobilization and agglutination tests are performed in depression slides in which varying dilutions of antibody serum are added to viable epididymal guinea pig sperm samples in the presence of complement. These tests are not consistently reproducible in our hands. Another procedure is the precipitation test in which a specimen of antibody serum is added to one of antigenic material in a capillary tube. This test has the advantages of simplicity and rapidity of operation but suffers from a relative insensitivity and uncertainty when whole sperm cells are employed. Another method is the double-diffusion agar plate procedure in which varying dilutions of antibody sera are placed in wells surrounding a central well containing the antigen. The slides are read at intervals up to 4 days, then fixed and stained for preservation. Passive cutaneous anaphylaxis (PCA) is another of the tests. Here, dilutions of antibody sera are injected intracutaneously in the backs of guinea pigs or rabbits. After 2-6 hours, the animal is challenged intravenously or intracardially with antigen mixed with Evans Blue dye (T-1824) and the reaction is read up to 4 days later for immediate and delayed skin reactions. The most sensitive and reproducible test is the one with which we were able to show originally that ilea and uteri of guinea pigs sensitized with sperm responded by contracting when a challenging dose of sperm is administered in vitro. 9 • 11. 13 Our finding led to further critical work in uterine anaphylaxis 13 • 14. 33 and to the demonstration of the induction of infertility in female guinea pigs by injection of sperm. is In preparation for the ileal ~r uterine test, the animals are sensitized by injection of antigenic material (testis homogenate, spermatozoa, or purified extracts thereof) in adjuvant. About 3 weeks later, the animals are sacrificed, segments of the ileum and/or
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uterus are removed, the segments are suspended in a constant-temperature, oxygenated bath containing Locke's solution, and are challenged with nonspecific and specific antigen. The degree of contracture elicited can be used as a semiquantitative index of antibody or of antigen.
Procedures for Extracting Antigens from Testis and Sperm This section will be limited to brief mention of some of the methods we have used in making extractions from guinea pig testicles and sperm. Implicit in this discussion is the understanding that these and other technics are also being applied to human material. Extraction methods applied to testis, sperm, and ejaculate have been detailed in previous reports. 16 , 19-21,2:1,25 Additional technics involve acid, alkaline, and neutral extraction of homogenized testicles and spermatozoa. The supernates of the homogenates are placed on DEAE- ( diethylaminoethyl)-cellulose columns. Using mixing vessels that permit continuous gradient elution at varying pH, fractions are collected and pooled with respect to their absorbing peaks (using the Beckman DU spectrophotometer) as outlined previously.27 In a sample run conducted in this way, 5 pooled fractions were tested for antigenicity (employing the isolated ileum or uterus of the sensitized guinea pig) and for aspermatogenic potency. Another approach has been to subject intact, washed, epididymal sperm to ultrasound for varying periods up to 2 hours. The supernates and precipitates are tested for antigenicity using the isolated organ technic and aspermatogenesis tests. When any of the fractions collected are found to be antigenic, further extractions are applied, including the use of agents for the extraction of proteins, lipids, and sugars. The materials are then lyophilized and specimens further tested for antigenicity.
Identification Procedures A battery of procedures is applied to the many extracts obtained; for example, anionic and cationic column behavior, electrophoresis (Tselius, paper, and cellulose acetate), paper chromatography, infra-red spectroscopy, spectrophotoHuorimetry, enzymatic digestion, histolOgical staining, and elementary analyses. We cannot hope to detail our experiences with all of these approaches and what follows is a brief resume of a few. DEAE-Cellulose Column Elution of A.S.A. Five milligrams of an active preparation of our A.S.A. (Anti-Spermatogenic Antigen) extracted as noted previously21 was placed on a DEAE-cellulose column. The column is 2.5 em.
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O.D., 13.5 cm. high, and contains 10 gm. With continuous gradient elution, using a mixing vessel containing 500 ml. of 0.1 M phosphate buffer, pH 8.0, and a 250-ml. reservoir of 0.3 M NaH 2 P04 in a manner similar to that already described, 5 fractions representing pooled eluates with absorption characteristics in the 260-280 p. wave length were collected. Each of the fractions was tested for aspermatogenic activity and for immunizing characteristics, using the isolated ileum and uterus as test organs. Column separation of TABLE 1. Enzymatic Studies on A.S.A. (Anti-Spermatogenic Antigen) Using Guinea Pigs
No. animals injected
Avg. pairedWt. of testes (gm.)
Testis damage rating *
Ileal testf
10 uninjected
4.1
0
0
6
1.4
+4
+
2 4 4 2 2
1.2 3.0 1.8 3.2 2.0
+4 +1 +4 +1 +3
+ 0 + 0 +
2 4 4 4
1.3 3.0 4.0 2.5
+4 +1 0 +3
+ 0 0 +
2 4 4 4
1.1 1.4 1.3 1.5
+4 +4 +4 +4
+ + + +
4 4 4
1.6 1.3 1.5
+4 +4 +4
+ + +
4 4 4
1.5 1.2 1.4
+4 +4 +4
)
A.S.A. (mg.)
Amt. of enzyme ( flg.)
1 '
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1
RNase 1 10
lOt 100
lOOt DNase 1 10 100
lOOt Ficin 1 10 100
lOOt Lipase 10 100
lOOt lysozyme + /i-glucuronidase 10 100
+ + lOOt + * Higher values indicate greater damage: +1, depletion of sperm and spermatids; +4, all spermatogenic tissue depleted except spermatogonia. t Isolated ileum of sensitized animal challenged with specific antigen. ± indicates no contraction; 0, no antibody. t Inactivated.
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guinea pig testicular hyaluronidase was also performed. The fractions collected were assayed for hyaluronidase activity and then tested for antigenicity using the aspermatogenic and ileal tests. Enzymatic Digestion Experiments. RNase (Worthington), DNase (Worthington), ficin, lipase, lysozyme, and ,B-glucuronidase (all from Nutritional Biochemicals) in 10-JLg and l00-fLg amounts were incubated with 1-10 mg. of a potent preparation of A.S.A. as noted previously.26 Adult male guinea pigs were injected, as noted in Table 1, with each of the respective preparations, with A.S.A. alone, or with the heat-treated enzyme-A.S.A. in adjuvant. At sacrifice, examination for aspermatogenesis was made and ileal sensitivity tests were conducted. Ion-exchange Column Experiments. An ion-exchange column 3.0 X 65 mm. containing Bio-Rad's AG50WX8 (H+), 40-80 wet mesh, 1.7 mEq./ml. of resin bed, was treated with 14.815 mg. of a potent preparation of A.S.A. The rate of How was 6.5 mm./min. Fractions were collected relative to absorption peak at 274 in the U.V. This absorption peak follows Beer's law. Accordingly, the following were collected: Fraction I, 13.230 mg.; II, 1.350 mg.; III, 0.107 mg.; IV, 0.098 mg.-totaling 14.785 mg., indicating 99.8 per cent recovery of the original s'ample. All samples were tested for antigenicity using the isolated sensitized ileum. Another resin column-12 X 90 mm., Amberlite IR-120 (Rohm and Haas) H+, 16-50 wet mesh, 1.9 MEq./ml. of resin bed-was treated with 40 mg. of A.S.A. Contrary to the findings above, this column returned only about 30 per cent of the sample, according to absorption-concentration plots. Cellulose Acetate Electrophoresis. Ten milligrams of a potent preparation of A.S.A. was dissolved in 2 ml. of 0.9% NaCI solution. One drop (corresponding to about 0.05 ml.) of the antigen solution was placed on each of two cellulose acetate strips (5 X 18 cm.) which had been ruled off into l-cm. segments. The strips were run in the Shandon Universal Electrophoresis apparatus for 2 hours at 140 v under refrigeration. One strip was then stained for protein using thiazine red. The other strip was cut into l-cm. segments which were placed in individual tubes carrying numbers corresponding to the number of the segment, and eluted overnight with 1 ml. of 0.9% NaCI solution. The strips were removed and the eluates were tested for carbohydrate using the Molisch reaction. RESULTS
The impracticability of enumerating all of our results with the many different approaches alluded to above needs no emphasis. We will, therefore,
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direct attention to certain of the experimental findings that open vistas rather than villi of exciting exploration.
