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Immunization of the Guinea Pig with Homologous Testis and Sperm
Immunization of the Guinea Pig with Homologous Testis and Sperm YOSHIHIKO OTANI, M.D., and S.
IT
J. BEHRMAN, M.D., F.R.C.O.G.
60 years since Landsteiner ( 1899) and Methcnikoff ( 1900) fonnd that mammalian spermatozoa were antigenic. In the intervening years many workers have studied testes and spermatozoa from an immunologic standpoint but mainly with the use of heterologous extracts. Some have used homologous material but with varying degrees of success. The present studies are primarily concerned with reevaluating the feasibility of homologous immunization of the guinea pig with testes and sperm, and the study of the nature and type of the antibodies so produced as a necessary step to further investigations. HAS BEEN
METHODS Animals and Immunization Techniques
Virgin guinea pigs weighing about but not more than 500 gm. were used. The animals were maintained on a very adequate diet of greens and rat pellets and allowed water ad libitum. The meticulous care and feeding of the animals was considered an important part of the experiment. These animals were injected with homogenized adult guinea pig testis and epididymis in complete Freund's adjuvant. The testis and epididymis were homogenized at 50,000 rpm for 5 min. and then emulsified with a volume of adjuvant equal to the weight of the testis. These animals were injected with this antigen at seven sites (each site 0,5 ml.) on the back and nuchal region intradermally. This is designated as Type A immunization. Animals were sacrificed 21-24 days following the last injection by a sharp blow on the head. Blood was taken by heart puncture and the serum inactivated at 56°C. for 30 min. and used for the precipitin test. Type B immnnization consisted of 0.5 ml. at each of three sites repeated at 2-week intervals on two occasions with a total of 9 injections over 4 weeks. From the Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, Mich. This project supported by Grant 03618 from the Josiah Macy Jr. Foundation and Grant 04.110 from the Association for the Aid of Crippled Children. Presented at the Tenth Annual Meeting of the Pacific Coast Society for the Study of Sterility, held in Scottsdale, Ariz., October, 1962.
456
VoL. 14,No.4, 1963
TESTIS AND SPERM !MMUNIZA TION
457
Preparation of Antigens
The tissues used were testis, kidney, liver, and sperm from guinea pig, rat, and rabbit. The organs obtained were washed, homogenized with Tyrode's solution, and then diluted with the same solution to ~s concentration. The homogenates were kept in the refrigerator over night and then frozen and thawed once, and centrifuged at 2000 rpm for 20 min. As the challenging antigen 1 ml. of the supernate was used. The sperm was obtained as follows. After sacrificing a male animal as described above, the testis complete with epididymis and vas deferens was cleanly dissected and freed from blood as much as possible. With a 2-ml. syringe and a No. 22-gauge needle 0.9% saline solution was forced through the vas deferens. When the epididymis was distended by this pressure, it was slit and the sperm-containing fluid collected in a small glass container. The sperm were counted and 20 X 106 sperm per milliliter was used as the antigen. Monkey testis was obtained by fresh dissection. Human testis was obtained at autopsy from a 60-year-old male who died of pulmonary tuberculosis, and in whom no histologic evidence of pathology was found. The antigens were prepared the same way as for the guinea pig testes. Tests
Dale-Schultz Test. Both uterine horns were removed from the sacrificed animals and kept in Tyrode's solution at 37°C. The uterine horn was then fixed to a glass rod inferiorly and to a muscle lever superiorly. The latter recorded the movements of the uterus on a smoked paper kymograph moving at l 6 inch per minute. The entire horn was bathed in 100 ml. Tyrode's solution kept at 37°C. with a thermostat, while oxygen was bubbled through constantly. The uterus was allowed to contract spontaneously for at least 20 min. The first challenging antigen was then added and 20 min. allowed for response. After washing, the next antigen was added and this procedure repeated till all the organ extracts had been used. After a final washing and period of rest, 0.5 ml. of oxytocin was added. Where the initial spontaneous contractions of the uterus were large, it was possible to compare duration of these with the contractions induced by the challenging antigens. Where the spontaneous contractions were too small to be satisfactorily read, the amplitude of the contractions induced by the challenging antigens were compared with the amplitude of the contractions produced by the oxytocin. These were compared on a scale of 0-4, where 4 is maximal (Fig. 1). Precipitin Test. Antigens for this test were prepared as follows. Testes were homogenized with equal volumes of normal saline and diluted to }4 concentration with saline. The homogenate was refrigerated over night, frozen
458
OTANI
& BEHRMAN
FERTILITY
& STERILITY
and thawed 2 or 3 times, and centrifuged at 27,000 g for 20 min. at 0°C. The clear supernatant was used. About 0.3 mi. of serum obtained from the animal to be tested was placed in a series of small precipitin tubes and to each was added an equal amount of serially diluted antigen; the result was read in both at 2 and 3 hr. The presence of a "ring" was positive (Fig. 2). This method is similar to that described by Weil and Edwards.
® ®®
Sp
0
30
60
T
L
90
K
120
Adj
Oxy
150
TIME ( minutes )
Fig. 1. (Immunized: Antigen guinea pig testis No. 34) Dale-Schultz Test showing spontaneous contractions followed by alternate washes ( W) . Antigens used were sperm ( Sp) , testis ( T), liver (L), kidney ( K), adjuvant ( Adf), and 0.5 ml. of oxytocin (Oxy).
Agglutination Test. All animals used for this test were immunized by the Type B method. Vaginal fluid was obtained by carefully inserting small cotton plugs into the vagina and leaving them there 3-4 hours. This was repeated with another plug after which both were put into a test tube containing 2 ml. of saline, shaken for 10 min., and centrifuged at 2000 rpm for 10 min. The clear supernatant was inactivated and used for the test. Uterine fluids were obtained by removing the whole uterus. It was kept as a vascular as possible by the technic of removal and soaking up of the blood between two gauze flats. The uterine cavity was washed with 2 ml. of normal saline twice, the combined washings being inactivated and used for the test. The test used was the macroscopic test of Kibrick modified by us in the following fashion: The sperm obtained was diluted with normal saline to a density of 10 X 106 sperm per milliliter. This dilution was mixed with an equal volume of 10% gelatin in normal saline and used for the antigen. Serial dilutions of inactivated serum were made and 0.3 ml. of each dilution was placed in a precipitin tube to which was added an equal amount of antigen
VoL. 14, No. 4, 1963
TESTIS AND SPERM IMMUNIZATION
459
and the mixture incubated at 37°C. Agglutination was evidenced by the appearance of white clumps (Fig. 3). These were sought at each dilution in the immune sera tested before agglutination occurred in the lowest dilution of the inactivated normal serum. Thus it was necessary to use time and dilution of agglutination response as an index for the presence of effective antibodies. Agglutination occurred in normal guinea pig serum in about 10-20 min.