Hyaluronidase The finding that guinea pig testicular hyaluranidase is So' different antigenically fram bovine testicular hyaluranidase and staphylacaccal hyaluronidase that nO' cross-reactians between them were seen led to' further efforts with the guinea pig testicular hyaluranidase. The fallawing interesting observation was made. One af aur purified preparatians af guinea pig testicular hyaluranidase (assaying at 500 TR U per milligram pratein N), placed an a DEAE-cellulase calumn, gave a Fractian II eluate with high hyaluranidase activity, while Fractian I eluate was devaid af hyaluronidase activity. After sensitizing guinea pigs with bath fractians, it was faund that the twa fractians were antigenically different, since cross-reactians were absent; that Fraction I (devoid af hyaluranidase activity) induced aspermatagenesis (Fig. 2); and that Fractian II did nat (Fig. 1). It is knawn that hyaluronidase is a family of enzymes rather than a discrete entity. It is passible, therefare, that a portian af the enzyme camplex that may have came through in Fractian I was inactivated. In this case, because aur anly test far the enzyme is a biochemical functianal ane, we cauld not gainsay this passibility; nor will activity rates determined far Fractian II allaw us to' negate the passibility, because activity cauld be a representatian af increased concentratian af the enzyme. Until recavery experiments are made pas sible by procedures based upon ather than functianal tests, this problem will await resolutian. At this time, then, these experiments lead us to' infer that: 1. our antispermatagenic antigen is nat hyaluronidase, but that 2. hyaluronidase is an antigenic canstituent of spermatazaa, and 3. it is passible to separate these two antigenic canstituents of spermatazaa by cellulosecolumn treatment.
+
Experiments on Enzymatic Degradation of A.S.A. In Table 1 are presented the results af treatment af aur A.S.A. with various enzymes. As can be deduced from the table, both ribonuclease and desaxyribonuclease inactivated the antigen to' some degree while other enzymes that digest prateins (ficin), fats (lipase), or carbahydrates (lysozyme and ,B-glucuranidase) did not. This permits the inference that our antispermatogenic antigenic material is closely assaciated with bath nucleic acids. Repetition af this experimentatian, using Fractian I of cellulase-column-separated
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material (see above) led to the same results. At this time, however, we do not know precisely how the nucleic acids are involved in the association with the antigenic material. This is partly due to the variability attendant upon the experiments with DNase and RNase that may be attributable to the different degrees of purity of these enzymes. Moreover, the possibility must be considered that the nucleic acids may not be the direct antigenic materials, but rather form hapten or "schlepper" complexes with another unit to form the complete antigen. Variability could thus be due to availability to the enzymes of the haptenic portion of the complex. The nucleic acids could exist as single stranded forms (rather than the usual doublestranded) protected by the core moiety of the complete antigen.
Ion-exchange Column Experiments When the fractions obtained (as described under Ion-exchange Column Fig. 1. Section of a testis of a guinea pig injected 60 days previously with Fraction II of DEAE-cellulose column elution of A.S.A. in adjuvant. Photograph reveals that the injection of this material had no effect on the recipient animars testes since this is the normal histology. This figure illustrates all 0 reactions. (x150)
Fig. 2. Section of a testis of a guinea pig injected 60 days previously with 1 mg. of Fraction I of DEAE-cellulose column elution of A.S.A. in adjuvant. Spermatogonia and Sertoli cells remain. All other spermatogenic elements are absent. This figure illustrates all +4 reactions. (x150)
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Experiments, above) were tested with the isolated ilea of sensitized animals, it was found that all 4 fractions were active. These fractions are currently under investigation for chemical and immunological identity. At this time, it can be said that Fraction 4 is different from 3 fractions isolated by cellulose column treatment of A.S.A. (Fig. 3). Moreover, female guinea pigs are successfully sensitized with at least Fraction 1 (Fig. 4).
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Fig. 3. Kymograph recording of reactions of a guinea pig ileum sensitized with Fraction 4 antigen obtained from ion-exchange column elution of A.S.A. At 1, 2 and 3, three fractions obtained by cellulose column treatment of guinea pig sperm antigenic material do not cause contracture of the ileum. At 4, the ileum contracts when provoked with the specific antigen. At 5, and 6, further challenging doses of the specific antigen elicit diminishing responses until at 7, the ileum does not contract to the specific antigen, indicating desensitization. At W, in each instance the bath is flushed 5-10 times in attempts to regain base line and to wash out the antigen. At S, the drum is stopped for 5 min. to allow time for a new base line to be established.
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Fig. 4. Kymograph recording of the reactions of the uterus of a guinea pig sensitized with Fraction 1 of ion-exchange column treated A.S.A. After the innate rhythm of contraction-relaxation, specific antigen, at 1, causes a strong contracture which is not relieved by washings (at W) or time (drum is stopped at 3:43 P.M. for 15 min. and again at S for 15 min.). Further additions of specific antigen (2 and 3) are without effect.
Cellulose Acetate Electrophoresis Staining of one strip of cellulose acetate containing A.S.A. indicated the presence of protein. Figure 5 shows the band of protein resolved. The mobility of this protein is characteristically low: it moved about 2-3 cm. from the source in a 2-hour run. Other experiments, done with Tselius electrophoresis, support the observation that the protein has a mobility reminiscent of serum a globulin. Eluates of the duplicate cellulose acetate strip (as noted under Ionexchange Column Experiments, above) when reacted in the Molisch test, Fig. 5. Electrophoretic run of purified antispermatogenic antigen spotted on cellulose acetate at Position 9. A 2-hour run at 140 v, with subsequent staining revealed a protein component had migrated 23 cm. to position 6.5-7.5.
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Fig. 6. Tubes containing eluates of I-cm. segments of a cellulose strip run in duplicate with that shown in Fig 5. Reaction with Molisch test shows carbohydrate component in A.S.A. as noted in fifth tube from left, corresponding to Segment 8 of cellulose strip.
gave clear-cut results: only one tube, representing the eluate from Segment 8 gave a strong positive test for carbohydrate, as shown in Fig. 6. The course of development of full color is worthy of note since this occurred approximately 4 hours after the reaction mixture was made. This result is instructive because the indication is that a simple sugar is not involved. Supporting evidence is readily inferred from the fact that the extraction procedure employs several dialysis runs and simple sugars would not have been retained. The tardiness of chromogenic development may also imply the presence of a complex polysaccharide and, possibly, that the polysaccharide is complexed with a carrier molecule from which it must be dissociated before reaction with the test ingredients can occur. We infer, for the moment, that if a carrier is present, it is probably not the protein detected in the duplicate strip: the protein migrated from Segment 9 to Segments 6.5-7.5 with good resolution and little or no trailing. The polysaccharide was found in high concentration only in Segment 8. Further studies are being made on these 2 fractions and the results will be forthcoming soon. At this time we can say that both the protein and the polysaccharide are antigenic. DISCUSSION
One of the goals of our long-range studies is to isolate and identify chemically and immunologically as many different antigens as exist in or are associated with spermatozoa. At this time we have some evidence for the existence of four antigenic ally different moieties: one is capable of hydrolyzing hyaluronic acid, leading to the conclusion that this antigen is hyaluronidase; another is associated with nucleic acids since its antigenic activity (ability to induce aspermatogenesis) is destroyed by treatment with nucleic acid enzymes; a third is a protein with a very slow mobility in electrophoresis; the fourth possesses the characteristics of a polysaccharide. Without excluding the possibility of other antigens yet to be found in spermatozoa, we will restrict our discussion to those mentioned above.
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Hyaluronidase is a promising antigen for studies of infertility and for conferring sterility. It is normally present on spermatozoa and readily released therefrom. We have demonstrated that hyaluronidase exhibits speciesspecificity.20.23 Currently, we are attempting to ascertain if hyaluronidase also exhibits organ-specificity. Interest in the latter is high because, if a vaccine containing hyaluronidase is to be employed to confer infertility, it would be most instructive to assure that organ cross-reactions would not occur. It is equally important to know of organ-specificity of hyaluronidase in certain immunologically based clinical cases of infertility because this would help to explain the genesis of such infertility. At this writing, we can only report an inability to find hyaluronidase in any parenchymatous organ of the guinea pig. If it should be demonstrated that no other tissue of the body contains hyaluronidase, concern about organ interaction would be considerably diminished if not abolished, and interest in this enzyme in infertility would be heightened. The nucleic acid-antigenic fraction is also intriguing, as the notations made in the respective section indicate. Further discussion of this topic will be postponed until more information can be gathered. The electrophoretically slow-moving protein will also be discussed at a future date. More can be said of the polysaccharide antigen. We have reported in some detail concerning this antigen. 19 • 21. 22 At this time the following remarks may be meaningful. The polysaccharide appears to be unique, as is indicated by our inability to extract any similar material from other organs (liver, kidney, brain) of the guinea pig. This organ specificity once again seems promising in studies of infertility for the reasons mentioned above. We can say that the polysaccharide appears to be quite closely bound to sperm: tests run on fresh, viable, epididymal sperm give very little reaction; freezing and thawing of such cells results in some release; the release of the greatest amount is obtained after extraction21 or, as we have found more recently, by application of ultrasound for 1-2 hours. With the latter technic it may now be feasible to procure considerable amounts of the antigen in relatively short time with small effort and little equipment. Further work along this line is progressing. With respect to the procurement of antigenic material from spermatogenic tissue, it is worth more than a passing mention to note that in collaboration with Dr. Russel T. Jordan of the National JeWish Hospital, we are accomplishing culture of testicular tissue. Some of the cultures have been maintained for more than 2 months (Fig. 7). We hope to determine the feasibility of using the culture fluid and the cells as an antigen bank. While considerable attention is being paid to the antigenic materials
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Fig. 7. Guinea pig testicular cells growing in tissue culture. (X1440)
obtainable from spermatozoa, other closely related problems are not being ignored. For example, the participation of the adjuvant in the sensitization process 17 is still under active investigation24 because of the need for precise definition of the vehicle in which the antigen is to be administered. Also of vital importance is the knowledge of the fate of the injected antigen and the kind and location of the resultant antibody. These two areas are also being explored. It has been found, for example, that it is possible to induce aspermatogenesis with the use of fluorescent-labeled spermatozoa28 and to elicit anaphylactic contracture of the ilea of aspermatogenic guinea pigs with fluorescent-tagged sperm. Future reports will record these results in detail. These, in brief are some of the procedures and results obtained in our progress to the isolation and identification of spermatogenic antigens. It is our hope that the information may find application in problems of human fertility and infertility. SUMMARY
Application of a variety of procedures has resulted in the isolation and tentative identification of at least four antigens from guinea pig spermatozoa. One is a hyaluronidase, another is a nucleic acid-containing moiety, a third is a protein, and a fourth is a polysaccharide. Pursuit of these lines of endeavor is aimed at elucidation of certain immunolOgically based clinical cases of infertility and at control of fertility by immune mechanisms.
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ADDENDUM
Mter preparation of this manuscript, an important result worthy of mention was obtained: Guinea pig testicular cells grown in tissue culture for more than 2 months were harvested and employed in challenging three isolated segments of an ileum of an animal sensitized with homologous testis. The anaphylactic contractures obtained are excellent evidence that the testicular cells from these tissue cultures continued to synthesize antigen. Insofar as we are aware, this is the first such demonstration and our hopes of maintaining testicular banks seem capable of realization.
REFERENCES 1. AVERY, O. T., GoEBEL, W. F., and BABERS, F. H. Chemo-immunological studies on conjugated carbohydrate-proteins. J. Exper Med. 55:769, 1932. 2. DARWIN, C. The Descent of Man and Selection in Relation to Sex. 2d ed. Appleton, N. Y., 1898, pp.189-192. 3. FARNUM, C. G. The biologic test for semen. J.A.M.A. 27:1721, 1901. 4. FREUND, J., LIPTON, M. M., and THOMPSON, G. E. Aspermatogenesis in the guinea pig induced by testicular tissue and adjuvants. J. Exper. Med. 97:711, 1953. 5. FREUND, J., THOMPSON, G. E., and LIPTON, M. M. Aspermatogenesis, anaphylaxis, and cutaneous sensitization induced in the guinea pig by homologous testicular extract. J. Exper. Med. 101 :591, 1955. 6. HEXTOEN, L., and MANLY, L. S. Specific precipitin reaction of semen. J. Infect. Dis. 32: 167, 1923. 7. HENLE, W. The specificity of some mammalian spermatozoa. J. Immunol. 34: 325,1938. 8. HENLE, W., HENLE, G., and CHAMBERS, L. A. Studies on the antigenic structure of some mammalian spermatozoa. J. Exper. Med. 68:335, 1938. 9. KATSH, S. In vitro demonstration of uterine anaphylaxis in guinea pigs sensitized with homologous testis or sperm. Nature 180:1047, 1957. 10. KATSH, S. Demonstration in vitro of anaphylactoid response of the uterus and ileum of guinea pigs injected with testis or sperm. J. Exper. Med. 107:95, 1958. 11. KATSH, S. Host-graft interrelationship and the effects of injections of organ homogenates and of cells upon the testes of experimental animals. Ann. New York Acad. Sc. 73:698, 1958. 12. KATSH, S., and BISHOP, D. W. The effects of homologous testicular and brain and heterologous testicular homogenates combined with adjuvant upon the testes of guinea pigs. J. Embryol. & Exper. Morph. 6:94,1958. 13. KATSH, S. In vitro anaphylactic and non-anaphylactic responses of organs of immunized guinea pigs. J. Pharmacal. & Exp. Therap. 124:86, 1958. 14. KATSH, S., and MARSHALL, J. M. Electrical and mechanical responses of uterine smooth muscle during anaphylaxis in vitro. Am. J. Physiol. 196:39, 1959. 15. KATSH, S. Immunology, fertility and infertility: a historical survey. Am. J. Obst. & Gynec. 77:946, 1959. 16. KATSH, S. Acidfastness of sperm. Anat. Rec. 133:397, 1959. 17. KATSH, S. The contribution of the bacterial components of adjuvant in the induction of aspermatogenesis and in the sensitization of the ilea of guinea pigs. Internat. Arch. Allergy 15:172, 1959.