Fig. 2. Precipitin test, illustrating presence of "ring" in first two tubes.
Fig. 3. Sperm agglutination test. White clumps represent agglutinated sperm. The two right-hand tubes are controls.
460
OTANI
&
FE!\TILITY
BEHRMAN
& STERILITY
RESULTS ~xperiment 1
(Dale-Schultz Test Using Guinea Pig, Rat, and Rabbit Organ Extracts)
From Tables 1, 2, and 3, it will be seen that the nonimmunized or control group gave no response to either liver, adjuvant, sperm, or testis of any of the TABLE 1. Results of Dale-Schultz Test Using Guinea Pig Organ Extracts Guinea Pig
Animal
No.
.,
.
.~
c:
" E !
.,
.. ·- .. N
Sperm
Adjuvall' Testis
Liver
Kidney
frecipitin Test
22
4
2
4
3
0
0
I
0
0
0
4
23
3
3
3
3
0
0
3
4
0
0
2
24
0
I
I
I
0
0
2
I
0
0
4
26
I
0
I
I
0
0
I
3
0
0
4
0
0
0
31
I
I
I
I
0
0
I
2
32
0
0
I
3
0
0
0
0
0
0
4
34
I
3
3
3
0
0
0
0
0
0
16 0
16
0
0
0
0
0
0
0
0
0
0
17
0
0
0
0
0
0
3
3
0
0
0
18
0
0
0
I
0
0
3
2
0
0
0
c:_
"o e~
e-::
-o
~0 0
z
19
0
0
0
I
4
2
4
4
0
I
0
29
0
0
0
0
0
0
4
4
0
0
0
30
0
0
0
0
0
0
0
0
0
0
0
TABLE 2. Results of Dale-Schultz Test Using Rat Organ Extracts Rat
Animal
.,..
Precipitin Adjuvant
No.
Sperm
Testis
Liver
Kidney
35
0
0
0
0
0
0
0
0
0
0
2
0
36
0
0
2
I
0
0
0
0
0
0
0
2
37
I
0
2 . 2
0
0
0
I
0
0
4
I
G.P. Rat
N
·c:
38
3
3
3
3
0
0
0
4
0
0
2
0
E
39
0
0
0
I
0
0
0
I
0
0
2
0
40
I
I
I
0
0
0
0
0
0
0
4
I
41
0
0
0
3
0
0
0
I
0
0
0
0
27
0
0
0
0
0
0
0
0
0
0
0
0 0
"E
.,..
33
I
0
0
0
0
0
2
3
0
0
0
e.::
42
0
0
0
0
0
0
0
0
0
0
0
0
t8 c:
46
0
0
0
0
0
0
I
0
0
0
0
0
z
47
0
0
0
0
0
0
I
3
0
0
0
oj
N
·c..!! "0
E c: 0
VoL. 14, No.4, 1963
461
TESTIS AND SPERM IMMUNIZATION
three animals used. It will be noted that the kidney, however, did give responses in both the immunized and nonimmunized group. On this basis it would seem that the response from this organ is not an antigen-antibody response, but rather a smooth-muscle contraction in response to some polypeptide such as rennin or substance P contained in the kidney, as suggested by Pernow. The.immunized animals almost always gave response to sperm or testes only, but in varying intensity. Generally the strength of response was much higher with guinea pig extract and less so with rat or rabbit. Experiment 2 (Dale-Schultz Test Using Human and Monkey Testes)
In 10 tests with human testes and 6 monkey testes only 2 (one animal) tests gave a response; the rest were negative. Using guinea pig testes very strong responses were obtained. There thus appears to be no cross reaction between guinea-pig and human or monkey testes (Table 4). TABLE 3. Results of Dale-Schultz Test Using Rabbit Organ Extracts A"imol No.
..,., N
·;:
" E !
..,.,
-~!! "0
E!: E :i
7U
Rabbit
Precipitin Adjuvant
Sperm
Testis
20
0
I
0
21
0
0
43
I
G.P. Rob.
Liver
Kidney
I
0
0
3
2
0
0
2
0
0
0
0
0
3
3
0
0
0
0
0
I
0
0
0
0
0
0
0
4
I
44
3
I
3
I
0
0
4
4
0
0
4
2
45
0
0
3
3
0
0
3
3
0
0
8
2
50
0
0
3
3
0
0
I
I
0
0
0
0
25
0
0
0
0
0
0
2
3
0
0
0
0
48
0
0
0
0
0
0
0
0
0
0
0
0
49
0
0
0
0
0
0
4
4
0
0
0
0
c:
53
0
0
0
0
0
0
3
3
0
0
0
0
z
54
0
0
0
0
0
0
3
3
0
0
0
0
0
TABLE 4. Results of Dale-Schultz Test Using Human and Monkey Testis Animal Human Mo"key G.P. No. Testis Testis Testis
..,
~
·;:
"E
§
51
0
0
52
0
0
- -
57
0
0
3
I
58
0
0
0
59
0
0
0
-
Precipitin Human Monkey
G.P.
3
2
0
0
3
3
0
0
4
3
3
0
0
2
0
4
3
0
0
4
0
3
4
0
0
2
8
462
OTANI
&
BEHRMAN
FERTILITY
&
STERILITY
Experiment 3 (Desensitization)
Usinguterifrom animals who had the Type B immunization, repeated challenges with guinea pig testis as the antigen were made. In Fig. 4 it will be observed that consecutive challenges resulted in progressively smaller responses until no response was obtained. This varied from 4 to 8 responses. Figure 5 shows another type of response, where no reaction was observed after the second challenge. Experiment 4 (Precipitin Tests)
The precipitin test was almost always positive when the guinea pig testis was used as the antigen (Tables 1, 2, 3), although the titres were rather low. When rat and rabbit testes were used as antigens, positive responses were also obtained but at much lower titres. Since cross reactions were observed, further experimentation using the precipitin test was employed. Animals were immunized by the Type B meth-
®
® Fig. 4. Dale-Schultz Test. Repeated challenges with guinea pig testis as antigen (T 1 , T 2, T 3 , etc.) with consecutively smaller responses.