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18. KATSH, S. Infertility in female guinea pigs induced by injection of homologous sperm. Am. J. Obst. & Gynec. 78:276, 1959. 19. KATSH, S. Localization of antispermatogenic factor in guinea pig testicles. Anat. Rec. 134:590, 1959. 20. KATSH, S. Anaphylactogenicity of hyaluronidase and an interspecies difference in the antigenicity of testicular hyaluronidase. Anat. Rec. 134:590, 1959. 21. KATSH, S. Localization and identification of antispermatogenic factor in guinea pig testicles. Internat. Arch. Allergy 16:241, 1960. 22. KATSH, S. Antigen-antibody reactions in relation to reproduction. J. Dairy Sc. suppl. 43:84, 1960. 23. KATSH, S. The anaphylactogenicity of testicular hyaluronidase and a species difference in testicular hyaluronidase demonstrated by isolated organ anaphylaxis. Internat. Arch. Allergy 17:70, 1960. 24. KATSH, S. Mediation of immunologically-induced aspermatogenesis by a nonacidfast bacterium. Anat. Rec. 138:359, 1960. 25. KATSH, S. Certain aspects of the pharmacology and immunology of human ejaculate relating to problems of fertility and infertility. Internat. J. Pertil. (In press) 26. KATSH, S., GAAL, T., and FLOYD, K. Nucleic acid nature of antispermatogenic antigen from guinea pig testicles. Anat. Rec. 137:370, 1960. 27. KATSH, S., GAAL, T., and FLOYD, K. DEAE-cellulose column separation of antispermatogenic antigen. Anat. Rec. 137:369, 1960. 28. KATSH, S., and WILLSON, J. T. The induction of aspermatogenesis by immunization with fluorescent-tagged sperm. Anat. Rec. 136:317, 1960. 29. LANDSTEINER, K. Zur Kenntnis der Spezifisch auf Blutkarperchen wirkenden Sera. Zentralbl. Bakt. 25:546, 1899. 30. LANDSTEINER, K. The Specificity of Serological Reactions. Thomas, Springfield, nt,1936. 31. LEWIS, J. H. The antigenic relationship of the alcohol-soluble fractions of brain and testicle. J. Immunol. 27:473,1934. 32. LEWIS, J. H. The antigenic relationship of alcohol-soluble substances of corpus luteum to those of testis and brain. Am. J. Path. 17:725, 1941. 33. MARSHALL, J. M., and KATSH, S. Transmembrane potentials in isolated nonpregnant guinea pig uteri. Ped. Proc. 17:105, 1958. 34. METALNIKOFF, S. Etudes sur la spermatoxine. Ann. Inst. Pasteur 14:577, 1900. 35. METCHNIKOFF, E. Recherches sur !'influence de l'organisme sur les toxines: sur la spermatoxine et l'antispermatoxine. Ann. Inst. Pasteur 14:1, 1900. 36. MUDD, S., and MUDD, E. The specificity of mammalian spermatozoa with special reference to electrophoresis as a means of serological differentiation. J. Immunol. 17:39, 1929. 37. PFEIFFER, H. Beitrage zur Lasung des biologisch-forensischen Problems der Unterscheidung von Sperma-Eiweiss gegeniiber den anderen Eiweissartem derselben Spezies durch die Prazipitinmethode. Wien. klin. Wchnschr. 18:637, 1905. 38. POMMERENKE, W. T. Effects of sperm injections into female rabbits. Physiol. Zool. 1:97, 1928. 39. RUMKE, P. Auto-antilichamen tegen spermatozoen als oorzaak van omvruchtbaarheid bij de man. Doctoral dissertation from Central Laboratorium van de Bloedtransfusiedienst, Amsterdam, Netherlands, 1959. 40. VOISIN, G., DELAUNAY, A., and BARBER, M. Sur des lesions testiculaires provoquees chez Ie cobaye par iso-et-autosensibilisation. Ann. Inst. Pasteur 81 :48, 1951. 41. VOISIN, G., and DELAUNAY, A. Sur les lesions testiculaires observees chez des animaux soumis a des injections de substances adjuvantes, seules on melangees avec des extraits de tissue homologues. Ann. Inst. Pasteur 89:307, 1955.
T
will present a brief review of our previous efforts and a report of our current attempts to elucidate the chemistry of spermatozoal antigens. The subject of this paper may have had its beginnings, by inference at least, in the Bible (Sarah able to conceive only after long continence) or in later writings such as Darwin's Descent of Man (the profligacy of women accounting for their infertility), the inference being that exposure to sperm had resulted in sterility. For us, however, the beginning must be dated from 1899 to 1900 when Landsteiner,29 Metchnikoff, and Metalnikoff first reported that injections of spermatozoa or testicular extracts into experimental animals led to antibody production. In the initial illumination of this revelation, much effort was directed toward accomplishing infertility by injections of testicular tissue or sperm; success in those ventures was elusive, however. During the intervening years, it has been amply demonstrated that spermatozoa are antigenic and that the antigenic nature of spermatozoa is remarkably species-selective and organ-selective if not species-specific and organ-specific. The following synopsis of reports on the subject is given not for the value of the findings per se, but rather for their fruitful implications. Farnum injected semen or testicular material of the dog, bull, or man into experimental animals and reported that the resultant antibodies were specific for each kind of material. There was no cross-reaction to indicate similarity of antigenicity: this was an indication of species-specificity. Pfeiffer injected IDS ARTICLE
From the University of Colorado Medical Center, Denver Colo. Presented at the Seventeenth Annual Meeting of the American Society for the ~tudy of Sterility, Apr. 21-23, 1961, Bal Harbour, Fla. It is a pleasure to acknowledge the continuing encouragement and financial support of Drs. Warren O. Nelson and Sheldon J. Segal of the Population Council, Inc. 522
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bull spenn into rabbits and reported that the antiserum reacted only with bovine testis and negligibly, if at all, with extracts of other bovine organs. This was an indication of organ-specificity. Hektoen and Manly injected human seminal fluid and spenn as well as swine, bovine, and equine seminal fluid into rabbits and reported species- and semen-specific precipitins. Pommerenke found cross-reactions between rabbit and rat spenn: the serum of rabbits injected with rabbit spenn was toxic for rat spenn and vice versa. Mudd and Mudd demonstrated that the spenn of man, guinea pig, bull, and ram induced species-specific antibodies in rabbits. However, cross-reactions between bull and ram spenn and their corresponding antisera were noted. These and other experiments led to the conclusion that mammalian spenn possessed both species- and tissue-specificity. The absoluteness of species and tissue specificity was modified by Lewis31 who disclosed that antitesticle sera reacted strongly with brain, but not with liver, lung, heart, spleen, kidney, or ovary; and that testicle and brain have a marked organ- and species-selectivity, but not absolute specificity. Henle7 supported the contention that species-specificity of spennatozoa was of a dominant rather than an absolute nature, for he found cross-reactions between spenn of different species. For example, there was strong antigenic resemblance between bull and sheep spenn, but less reactivity between bull and human spenn. Henle et al.,s using bull spenn, found: 1. heat-labile, head-specific and tail-specific antigens and a heat-stable antigen common to both heads and tails that was species-specific; 2. three different cross-reacting antigens, two of which were in the heads and one in the tails; 3. one head antigen that was not active in the native cell (discovered after rupture of the spenn). Lewis32 demonstrated that not only did antitestis sera react with brain tissue but also that antisera against alcoholic extracts of these two organs had the same antigenic properties. Moreover, both organs showed otherwise complete organ specificity except for cross-reactions with corpus luteum. Voisin et al. 4o• 41 and Freund and colleagues4 • II reported that homologous testicular tissue in adjuvant could induce aspennatogenesis in the guinea pig. The latter workers also found that testis or spenn from the bull, rabbit, hamster, or sheep failed to elicit testicular damage in the guinea pig. Freund and co-workers recorded experiments in which aspennatogenesis, anaphylaxiS, and cutaneous sensitization were induced in the guinea pig by homologous testicular extract. II We have also reported a species-specificity among the spenn of several animal fonns and a speCies-selectivity among the spenn of other fonns, using the isolated ileum or uterus of the sensitized guinea pig and aspennatogenesis as test systems. lO
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At this point it is well to call to mind our reason for setting forth the information regarding the antigenic specmcity of sperm. What is the signmcance to be extracted from the above studies? Elicitation of antibody production (in a competent biological system) is a function of antigenic stimulation. The reaction between antigen and its corresponding antibody is so specific as to constitute one of the most discriminating analytical tools available to biologists. Indeed, antibodies can be employed, for example, to distinguish between the a and f3 glucosides of glucose1 and the 3 stereoisomers (d-, 1- and meso-) of tartaric acid. 30 There are many and noteworthy instances of the remarkable ability of antibodies to detect quite minor differences between antigens. But the points we wish to establish are simply that antigen-antibody interactions can be exquisitely and faithfully specmc; that cross-reactions denote a degree of antigenic similarity; and, that nonreactivity implies antigenic dissimilarity. Now, an antigen is nothing more than a chemical moiety capable of eliciting antibody production to that moiety. Applying this background to sperm, the clear and simple conclusion is that the degree of reactivity and the lack of reactivity of different spermatozoa are referrable to the degree of similarity and dissimilarity, respectively, of the chemical moieties of the sperm cells that are responsible for the antibody produced. This reasoning has directed our experimental investigations, for it was clear that a major advance in an understanding of immunologically-based clinical cases of infertility in human males and females awaits a firm knowledge of the chemical identity of the antigens involved. Equally, it was recognized that any concerted attempt to control fertility (as in population problems) by immune methods would be suspended until the proposed antigenic materials could be identified. MATERIALS AND METHODS
Sensitizing Procedures While many procedures and a great variety of laboratory animals have been employed, for the purpose of this discussion a brief description of a few approaches will suffice. Induction of Aspermatogenesis. This technic has been described in detail elsewhere. 4 , 5,11,12,17 Briefly, the testis homogenate (in 0.9% NaCI), the epididymal spermatozoal suspension (in saline), or the purified material (in saline) is emulsmed with an equal volume of the Freund-type adjuvant so as to form a stiff water-in-oil emulsion. The emulsion is injected intracutaneously in several sites in the nuchal region of male guinea pigs. At intervals
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from one month following injection, the animals are sacrificed, the blood is taken for subsequent serological examination, the ileum is removed and placed in Locke's solution for later anaphylactic tests, and the testes are removed, weighed, sectioned and stained for histological study. The degree of testicular damage is rated from 0 to 4 depending upon the degree of depletion of spermatogenic tissue. Obtaining Sperm Antisera. Male and female guinea pigs and rabbits are injected intradermally with testicular homogenates, washed epididymal sperm, or purified extracts thereof in adjuvant or in saline. Cardiac blood is drawn at intervals and the sera tested for antibody, using spermatozoa or purified extracts as antigens.
Procedures for Detecting Antisperm Antibodies A battery of tests is employed to detect antibody to sperm and extracted sperm antigens. Some of these are briefly as follows. Sperm-immobilization and agglutination tests are performed in depression slides in which varying dilutions of antibody serum are added to viable epididymal guinea pig sperm samples in the presence of complement. These tests are not consistently reproducible in our hands. Another procedure is the precipitation test in which a specimen of antibody serum is added to one of antigenic material in a capillary tube. This test has the advantages of simplicity and rapidity of operation but suffers from a relative insensitivity and uncertainty when whole sperm cells are employed. Another method is the double-diffusion agar plate procedure in which varying dilutions of antibody sera are placed in wells surrounding a central well containing the antigen. The slides are read at intervals up to 4 days, then fixed and stained for preservation. Passive cutaneous anaphylaxis (PCA) is another of the tests. Here, dilutions of antibody sera are injected intracutaneously in the backs of guinea pigs or rabbits. After 2-6 hours, the animal is challenged intravenously or intracardially with antigen mixed with Evans Blue dye (T-1824) and the reaction is read up to 4 days later for immediate and delayed skin reactions. The most sensitive and reproducible test is the one with which we were able to show originally that ilea and uteri of guinea pigs sensitized with sperm responded by contracting when a challenging dose of sperm is administered in vitro. 9 • 11. 13 Our finding led to further critical work in uterine anaphylaxis 13 • 14. 33 and to the demonstration of the induction of infertility in female guinea pigs by injection of sperm. is In preparation for the ileal ~r uterine test, the animals are sensitized by injection of antigenic material (testis homogenate, spermatozoa, or purified extracts thereof) in adjuvant. About 3 weeks later, the animals are sacrificed, segments of the ileum and/or
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uterus are removed, the segments are suspended in a constant-temperature, oxygenated bath containing Locke's solution, and are challenged with nonspecific and specific antigen. The degree of contracture elicited can be used as a semiquantitative index of antibody or of antigen.