I
r,
Fig. 5. Dale-Schultz Test. Repeated challenges with guinea pig testis as antigen, with no response after second challenge.
0
30
60 TIME (minutes)
90
120
VoL. 14, No.4, 1963
463
TESTIS AND SPERM IMMUNIZATION
od and antigens of rat, rabbit, and guinea-pig testes, and kidney and liver were prepared as described. From Table 5 it will be observed that liver and kidney of all animals failed to give a positive test, as did human or monkey testes. While rat and rabbit testes gave positive tests up to dilutions of 1:4, only guinea pig testes gave responses in dilutions up to 1:16. Experiment 5 ( Sperm Agglutinin Test in Serum Genital Tract)
The animals were immunized by the Type B method and the vaginal and uterine fluids obtained as described. Six animals were sacrificed to obtain uteri; three were removed with the cervix intact and three were removed TABLE 5. Precipitin Test: Type B Immunized Cross Reaction for Various Organs and Animals animal NO
68
~n n
I
2
G-P
T L K
+ ±
+
-
-
Rat
T L K
+
+
Rabbit
T L K
+
Human Testis Monkey Testis
69
70
-
-
4
8
16
32
64
NaCI
±
-
-
-
-
+
+
--
-
-
-
+
±
-
-
-
-
+
-
-
-
-
-
-
-
-
+
+
-
-
-
±
G-P
T L K
+ -
+
-
-
-
Rat
T L K
+ -
-
+ -
-
-
-
-
Rabbit
T L K
+
±
-
-
-
-
+
+
-
-
-
-
Human Testis
-
Monkey Testis
-
-
G-P
T L K
+
+
+
Rat
T L K
+
+
+
Rabbit
T L K
Human Testis Mankey Testis
-
-
-
-
+
+
-
-
-
-
-
±
-
-
-
-
-
-
-
-
-
±
464
OTANI
& BEHRMAN
FERTILITY
& STERILITY
TABLE 6. Type B Immunized Agglutinin Test in Serum and Genital Tract animal ~n N2 m
.
56
Serum Ut. Wash . Vag. Secretion
60
Ut.
s.
0
L&J
61
N
z
:::> :I!!
::!
v. s.
Ut. V.
s. 63
Ut. V.
64
Ut.
s.
66
Ut.
55
Ut.
0 L&J
N
z:::> I
z 0 z
v. s.
* *
.. *
Ut.
65
Ut.
67
Ut.
4
++ + + ++ + + ++ + + ++ + + ++ + + ++ + +
++ + + ++
-
± -
v. s.
62
:I!!
!
v. s.
..
2
v. s.
v. s.
±
+ ++
+
+ ++ + + ++
-
+ ++ + +
-
± ---
-- ---
±
v.
8
16
32
64 128 256 512 1024 NaCI
++ ++ ++ +
+
- + + - - ++ ++ + + + - - -- - + + ++ + + + + - - - + - - - ++ ++ + + + + - - + + - - ++ ++ + + + - - - + - - - ++ + + + + ± - - + + - - - - -- -- -- - - - - - - - - - - - - - - -- --
-
+
+
±
+
+
- -
+
+
- -
+
+
±
-
+
+
-
-
+
±
- -
(-)
-
·I Without cervix
*
With cervix
supracervically. T;:tble 6 shows the results of the sperm agglutination te~t~ of the serum, uterine washings, and vaginal fluids run in dilutions and em•. · pared with saline and nonimmunized controls. The titres in the serum were very high-up to 1:512. The vaginal and uterine washings also contained agglutinins, but to a much lesser extent. Of interest is that washings taken from the uteri removed complete with the cervix contained a somewhat higher titre of agglutinin than did the washings from the supracervically removed uteri. It is quite obvious too that this test is only interpreted as having qualitative rather than quantitative significance, as the methods for obtaining vaginal and uterine fluids were different and there were varying amounts of fluid obtained from the different 2nimals. The significant finding is that agglutinins could be obtained in the serum, uterine, and vaginal washings of immunized animals, a fact which is important in our future experimentation. The agglutinated sperm in the immune serum when examined micro-
VoL. 14, No. 4, 1963
TESTIS AND SPERM IMMUNIZATION
465
scopically revealed many types of agglutination; e.g., tail-to-tail, tail-to-head, and head-to-head. However, the latter type definitely predominated. DISCUSSION
On general principles it would seem feasible that one of the causes of infertility could be antibody formation by the female against her husband's sperm per se, or even on the basis of agglutination on the basis of ABO (H) blood incompatibility. 5 If this proves to be so, it is then entirely feasible to induce partial or permanent sterility in a female by inducing circulating antibodies to these sperm. The enormous possibilities of this immunologic and biologic method of birth control, therefore, is quite obvious. With the inevitable pitfalls that exist between loose rationalization and actual fact it becomes mandatory to start at a basic level of investigation and progressively develop this theme; such is the purpose of this work and the presentations to follow. One of the most sensitive tests of antigen-antibody reaction is the uterine response devised by Schultz and Dale. It is actually a test of anaphylaxis in vitro. Whereas it is an extremely sensitive test and in some instances specific, more frequently it is sensitive to a group of substances with similar antigenicity. In our experiments with a homologous testis and epididymal sperm the Dale-Schultz test was almost always strongly positive. This has also been shown by Katsch. However, cross reactions, though much weaker, were observed between rat and rabbit testis and sperm, but not between it and human and monkey testis, nor between it and liver or kidney. Thus there is some similarity of antigenicity between testes and sperm of similar species. This fact is also borne out in the precipitin tests. We believe that the uterine contractions induced were due to an antigen-antibody reaction, and more importantly, that the antibody is a tissue-fixing one. This seems to be borne out by the desensitization experiments where repeated challenges by the antigen resulted in successively smaller uterine contractions till, in some cases, no further uterine contraction could be elicited. Katsch also postulated that if the uterus was sufficiently sensitized and continues to be maintained by further copulations, that possibly the fertility of the female could be affected since, in the strongly contracted uterus, implantation of the blastocyst could be inhibited or prevented. This is a most provocative thought but it should be appreciated that Eliasson4 • 5 has extracted a substance, prostaglandin, from the human semen that is capable of stimulating the human nonpregnant uterus at the time of ovulation for the purpose presumably of facilitating sperm migration; yet, fertility is possible. However, Katsch's postulate cannot be ignored.· Kerr7 • 8 induced Trichomanas abortus antibodies in the cow, which became tissue-fixed in the uterus. On introducing the organism into