Procedures for Extracting Antigens from Testis and Sperm This section will be limited to brief mention of some of the methods we have used in making extractions from guinea pig testicles and sperm. Implicit in this discussion is the understanding that these and other technics are also being applied to human material. Extraction methods applied to testis, sperm, and ejaculate have been detailed in previous reports. 16 , 19-21,2:1,25 Additional technics involve acid, alkaline, and neutral extraction of homogenized testicles and spermatozoa. The supernates of the homogenates are placed on DEAE- ( diethylaminoethyl)-cellulose columns. Using mixing vessels that permit continuous gradient elution at varying pH, fractions are collected and pooled with respect to their absorbing peaks (using the Beckman DU spectrophotometer) as outlined previously.27 In a sample run conducted in this way, 5 pooled fractions were tested for antigenicity (employing the isolated ileum or uterus of the sensitized guinea pig) and for aspermatogenic potency. Another approach has been to subject intact, washed, epididymal sperm to ultrasound for varying periods up to 2 hours. The supernates and precipitates are tested for antigenicity using the isolated organ technic and aspermatogenesis tests. When any of the fractions collected are found to be antigenic, further extractions are applied, including the use of agents for the extraction of proteins, lipids, and sugars. The materials are then lyophilized and specimens further tested for antigenicity.
Identification Procedures A battery of procedures is applied to the many extracts obtained; for example, anionic and cationic column behavior, electrophoresis (Tselius, paper, and cellulose acetate), paper chromatography, infra-red spectroscopy, spectrophotoHuorimetry, enzymatic digestion, histolOgical staining, and elementary analyses. We cannot hope to detail our experiences with all of these approaches and what follows is a brief resume of a few. DEAE-Cellulose Column Elution of A.S.A. Five milligrams of an active preparation of our A.S.A. (Anti-Spermatogenic Antigen) extracted as noted previously21 was placed on a DEAE-cellulose column. The column is 2.5 em.
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O.D., 13.5 cm. high, and contains 10 gm. With continuous gradient elution, using a mixing vessel containing 500 ml. of 0.1 M phosphate buffer, pH 8.0, and a 250-ml. reservoir of 0.3 M NaH 2 P04 in a manner similar to that already described, 5 fractions representing pooled eluates with absorption characteristics in the 260-280 p. wave length were collected. Each of the fractions was tested for aspermatogenic activity and for immunizing characteristics, using the isolated ileum and uterus as test organs. Column separation of TABLE 1. Enzymatic Studies on A.S.A. (Anti-Spermatogenic Antigen) Using Guinea Pigs
No. animals injected
Avg. pairedWt. of testes (gm.)
Testis damage rating *
Ileal testf
10 uninjected
4.1
0
0
6
1.4
+4
+
2 4 4 2 2
1.2 3.0 1.8 3.2 2.0
+4 +1 +4 +1 +3
+ 0 + 0 +
2 4 4 4
1.3 3.0 4.0 2.5
+4 +1 0 +3
+ 0 0 +
2 4 4 4
1.1 1.4 1.3 1.5
+4 +4 +4 +4
+ + + +
4 4 4
1.6 1.3 1.5
+4 +4 +4
+ + +
4 4 4
1.5 1.2 1.4
+4 +4 +4
)
A.S.A. (mg.)
Amt. of enzyme ( flg.)
1 '
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1
RNase 1 10
lOt 100
lOOt DNase 1 10 100
lOOt Ficin 1 10 100
lOOt Lipase 10 100
lOOt lysozyme + /i-glucuronidase 10 100
+ + lOOt + * Higher values indicate greater damage: +1, depletion of sperm and spermatids; +4, all spermatogenic tissue depleted except spermatogonia. t Isolated ileum of sensitized animal challenged with specific antigen. ± indicates no contraction; 0, no antibody. t Inactivated.
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guinea pig testicular hyaluronidase was also performed. The fractions collected were assayed for hyaluronidase activity and then tested for antigenicity using the aspermatogenic and ileal tests. Enzymatic Digestion Experiments. RNase (Worthington), DNase (Worthington), ficin, lipase, lysozyme, and ,B-glucuronidase (all from Nutritional Biochemicals) in 10-JLg and l00-fLg amounts were incubated with 1-10 mg. of a potent preparation of A.S.A. as noted previously.26 Adult male guinea pigs were injected, as noted in Table 1, with each of the respective preparations, with A.S.A. alone, or with the heat-treated enzyme-A.S.A. in adjuvant. At sacrifice, examination for aspermatogenesis was made and ileal sensitivity tests were conducted. Ion-exchange Column Experiments. An ion-exchange column 3.0 X 65 mm. containing Bio-Rad's AG50WX8 (H+), 40-80 wet mesh, 1.7 mEq./ml. of resin bed, was treated with 14.815 mg. of a potent preparation of A.S.A. The rate of How was 6.5 mm./min. Fractions were collected relative to absorption peak at 274 in the U.V. This absorption peak follows Beer's law. Accordingly, the following were collected: Fraction I, 13.230 mg.; II, 1.350 mg.; III, 0.107 mg.; IV, 0.098 mg.-totaling 14.785 mg., indicating 99.8 per cent recovery of the original s'ample. All samples were tested for antigenicity using the isolated sensitized ileum. Another resin column-12 X 90 mm., Amberlite IR-120 (Rohm and Haas) H+, 16-50 wet mesh, 1.9 MEq./ml. of resin bed-was treated with 40 mg. of A.S.A. Contrary to the findings above, this column returned only about 30 per cent of the sample, according to absorption-concentration plots. Cellulose Acetate Electrophoresis. Ten milligrams of a potent preparation of A.S.A. was dissolved in 2 ml. of 0.9% NaCI solution. One drop (corresponding to about 0.05 ml.) of the antigen solution was placed on each of two cellulose acetate strips (5 X 18 cm.) which had been ruled off into l-cm. segments. The strips were run in the Shandon Universal Electrophoresis apparatus for 2 hours at 140 v under refrigeration. One strip was then stained for protein using thiazine red. The other strip was cut into l-cm. segments which were placed in individual tubes carrying numbers corresponding to the number of the segment, and eluted overnight with 1 ml. of 0.9% NaCI solution. The strips were removed and the eluates were tested for carbohydrate using the Molisch reaction. RESULTS
The impracticability of enumerating all of our results with the many different approaches alluded to above needs no emphasis. We will, therefore,
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direct attention to certain of the experimental findings that open vistas rather than villi of exciting exploration.
Hyaluronidase The finding that guinea pig testicular hyaluranidase is So' different antigenically fram bovine testicular hyaluranidase and staphylacaccal hyaluronidase that nO' cross-reactians between them were seen led to' further efforts with the guinea pig testicular hyaluranidase. The fallawing interesting observation was made. One af aur purified preparatians af guinea pig testicular hyaluranidase (assaying at 500 TR U per milligram pratein N), placed an a DEAE-cellulase calumn, gave a Fractian II eluate with high hyaluranidase activity, while Fractian I eluate was devaid af hyaluronidase activity. After sensitizing guinea pigs with bath fractians, it was faund that the twa fractians were antigenically different, since cross-reactians were absent; that Fraction I (devoid af hyaluranidase activity) induced aspermatagenesis (Fig. 2); and that Fractian II did nat (Fig. 1). It is knawn that hyaluronidase is a family of enzymes rather than a discrete entity. It is passible, therefare, that a portian af the enzyme camplex that may have came through in Fractian I was inactivated. In this case, because aur anly test far the enzyme is a biochemical functianal ane, we cauld not gainsay this passibility; nor will activity rates determined far Fractian II allaw us to' negate the passibility, because activity cauld be a representatian af increased concentratian af the enzyme. Until recavery experiments are made pas sible by procedures based upon ather than functianal tests, this problem will await resolutian. At this time, then, these experiments lead us to' infer that: 1. our antispermatagenic antigen is nat hyaluronidase, but that 2. hyaluronidase is an antigenic canstituent of spermatazaa, and 3. it is passible to separate these two antigenic canstituents of spermatazaa by cellulosecolumn treatment.