466
OTANI
& BEHRMAN
FERTILITY
&
STERILITY
the uterine cavity strong myoclonic contractions of the uterus were induced after 2 hr. Thus the role of uterine hypercontractility as well as direct sperm · inhibition must be entertained. The precipitin reaction obtained was in all cases much weaker than the Dale-Schultz response and without any parallel correlation. The only direct correlation was evidence of cross reaction between guinea pig, rat, and rabbit testis and sperm, and no cross reaction between guinea pig and human or monkey testis. There were also no cross reactions between testis and kidney or liver. The presence of sperm agglutinins in serum of animals heterologously immunized has been reported. Recently, Edwards, after intramuscular injection of rabbits with semen and adjuvant, reported that sperm agglutinins (by the hanging-drop method) and sperm-immobilizing antibodies as well as precipitins could be detected in the sperm but not in the genital tract. Strauss claimed that heterologous antibodies in the serum seem to pass into the vaginal fluids, and McCartney reported sperm immobilizing antibodies and agglutinins in vaginal fluid of rats immunized with rat sperm. Kiddy et al. reported that immune sera of heifers immunized with rabbit sperm caused tail-to-tail agglutination of rabbit sperm in contrast to head-to-head type produced by normal sera. Rumke and Hellinga observed all types of agglutination and stated that no correlation could be demonstrated between the type of agglutination, the immobilization effect, the titre of antibody, and the clinical picture. In our sperm agglutinin tests high titres were uniformly obtained in the sera, but low in the uterine washings and vaginal fluid. Therefore, we believe serum agglutinins were induced and that these homologously induced agglutinins are capable of passing into the genital tract. Where the cervix was included in the uterine washings a somewhat higher titre was found but no explanation for this can be given at this time. Microscopic examination showed predominantly head-to-head type of agglutination. We, therefore, feel sufficiently encouraged that is is possible to induce agglutinins and precipitins in the serum to homologous testis and epididymal sperm in the guinea pig, that these are passed into the genital fluids, and that the antibodies tend to fix in the uterus. The next important step is to reproduce the same experiments by transvaginal immunization and then to show that the presence of these antibodies will, or does, in fact impair or reduce fertility by mating experiments. This investigation is now in progress and will be the subject of future communications. SUMMARY
1. Immunization of the guinea pig with homologous testis and epididymal sperm was done by two methods described as Types A and B.
VoL. 14, No. 4, 1963
TESTIS AND SPERM IMMUNIZATION
467
2. Proof of immunization was tested by the Dale-Schultz, precipitin, and agglutinin tests. ~· Cross reactions were demonstrated between guinea pig, rat, and rabbit testes, but not between them and human or monkey testes. 4. It is postulated that homologous sperm agglutinins pass from the serum into the uterine and vaginal fluids and that the agglutination is mainly of the head-to-head type. 5. It is also suggested that the sperm antibodies are tissue-fixed in the uterus. Department of Obstetrics and Gynecology University of Michigan Medical Center Ann Arbor, Mich.
REFERENCES 1. BEHRMAN, S. J., BuETTNER-JANUSCH, J., HEGLER, R., GERSHOWITZ, H., and TEw, W. L. ABO(H) blood incompatibility as a cause for infertility. Am. f. Obst. & Gynec. 79(5) :847, 1960. 2. DALE, H. H. The anaphylactic reaction of plain muscle in the guinea pig. Jour. Pharmacal. & Exper. Therapy 4:167, 1913. 3. EDWARDS, R. G. Antigenicity of rabbit semen, bull semen and egg yolk after intravaginal or intramuscular injection into female rabbits. /. Repro. & Fertil. 1:385, 1960. 4. ELIASSON, R. Studies on prostaglandin. Acta. physiol. scandinav. 46:158, 1959. 5. ELIASSON, R., and PossE, N. Effect of prostaglandin on the non-pregnant human uterus in vivo. Acta. Obst. et Gynec. scandinav. 89:112, 1960. 6. KATSCH, S. Demonstration in vitro of anaphylactoid response of the uterus and ileum of guinea pigs injected with testis or sperm. f. Exper. Med. 107:95, 1958. 7. KERR, W. R., and RoBERTSON, M. Active and passive sensitization of the uterus of the cow in vivo against trichomonas foetus antigen and the evidence for the local production of antibody in that site. J. Hyg. 51:405, 1953. 8. KERR, W. R. Immobilization and agglutination of trichomonas fetus. Brit. Vet. J. 111: 169, 1955. 9. KrBRICK, S., and BELDING, D. L. A modified macroscopic agglutination test. Fertil. & Steril. 8:419, 1952. 10. KIDDY, C ..A., STONE, W. H., and CASIDA, L. E. Immunologic studies on fertility and sterility. II. Effects of treatment of semen with antibodies on fertility in rabbits. f. Immunol. 82:125, 1959. 11. McCARTNEY, J. Mechanism of sterilization of female rat from injections of spermatozoa. Am.]. Physiol. 66:404, 1923. 12. PERNOW, B. Substance P distribution in the digestive tract. Acta physiol. scandinav. 24:97, 1951. 13. RuMKE, P. H., and HELLINGA, G. Auto-antibodies against spermatozoa in sterile men. Am. ]. Clin. Path. 82:357, 1959. 14. ScHULTZ, W. H. The reaction of smooth muscle of the guinea pig sensitized with horse serum. f. Pharmacol. & Exp. Therapy 1:549, 1910. 15. STRAUSS, E. K. Occurrence of antibody in human vaginal mucus. Proc. Soc. Exper. Biol. & Med. 106:617, 1961. 16. WEIL, A. J. Antigen of the adnexal glands of the male genital tract. Fertil. and Steril. 12:538, 1961.
IT
J. BEHRMAN, M.D., F.R.C.O.G.
60 years since Landsteiner ( 1899) and Methcnikoff ( 1900) fonnd that mammalian spermatozoa were antigenic. In the intervening years many workers have studied testes and spermatozoa from an immunologic standpoint but mainly with the use of heterologous extracts. Some have used homologous material but with varying degrees of success. The present studies are primarily concerned with reevaluating the feasibility of homologous immunization of the guinea pig with testes and sperm, and the study of the nature and type of the antibodies so produced as a necessary step to further investigations. HAS BEEN
METHODS Animals and Immunization Techniques
Virgin guinea pigs weighing about but not more than 500 gm. were used. The animals were maintained on a very adequate diet of greens and rat pellets and allowed water ad libitum. The meticulous care and feeding of the animals was considered an important part of the experiment. These animals were injected with homogenized adult guinea pig testis and epididymis in complete Freund's adjuvant. The testis and epididymis were homogenized at 50,000 rpm for 5 min. and then emulsified with a volume of adjuvant equal to the weight of the testis. These animals were injected with this antigen at seven sites (each site 0,5 ml.) on the back and nuchal region intradermally. This is designated as Type A immunization. Animals were sacrificed 21-24 days following the last injection by a sharp blow on the head. Blood was taken by heart puncture and the serum inactivated at 56°C. for 30 min. and used for the precipitin test. Type B immnnization consisted of 0.5 ml. at each of three sites repeated at 2-week intervals on two occasions with a total of 9 injections over 4 weeks. From the Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, Mich. This project supported by Grant 03618 from the Josiah Macy Jr. Foundation and Grant 04.110 from the Association for the Aid of Crippled Children. Presented at the Tenth Annual Meeting of the Pacific Coast Society for the Study of Sterility, held in Scottsdale, Ariz., October, 1962.
456
VoL. 14,No.4, 1963
TESTIS AND SPERM !MMUNIZA TION
457
Preparation of Antigens
The tissues used were testis, kidney, liver, and sperm from guinea pig, rat, and rabbit. The organs obtained were washed, homogenized with Tyrode's solution, and then diluted with the same solution to ~s concentration. The homogenates were kept in the refrigerator over night and then frozen and thawed once, and centrifuged at 2000 rpm for 20 min. As the challenging antigen 1 ml. of the supernate was used. The sperm was obtained as follows. After sacrificing a male animal as described above, the testis complete with epididymis and vas deferens was cleanly dissected and freed from blood as much as possible. With a 2-ml. syringe and a No. 22-gauge needle 0.9% saline solution was forced through the vas deferens. When the epididymis was distended by this pressure, it was slit and the sperm-containing fluid collected in a small glass container. The sperm were counted and 20 X 106 sperm per milliliter was used as the antigen. Monkey testis was obtained by fresh dissection. Human testis was obtained at autopsy from a 60-year-old male who died of pulmonary tuberculosis, and in whom no histologic evidence of pathology was found. The antigens were prepared the same way as for the guinea pig testes. Tests
Dale-Schultz Test. Both uterine horns were removed from the sacrificed animals and kept in Tyrode's solution at 37°C. The uterine horn was then fixed to a glass rod inferiorly and to a muscle lever superiorly. The latter recorded the movements of the uterus on a smoked paper kymograph moving at l 6 inch per minute. The entire horn was bathed in 100 ml. Tyrode's solution kept at 37°C. with a thermostat, while oxygen was bubbled through constantly. The uterus was allowed to contract spontaneously for at least 20 min. The first challenging antigen was then added and 20 min. allowed for response. After washing, the next antigen was added and this procedure repeated till all the organ extracts had been used. After a final washing and period of rest, 0.5 ml. of oxytocin was added. Where the initial spontaneous contractions of the uterus were large, it was possible to compare duration of these with the contractions induced by the challenging antigens. Where the spontaneous contractions were too small to be satisfactorily read, the amplitude of the contractions induced by the challenging antigens were compared with the amplitude of the contractions produced by the oxytocin. These were compared on a scale of 0-4, where 4 is maximal (Fig. 1). Precipitin Test. Antigens for this test were prepared as follows. Testes were homogenized with equal volumes of normal saline and diluted to }4 concentration with saline. The homogenate was refrigerated over night, frozen
458
OTANI
& BEHRMAN
FERTILITY
& STERILITY
and thawed 2 or 3 times, and centrifuged at 27,000 g for 20 min. at 0°C. The clear supernatant was used. About 0.3 mi. of serum obtained from the animal to be tested was placed in a series of small precipitin tubes and to each was added an equal amount of serially diluted antigen; the result was read in both at 2 and 3 hr. The presence of a "ring" was positive (Fig. 2). This method is similar to that described by Weil and Edwards.
® ®®
Sp
0
30
60
T
L
90
K
120
Adj
Oxy
150
TIME ( minutes )
Fig. 1. (Immunized: Antigen guinea pig testis No. 34) Dale-Schultz Test showing spontaneous contractions followed by alternate washes ( W) . Antigens used were sperm ( Sp) , testis ( T), liver (L), kidney ( K), adjuvant ( Adf), and 0.5 ml. of oxytocin (Oxy).
Agglutination Test. All animals used for this test were immunized by the Type B method. Vaginal fluid was obtained by carefully inserting small cotton plugs into the vagina and leaving them there 3-4 hours. This was repeated with another plug after which both were put into a test tube containing 2 ml. of saline, shaken for 10 min., and centrifuged at 2000 rpm for 10 min. The clear supernatant was inactivated and used for the test. Uterine fluids were obtained by removing the whole uterus. It was kept as a vascular as possible by the technic of removal and soaking up of the blood between two gauze flats. The uterine cavity was washed with 2 ml. of normal saline twice, the combined washings being inactivated and used for the test. The test used was the macroscopic test of Kibrick modified by us in the following fashion: The sperm obtained was diluted with normal saline to a density of 10 X 106 sperm per milliliter. This dilution was mixed with an equal volume of 10% gelatin in normal saline and used for the antigen. Serial dilutions of inactivated serum were made and 0.3 ml. of each dilution was placed in a precipitin tube to which was added an equal amount of antigen
VoL. 14, No. 4, 1963
TESTIS AND SPERM IMMUNIZATION
459
and the mixture incubated at 37°C. Agglutination was evidenced by the appearance of white clumps (Fig. 3). These were sought at each dilution in the immune sera tested before agglutination occurred in the lowest dilution of the inactivated normal serum. Thus it was necessary to use time and dilution of agglutination response as an index for the presence of effective antibodies. Agglutination occurred in normal guinea pig serum in about 10-20 min.