+
Experiments on Enzymatic Degradation of A.S.A. In Table 1 are presented the results af treatment af aur A.S.A. with various enzymes. As can be deduced from the table, both ribonuclease and desaxyribonuclease inactivated the antigen to' some degree while other enzymes that digest prateins (ficin), fats (lipase), or carbahydrates (lysozyme and ,B-glucuranidase) did not. This permits the inference that our antispermatogenic antigenic material is closely assaciated with bath nucleic acids. Repetition af this experimentatian, using Fractian I of cellulase-column-separated
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material (see above) led to the same results. At this time, however, we do not know precisely how the nucleic acids are involved in the association with the antigenic material. This is partly due to the variability attendant upon the experiments with DNase and RNase that may be attributable to the different degrees of purity of these enzymes. Moreover, the possibility must be considered that the nucleic acids may not be the direct antigenic materials, but rather form hapten or "schlepper" complexes with another unit to form the complete antigen. Variability could thus be due to availability to the enzymes of the haptenic portion of the complex. The nucleic acids could exist as single stranded forms (rather than the usual doublestranded) protected by the core moiety of the complete antigen.
Ion-exchange Column Experiments When the fractions obtained (as described under Ion-exchange Column Fig. 1. Section of a testis of a guinea pig injected 60 days previously with Fraction II of DEAE-cellulose column elution of A.S.A. in adjuvant. Photograph reveals that the injection of this material had no effect on the recipient animars testes since this is the normal histology. This figure illustrates all 0 reactions. (x150)
Fig. 2. Section of a testis of a guinea pig injected 60 days previously with 1 mg. of Fraction I of DEAE-cellulose column elution of A.S.A. in adjuvant. Spermatogonia and Sertoli cells remain. All other spermatogenic elements are absent. This figure illustrates all +4 reactions. (x150)
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Experiments, above) were tested with the isolated ilea of sensitized animals, it was found that all 4 fractions were active. These fractions are currently under investigation for chemical and immunological identity. At this time, it can be said that Fraction 4 is different from 3 fractions isolated by cellulose column treatment of A.S.A. (Fig. 3). Moreover, female guinea pigs are successfully sensitized with at least Fraction 1 (Fig. 4).
r ,
l
r
~
l
r
Fig. 3. Kymograph recording of reactions of a guinea pig ileum sensitized with Fraction 4 antigen obtained from ion-exchange column elution of A.S.A. At 1, 2 and 3, three fractions obtained by cellulose column treatment of guinea pig sperm antigenic material do not cause contracture of the ileum. At 4, the ileum contracts when provoked with the specific antigen. At 5, and 6, further challenging doses of the specific antigen elicit diminishing responses until at 7, the ileum does not contract to the specific antigen, indicating desensitization. At W, in each instance the bath is flushed 5-10 times in attempts to regain base line and to wash out the antigen. At S, the drum is stopped for 5 min. to allow time for a new base line to be established.
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Fig. 4. Kymograph recording of the reactions of the uterus of a guinea pig sensitized with Fraction 1 of ion-exchange column treated A.S.A. After the innate rhythm of contraction-relaxation, specific antigen, at 1, causes a strong contracture which is not relieved by washings (at W) or time (drum is stopped at 3:43 P.M. for 15 min. and again at S for 15 min.). Further additions of specific antigen (2 and 3) are without effect.
Cellulose Acetate Electrophoresis Staining of one strip of cellulose acetate containing A.S.A. indicated the presence of protein. Figure 5 shows the band of protein resolved. The mobility of this protein is characteristically low: it moved about 2-3 cm. from the source in a 2-hour run. Other experiments, done with Tselius electrophoresis, support the observation that the protein has a mobility reminiscent of serum a globulin. Eluates of the duplicate cellulose acetate strip (as noted under Ionexchange Column Experiments, above) when reacted in the Molisch test, Fig. 5. Electrophoretic run of purified antispermatogenic antigen spotted on cellulose acetate at Position 9. A 2-hour run at 140 v, with subsequent staining revealed a protein component had migrated 23 cm. to position 6.5-7.5.
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Fig. 6. Tubes containing eluates of I-cm. segments of a cellulose strip run in duplicate with that shown in Fig 5. Reaction with Molisch test shows carbohydrate component in A.S.A. as noted in fifth tube from left, corresponding to Segment 8 of cellulose strip.
gave clear-cut results: only one tube, representing the eluate from Segment 8 gave a strong positive test for carbohydrate, as shown in Fig. 6. The course of development of full color is worthy of note since this occurred approximately 4 hours after the reaction mixture was made. This result is instructive because the indication is that a simple sugar is not involved. Supporting evidence is readily inferred from the fact that the extraction procedure employs several dialysis runs and simple sugars would not have been retained. The tardiness of chromogenic development may also imply the presence of a complex polysaccharide and, possibly, that the polysaccharide is complexed with a carrier molecule from which it must be dissociated before reaction with the test ingredients can occur. We infer, for the moment, that if a carrier is present, it is probably not the protein detected in the duplicate strip: the protein migrated from Segment 9 to Segments 6.5-7.5 with good resolution and little or no trailing. The polysaccharide was found in high concentration only in Segment 8. Further studies are being made on these 2 fractions and the results will be forthcoming soon. At this time we can say that both the protein and the polysaccharide are antigenic. DISCUSSION
One of the goals of our long-range studies is to isolate and identify chemically and immunologically as many different antigens as exist in or are associated with spermatozoa. At this time we have some evidence for the existence of four antigenic ally different moieties: one is capable of hydrolyzing hyaluronic acid, leading to the conclusion that this antigen is hyaluronidase; another is associated with nucleic acids since its antigenic activity (ability to induce aspermatogenesis) is destroyed by treatment with nucleic acid enzymes; a third is a protein with a very slow mobility in electrophoresis; the fourth possesses the characteristics of a polysaccharide. Without excluding the possibility of other antigens yet to be found in spermatozoa, we will restrict our discussion to those mentioned above.