Fig. 2. Precipitin test, illustrating presence of "ring" in first two tubes.
Fig. 3. Sperm agglutination test. White clumps represent agglutinated sperm. The two right-hand tubes are controls.
460
OTANI
&
FE!\TILITY
BEHRMAN
& STERILITY
RESULTS ~xperiment 1
(Dale-Schultz Test Using Guinea Pig, Rat, and Rabbit Organ Extracts)
From Tables 1, 2, and 3, it will be seen that the nonimmunized or control group gave no response to either liver, adjuvant, sperm, or testis of any of the TABLE 1. Results of Dale-Schultz Test Using Guinea Pig Organ Extracts Guinea Pig
Animal
No.
.,
.
.~
c:
" E !
.,
.. ·- .. N
Sperm
Adjuvall' Testis
Liver
Kidney
frecipitin Test
22
4
2
4
3
0
0
I
0
0
0
4
23
3
3
3
3
0
0
3
4
0
0
2
24
0
I
I
I
0
0
2
I
0
0
4
26
I
0
I
I
0
0
I
3
0
0
4
0
0
0
31
I
I
I
I
0
0
I
2
32
0
0
I
3
0
0
0
0
0
0
4
34
I
3
3
3
0
0
0
0
0
0
16 0
16
0
0
0
0
0
0
0
0
0
0
17
0
0
0
0
0
0
3
3
0
0
0
18
0
0
0
I
0
0
3
2
0
0
0
c:_
"o e~
e-::
-o
~0 0
z
19
0
0
0
I
4
2
4
4
0
I
0
29
0
0
0
0
0
0
4
4
0
0
0
30
0
0
0
0
0
0
0
0
0
0
0
TABLE 2. Results of Dale-Schultz Test Using Rat Organ Extracts Rat
Animal
.,..
Precipitin Adjuvant
No.
Sperm
Testis
Liver
Kidney
35
0
0
0
0
0
0
0
0
0
0
2
0
36
0
0
2
I
0
0
0
0
0
0
0
2
37
I
0
2 . 2
0
0
0
I
0
0
4
I
G.P. Rat
N
·c:
38
3
3
3
3
0
0
0
4
0
0
2
0
E
39
0
0
0
I
0
0
0
I
0
0
2
0
40
I
I
I
0
0
0
0
0
0
0
4
I
41
0
0
0
3
0
0
0
I
0
0
0
0
27
0
0
0
0
0
0
0
0
0
0
0
0 0
"E
.,..
33
I
0
0
0
0
0
2
3
0
0
0
e.::
42
0
0
0
0
0
0
0
0
0
0
0
0
t8 c:
46
0
0
0
0
0
0
I
0
0
0
0
0
z
47
0
0
0
0
0
0
I
3
0
0
0
oj
N
·c..!! "0
E c: 0
VoL. 14, No.4, 1963
461
TESTIS AND SPERM IMMUNIZATION
three animals used. It will be noted that the kidney, however, did give responses in both the immunized and nonimmunized group. On this basis it would seem that the response from this organ is not an antigen-antibody response, but rather a smooth-muscle contraction in response to some polypeptide such as rennin or substance P contained in the kidney, as suggested by Pernow. The.immunized animals almost always gave response to sperm or testes only, but in varying intensity. Generally the strength of response was much higher with guinea pig extract and less so with rat or rabbit. Experiment 2 (Dale-Schultz Test Using Human and Monkey Testes)
In 10 tests with human testes and 6 monkey testes only 2 (one animal) tests gave a response; the rest were negative. Using guinea pig testes very strong responses were obtained. There thus appears to be no cross reaction between guinea-pig and human or monkey testes (Table 4). TABLE 3. Results of Dale-Schultz Test Using Rabbit Organ Extracts A"imol No.
..,., N
·;:
" E !
..,.,
-~!! "0
E!: E :i
7U
Rabbit
Precipitin Adjuvant
Sperm
Testis
20
0
I
0
21
0
0
43
I
G.P. Rob.
Liver
Kidney
I
0
0
3
2
0
0
2
0
0
0
0
0
3
3
0
0
0
0
0
I
0
0
0
0
0
0
0
4
I
44
3
I
3
I
0
0
4
4
0
0
4
2
45
0
0
3
3
0
0
3
3
0
0
8
2
50
0
0
3
3
0
0
I
I
0
0
0
0
25
0
0
0
0
0
0
2
3
0
0
0
0
48
0
0
0
0
0
0
0
0
0
0
0
0
49
0
0
0
0
0
0
4
4
0
0
0
0
c:
53
0
0
0
0
0
0
3
3
0
0
0
0
z
54
0
0
0
0
0
0
3
3
0
0
0
0
0
TABLE 4. Results of Dale-Schultz Test Using Human and Monkey Testis Animal Human Mo"key G.P. No. Testis Testis Testis
..,
~
·;:
"E
§
51
0
0
52
0
0
- -
57
0
0
3
I
58
0
0
0
59
0
0
0
-
Precipitin Human Monkey
G.P.
3
2
0
0
3
3
0
0
4
3
3
0
0
2
0
4
3
0
0
4
0
3
4
0
0
2
8
462
OTANI
&
BEHRMAN
FERTILITY
&
STERILITY
Experiment 3 (Desensitization)
Usinguterifrom animals who had the Type B immunization, repeated challenges with guinea pig testis as the antigen were made. In Fig. 4 it will be observed that consecutive challenges resulted in progressively smaller responses until no response was obtained. This varied from 4 to 8 responses. Figure 5 shows another type of response, where no reaction was observed after the second challenge. Experiment 4 (Precipitin Tests)
The precipitin test was almost always positive when the guinea pig testis was used as the antigen (Tables 1, 2, 3), although the titres were rather low. When rat and rabbit testes were used as antigens, positive responses were also obtained but at much lower titres. Since cross reactions were observed, further experimentation using the precipitin test was employed. Animals were immunized by the Type B meth-
®
® Fig. 4. Dale-Schultz Test. Repeated challenges with guinea pig testis as antigen (T 1 , T 2, T 3 , etc.) with consecutively smaller responses.
I
r,
Fig. 5. Dale-Schultz Test. Repeated challenges with guinea pig testis as antigen, with no response after second challenge.
0
30
60 TIME (minutes)
90
120
VoL. 14, No.4, 1963
463
TESTIS AND SPERM IMMUNIZATION
od and antigens of rat, rabbit, and guinea-pig testes, and kidney and liver were prepared as described. From Table 5 it will be observed that liver and kidney of all animals failed to give a positive test, as did human or monkey testes. While rat and rabbit testes gave positive tests up to dilutions of 1:4, only guinea pig testes gave responses in dilutions up to 1:16. Experiment 5 ( Sperm Agglutinin Test in Serum Genital Tract)
The animals were immunized by the Type B method and the vaginal and uterine fluids obtained as described. Six animals were sacrificed to obtain uteri; three were removed with the cervix intact and three were removed TABLE 5. Precipitin Test: Type B Immunized Cross Reaction for Various Organs and Animals animal NO
68
~n n
I
2
G-P
T L K
+ ±
+
-
-
Rat
T L K
+
+
Rabbit
T L K
+
Human Testis Monkey Testis
69
70
-
-
4
8
16
32
64
NaCI
±
-
-
-
-
+
+
--
-
-
-
+
±
-
-
-
-
+
-
-
-
-
-
-
-
-
+
+
-
-
-
±
G-P
T L K
+ -
+
-
-
-
Rat
T L K
+ -
-
+ -
-
-
-
-
Rabbit
T L K
+
±
-
-
-
-
+
+
-
-
-
-
Human Testis
-
Monkey Testis
-
-
G-P
T L K
+
+
+
Rat
T L K
+
+
+
Rabbit
T L K
Human Testis Mankey Testis
-
-
-
-
+
+
-
-
-
-
-
±
-
-
-
-
-
-
-
-
-
±
464
OTANI
& BEHRMAN
FERTILITY
& STERILITY
TABLE 6. Type B Immunized Agglutinin Test in Serum and Genital Tract animal ~n N2 m
.
56
Serum Ut. Wash . Vag. Secretion
60
Ut.
s.
0
L&J
61
N
z
:::> :I!!
::!
v. s.
Ut. V.
s. 63
Ut. V.
64
Ut.
s.
66
Ut.
55
Ut.
0 L&J
N
z:::> I
z 0 z
v. s.
* *
.. *
Ut.
65
Ut.
67
Ut.
4
++ + + ++ + + ++ + + ++ + + ++ + + ++ + +
++ + + ++
-
± -
v. s.
62
:I!!
!
v. s.
..
2
v. s.
v. s.
±
+ ++
+
+ ++ + + ++
-
+ ++ + +
-
± ---
-- ---
±
v.
8
16
32
64 128 256 512 1024 NaCI
++ ++ ++ +
+
- + + - - ++ ++ + + + - - -- - + + ++ + + + + - - - + - - - ++ ++ + + + + - - + + - - ++ ++ + + + - - - + - - - ++ + + + + ± - - + + - - - - -- -- -- - - - - - - - - - - - - - - -- --
-
+
+
±
+
+
- -
+
+
- -
+
+
±
-
+
+
-
-
+
±
- -
(-)
-
·I Without cervix
*
With cervix
supracervically. T;:tble 6 shows the results of the sperm agglutination te~t~ of the serum, uterine washings, and vaginal fluids run in dilutions and em•. · pared with saline and nonimmunized controls. The titres in the serum were very high-up to 1:512. The vaginal and uterine washings also contained agglutinins, but to a much lesser extent. Of interest is that washings taken from the uteri removed complete with the cervix contained a somewhat higher titre of agglutinin than did the washings from the supracervically removed uteri. It is quite obvious too that this test is only interpreted as having qualitative rather than quantitative significance, as the methods for obtaining vaginal and uterine fluids were different and there were varying amounts of fluid obtained from the different 2nimals. The significant finding is that agglutinins could be obtained in the serum, uterine, and vaginal washings of immunized animals, a fact which is important in our future experimentation. The agglutinated sperm in the immune serum when examined micro-
VoL. 14, No. 4, 1963
TESTIS AND SPERM IMMUNIZATION
465
scopically revealed many types of agglutination; e.g., tail-to-tail, tail-to-head, and head-to-head. However, the latter type definitely predominated. DISCUSSION
On general principles it would seem feasible that one of the causes of infertility could be antibody formation by the female against her husband's sperm per se, or even on the basis of agglutination on the basis of ABO (H) blood incompatibility. 5 If this proves to be so, it is then entirely feasible to induce partial or permanent sterility in a female by inducing circulating antibodies to these sperm. The enormous possibilities of this immunologic and biologic method of birth control, therefore, is quite obvious. With the inevitable pitfalls that exist between loose rationalization and actual fact it becomes mandatory to start at a basic level of investigation and progressively develop this theme; such is the purpose of this work and the presentations to follow. One of the most sensitive tests of antigen-antibody reaction is the uterine response devised by Schultz and Dale. It is actually a test of anaphylaxis in vitro. Whereas it is an extremely sensitive test and in some instances specific, more frequently it is sensitive to a group of substances with similar antigenicity. In our experiments with a homologous testis and epididymal sperm the Dale-Schultz test was almost always strongly positive. This has also been shown by Katsch. However, cross reactions, though much weaker, were observed between rat and rabbit testis and sperm, but not between it and human and monkey testis, nor between it and liver or kidney. Thus there is some similarity of antigenicity between testes and sperm of similar species. This fact is also borne out in the precipitin tests. We believe that the uterine contractions induced were due to an antigen-antibody reaction, and more importantly, that the antibody is a tissue-fixing one. This seems to be borne out by the desensitization experiments where repeated challenges by the antigen resulted in successively smaller uterine contractions till, in some cases, no further uterine contraction could be elicited. Katsch also postulated that if the uterus was sufficiently sensitized and continues to be maintained by further copulations, that possibly the fertility of the female could be affected since, in the strongly contracted uterus, implantation of the blastocyst could be inhibited or prevented. This is a most provocative thought but it should be appreciated that Eliasson4 • 5 has extracted a substance, prostaglandin, from the human semen that is capable of stimulating the human nonpregnant uterus at the time of ovulation for the purpose presumably of facilitating sperm migration; yet, fertility is possible. However, Katsch's postulate cannot be ignored.· Kerr7 • 8 induced Trichomanas abortus antibodies in the cow, which became tissue-fixed in the uterus. On introducing the organism into