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Hyaluronidase is a promising antigen for studies of infertility and for conferring sterility. It is normally present on spermatozoa and readily released therefrom. We have demonstrated that hyaluronidase exhibits speciesspecificity.20.23 Currently, we are attempting to ascertain if hyaluronidase also exhibits organ-specificity. Interest in the latter is high because, if a vaccine containing hyaluronidase is to be employed to confer infertility, it would be most instructive to assure that organ cross-reactions would not occur. It is equally important to know of organ-specificity of hyaluronidase in certain immunologically based clinical cases of infertility because this would help to explain the genesis of such infertility. At this writing, we can only report an inability to find hyaluronidase in any parenchymatous organ of the guinea pig. If it should be demonstrated that no other tissue of the body contains hyaluronidase, concern about organ interaction would be considerably diminished if not abolished, and interest in this enzyme in infertility would be heightened. The nucleic acid-antigenic fraction is also intriguing, as the notations made in the respective section indicate. Further discussion of this topic will be postponed until more information can be gathered. The electrophoretically slow-moving protein will also be discussed at a future date. More can be said of the polysaccharide antigen. We have reported in some detail concerning this antigen. 19 • 21. 22 At this time the following remarks may be meaningful. The polysaccharide appears to be unique, as is indicated by our inability to extract any similar material from other organs (liver, kidney, brain) of the guinea pig. This organ specificity once again seems promising in studies of infertility for the reasons mentioned above. We can say that the polysaccharide appears to be quite closely bound to sperm: tests run on fresh, viable, epididymal sperm give very little reaction; freezing and thawing of such cells results in some release; the release of the greatest amount is obtained after extraction21 or, as we have found more recently, by application of ultrasound for 1-2 hours. With the latter technic it may now be feasible to procure considerable amounts of the antigen in relatively short time with small effort and little equipment. Further work along this line is progressing. With respect to the procurement of antigenic material from spermatogenic tissue, it is worth more than a passing mention to note that in collaboration with Dr. Russel T. Jordan of the National JeWish Hospital, we are accomplishing culture of testicular tissue. Some of the cultures have been maintained for more than 2 months (Fig. 7). We hope to determine the feasibility of using the culture fluid and the cells as an antigen bank. While considerable attention is being paid to the antigenic materials
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Fig. 7. Guinea pig testicular cells growing in tissue culture. (X1440)
obtainable from spermatozoa, other closely related problems are not being ignored. For example, the participation of the adjuvant in the sensitization process 17 is still under active investigation24 because of the need for precise definition of the vehicle in which the antigen is to be administered. Also of vital importance is the knowledge of the fate of the injected antigen and the kind and location of the resultant antibody. These two areas are also being explored. It has been found, for example, that it is possible to induce aspermatogenesis with the use of fluorescent-labeled spermatozoa28 and to elicit anaphylactic contracture of the ilea of aspermatogenic guinea pigs with fluorescent-tagged sperm. Future reports will record these results in detail. These, in brief are some of the procedures and results obtained in our progress to the isolation and identification of spermatogenic antigens. It is our hope that the information may find application in problems of human fertility and infertility. SUMMARY
Application of a variety of procedures has resulted in the isolation and tentative identification of at least four antigens from guinea pig spermatozoa. One is a hyaluronidase, another is a nucleic acid-containing moiety, a third is a protein, and a fourth is a polysaccharide. Pursuit of these lines of endeavor is aimed at elucidation of certain immunolOgically based clinical cases of infertility and at control of fertility by immune mechanisms.
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ADDENDUM
Mter preparation of this manuscript, an important result worthy of mention was obtained: Guinea pig testicular cells grown in tissue culture for more than 2 months were harvested and employed in challenging three isolated segments of an ileum of an animal sensitized with homologous testis. The anaphylactic contractures obtained are excellent evidence that the testicular cells from these tissue cultures continued to synthesize antigen. Insofar as we are aware, this is the first such demonstration and our hopes of maintaining testicular banks seem capable of realization.
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18. KATSH, S. Infertility in female guinea pigs induced by injection of homologous sperm. Am. J. Obst. & Gynec. 78:276, 1959. 19. KATSH, S. Localization of antispermatogenic factor in guinea pig testicles. Anat. Rec. 134:590, 1959. 20. KATSH, S. Anaphylactogenicity of hyaluronidase and an interspecies difference in the antigenicity of testicular hyaluronidase. Anat. Rec. 134:590, 1959. 21. KATSH, S. Localization and identification of antispermatogenic factor in guinea pig testicles. Internat. Arch. Allergy 16:241, 1960. 22. KATSH, S. Antigen-antibody reactions in relation to reproduction. J. Dairy Sc. suppl. 43:84, 1960. 23. KATSH, S. The anaphylactogenicity of testicular hyaluronidase and a species difference in testicular hyaluronidase demonstrated by isolated organ anaphylaxis. Internat. Arch. Allergy 17:70, 1960. 24. KATSH, S. Mediation of immunologically-induced aspermatogenesis by a nonacidfast bacterium. Anat. Rec. 138:359, 1960. 25. KATSH, S. Certain aspects of the pharmacology and immunology of human ejaculate relating to problems of fertility and infertility. Internat. J. Pertil. (In press) 26. KATSH, S., GAAL, T., and FLOYD, K. Nucleic acid nature of antispermatogenic antigen from guinea pig testicles. Anat. Rec. 137:370, 1960. 27. KATSH, S., GAAL, T., and FLOYD, K. DEAE-cellulose column separation of antispermatogenic antigen. Anat. Rec. 137:369, 1960. 28. KATSH, S., and WILLSON, J. T. The induction of aspermatogenesis by immunization with fluorescent-tagged sperm. Anat. Rec. 136:317, 1960. 29. LANDSTEINER, K. Zur Kenntnis der Spezifisch auf Blutkarperchen wirkenden Sera. Zentralbl. Bakt. 25:546, 1899. 30. LANDSTEINER, K. The Specificity of Serological Reactions. Thomas, Springfield, nt,1936. 31. LEWIS, J. H. The antigenic relationship of the alcohol-soluble fractions of brain and testicle. J. Immunol. 27:473,1934. 32. LEWIS, J. H. The antigenic relationship of alcohol-soluble substances of corpus luteum to those of testis and brain. Am. J. Path. 17:725, 1941. 33. MARSHALL, J. M., and KATSH, S. Transmembrane potentials in isolated nonpregnant guinea pig uteri. Ped. Proc. 17:105, 1958. 34. METALNIKOFF, S. Etudes sur la spermatoxine. Ann. Inst. Pasteur 14:577, 1900. 35. METCHNIKOFF, E. Recherches sur !'influence de l'organisme sur les toxines: sur la spermatoxine et l'antispermatoxine. Ann. Inst. Pasteur 14:1, 1900. 36. MUDD, S., and MUDD, E. The specificity of mammalian spermatozoa with special reference to electrophoresis as a means of serological differentiation. J. Immunol. 17:39, 1929. 37. PFEIFFER, H. Beitrage zur Lasung des biologisch-forensischen Problems der Unterscheidung von Sperma-Eiweiss gegeniiber den anderen Eiweissartem derselben Spezies durch die Prazipitinmethode. Wien. klin. Wchnschr. 18:637, 1905. 38. POMMERENKE, W. T. Effects of sperm injections into female rabbits. Physiol. Zool. 1:97, 1928. 39. RUMKE, P. Auto-antilichamen tegen spermatozoen als oorzaak van omvruchtbaarheid bij de man. Doctoral dissertation from Central Laboratorium van de Bloedtransfusiedienst, Amsterdam, Netherlands, 1959. 40. VOISIN, G., DELAUNAY, A., and BARBER, M. Sur des lesions testiculaires provoquees chez Ie cobaye par iso-et-autosensibilisation. Ann. Inst. Pasteur 81 :48, 1951. 41. VOISIN, G., and DELAUNAY, A. Sur les lesions testiculaires observees chez des animaux soumis a des injections de substances adjuvantes, seules on melangees avec des extraits de tissue homologues. Ann. Inst. Pasteur 89:307, 1955.