466
OTANI
& BEHRMAN
FERTILITY
&
STERILITY
the uterine cavity strong myoclonic contractions of the uterus were induced after 2 hr. Thus the role of uterine hypercontractility as well as direct sperm · inhibition must be entertained. The precipitin reaction obtained was in all cases much weaker than the Dale-Schultz response and without any parallel correlation. The only direct correlation was evidence of cross reaction between guinea pig, rat, and rabbit testis and sperm, and no cross reaction between guinea pig and human or monkey testis. There were also no cross reactions between testis and kidney or liver. The presence of sperm agglutinins in serum of animals heterologously immunized has been reported. Recently, Edwards, after intramuscular injection of rabbits with semen and adjuvant, reported that sperm agglutinins (by the hanging-drop method) and sperm-immobilizing antibodies as well as precipitins could be detected in the sperm but not in the genital tract. Strauss claimed that heterologous antibodies in the serum seem to pass into the vaginal fluids, and McCartney reported sperm immobilizing antibodies and agglutinins in vaginal fluid of rats immunized with rat sperm. Kiddy et al. reported that immune sera of heifers immunized with rabbit sperm caused tail-to-tail agglutination of rabbit sperm in contrast to head-to-head type produced by normal sera. Rumke and Hellinga observed all types of agglutination and stated that no correlation could be demonstrated between the type of agglutination, the immobilization effect, the titre of antibody, and the clinical picture. In our sperm agglutinin tests high titres were uniformly obtained in the sera, but low in the uterine washings and vaginal fluid. Therefore, we believe serum agglutinins were induced and that these homologously induced agglutinins are capable of passing into the genital tract. Where the cervix was included in the uterine washings a somewhat higher titre was found but no explanation for this can be given at this time. Microscopic examination showed predominantly head-to-head type of agglutination. We, therefore, feel sufficiently encouraged that is is possible to induce agglutinins and precipitins in the serum to homologous testis and epididymal sperm in the guinea pig, that these are passed into the genital fluids, and that the antibodies tend to fix in the uterus. The next important step is to reproduce the same experiments by transvaginal immunization and then to show that the presence of these antibodies will, or does, in fact impair or reduce fertility by mating experiments. This investigation is now in progress and will be the subject of future communications. SUMMARY
1. Immunization of the guinea pig with homologous testis and epididymal sperm was done by two methods described as Types A and B.
VoL. 14, No. 4, 1963
TESTIS AND SPERM IMMUNIZATION
467
2. Proof of immunization was tested by the Dale-Schultz, precipitin, and agglutinin tests. ~· Cross reactions were demonstrated between guinea pig, rat, and rabbit testes, but not between them and human or monkey testes. 4. It is postulated that homologous sperm agglutinins pass from the serum into the uterine and vaginal fluids and that the agglutination is mainly of the head-to-head type. 5. It is also suggested that the sperm antibodies are tissue-fixed in the uterus. Department of Obstetrics and Gynecology University of Michigan Medical Center Ann Arbor, Mich.
REFERENCES 1. BEHRMAN, S. J., BuETTNER-JANUSCH, J., HEGLER, R., GERSHOWITZ, H., and TEw, W. L. ABO(H) blood incompatibility as a cause for infertility. Am. f. Obst. & Gynec. 79(5) :847, 1960. 2. DALE, H. H. The anaphylactic reaction of plain muscle in the guinea pig. Jour. Pharmacal. & Exper. Therapy 4:167, 1913. 3. EDWARDS, R. G. Antigenicity of rabbit semen, bull semen and egg yolk after intravaginal or intramuscular injection into female rabbits. /. Repro. & Fertil. 1:385, 1960. 4. ELIASSON, R. Studies on prostaglandin. Acta. physiol. scandinav. 46:158, 1959. 5. ELIASSON, R., and PossE, N. Effect of prostaglandin on the non-pregnant human uterus in vivo. Acta. Obst. et Gynec. scandinav. 89:112, 1960. 6. KATSCH, S. Demonstration in vitro of anaphylactoid response of the uterus and ileum of guinea pigs injected with testis or sperm. f. Exper. Med. 107:95, 1958. 7. KERR, W. R., and RoBERTSON, M. Active and passive sensitization of the uterus of the cow in vivo against trichomonas foetus antigen and the evidence for the local production of antibody in that site. J. Hyg. 51:405, 1953. 8. KERR, W. R. Immobilization and agglutination of trichomonas fetus. Brit. Vet. J. 111: 169, 1955. 9. KrBRICK, S., and BELDING, D. L. A modified macroscopic agglutination test. Fertil. & Steril. 8:419, 1952. 10. KIDDY, C ..A., STONE, W. H., and CASIDA, L. E. Immunologic studies on fertility and sterility. II. Effects of treatment of semen with antibodies on fertility in rabbits. f. Immunol. 82:125, 1959. 11. McCARTNEY, J. Mechanism of sterilization of female rat from injections of spermatozoa. Am.]. Physiol. 66:404, 1923. 12. PERNOW, B. Substance P distribution in the digestive tract. Acta physiol. scandinav. 24:97, 1951. 13. RuMKE, P. H., and HELLINGA, G. Auto-antibodies against spermatozoa in sterile men. Am. ]. Clin. Path. 82:357, 1959. 14. ScHULTZ, W. H. The reaction of smooth muscle of the guinea pig sensitized with horse serum. f. Pharmacol. & Exp. Therapy 1:549, 1910. 15. STRAUSS, E. K. Occurrence of antibody in human vaginal mucus. Proc. Soc. Exper. Biol. & Med. 106:617, 1961. 16. WEIL, A. J. Antigen of the adnexal glands of the male genital tract. Fertil. and Steril. 12:538, 1961.