ABSTRACT
Numerous efforts have demonstrated antibody production after injection of spermatozoa into animals of the same species. For example, Metalnikoff (1900) and Kennedy (1924) reported anti-guinea-pig sperm ‘toxins’ in guinea-pigs; McCartney (1923) noted anti-rat sperm ‘toxins’ in rats; Pfeiffer (1905), Dittler (1920), and Pommerenke (1928) demonstrated anti-rabbit ‘spermatotoxins’ in rabbits. Antibody production against heterologous sperm has also been disclosed: Mudd & Mudd (1929) injected human, guinea-pig, bull, and ram sperm into rabbits and reported that the resultant antibodies were species specific. The absoluteness of specificity, both organ and species, however, has been qualified by the study of Lewis (1934), who found that brain and testicles possess common antigens, and Henle (1938) has extended Mudd & Mudd’s (1929) observations on cross-reaction between sperm of closely related species. In the above-mentioned studies the methods for determining antisperm activity of antisera included complement-fixation, sperm-immobilization, agglutination, and precipitin tests.
Recently, Freund, Lipton, & Thompson (1953) employed adjuvants combined with homologous and autologous testicular homogenates to induce aspermatogenesis in guinea-pigs. The report of Freund and co-workers is intriguing, since the ability to induce a loss of tissue either by destruction of existing tissue or by prevention of proliferation is tantamount to control of such fundamental processes as growth and differentiation. In the case of induced aspermatogenesis it appears that only the spermatogenic elements are affected. The opportunity to study such a unique response seemed an exciting one, and was accentuated by reports that active immunization with brain (Freund et al., 1947) and with thyroid (Rose & Witebsky, 1956) can cause damage in the respective homologous organs. These conditions (aspermatogenesis, encephalomyelitis, and thyro pathy) are presumed to be effected by an auto-immune response. If this type of reaction should be found to apply to other organ systems, it might reveal an underlying concept of broad and fundamental significance; namely, that each tissue or organ might contain antigens which, under proper conditions, could lead to the morphological and/or functional alteration, and perhaps even to the destruction, of the organs from which the antigens are derived. This current study initiated efforts directed at investigating this concept.
After confirming the results of Freund et al. that homologous testicular preparations in adjuvant do induce aspermatogenesis in guinea-pigs, a broad programme was undertaken to gain information on the following points: (1) Is the antispermatogenic factor restricted to the seminiferous epithelium or does the nonspermatogenic portion of the testis also contain the agent? (2) Can heterologous testicular preparations or homologous brain homogenates be employed in similar fashion to affect spermatogenesis? (3) Is the reaction by which the testes are damaged an immunological process?
This report indicates that an antispermatogenic factor resides in the sperma-togenic tissue alone; that heterologous testicular preparations and homologous brain tissue do induce lesions in the testes; and that the weight of evidence is in favour of an immune basis as the mechanism whereby the testis is damaged.
MATERIALS AND METHODS
Forty-nine adult male guinea-pigs were treated as noted in Table 1. They were fed a complete diet including greens, and fresh water was available at all times. All of these animals gained weight and were apparently free of infection. Eight other guinea-pigs lost weight or developed infection during the experiments, and these animals were, therefore, excluded from this report.
The effects of injections of homologous and heterologous testicular homogenates and other materials on the testes of guinea-pigs

A weighed amount of the material to be injected was homogenized in a Ten Broeck (1931) grinder with an equal volume of a 0·9 per cent, solution of NaCl. Aliquots of the homogenates were emulsified in equal volumes of the Freund complete adjuvant (paraffin oil, Bayol F, and Mycobacterium butyricum", Freund, Lipton, & Thompson, 1953) with the aid of a syringe and a blunt-tipped 15-gauge needle. Between 0·5 and 1·0 ml. of the emulsion was injected intra-cutaneously in several (5-15) sites close to the nuchal region and along the middorsal line. When several courses of injection were made, approximately 2 weeks intervened between successive administrations. The animals were examined frequently, at which times the testes were palpated; severe testicular damage could be noted by reduction in size of the organ. Body-weights were recorded at each injection and at sacrifice. At the time of sacrifice, blood was withdrawn by cardiac puncture, and the sera were stored in a deep-freezer for later use; the testes were weighed, and fragments for biopsy, together with pieces of seminal vesicle and prostatic tissue, were placed in Bouin’s picro-formol or 10 per cent, neutral formol solution. In order to check the presence and motility of sperm, the epididymides were flushed of sperm by forcing 0·9 per cent, saline through the vas deferens using a 22-gauge needle and a 5-ml. syringe and slitting the epididymides when pressure had bulged the tubules. The sperm so obtained were examined microscopically for motility and density and were used immediately for sperm-immobilization and agglutination studies, or were stored in a deepfreezer for subsequent complement-fixation tests.
Histological examinations were made on sections of the organs, cut at 7 μ. and stained with hematoxylin-eosin or Heidenhain’s azan.
Histological interpretation of the testicular biopsies was based upon a rating scale in which ascending numerals indicate increasing order of damage in a manner similar to that utilized by Freund et al. (1953). Testicular injury was rated as follows: 0 represents the normal or control condition; 1 indicates a depletion of mature sperm and the presence of sloughed cells in the lumina of the seminiferous tubules; 2 refers to an increase in cellular material and debris in the lumina and a vacuolation of the cytoplasm in the primary spermatocytes; 3 refers to a general lack of secondary spermatocytes and the exfoliation of primary spermatocytes; 4 is the extreme condition in which there may be a complete or an almost complete absence of spermatogenic elements, including spermatogonia, which imparts to the tubules the appearance of vacuoles lined by Sertoli cells with or without their processes.
For complement-fixation tests the sera were inactivated at 60° C. for 3 minutes and absorbed with sheep cells. A block titration against sperm was carried out using fresh or frozen-thawed guinea-pig sperm according to the method of Osler, Strauss, & Mayer (1952). For sperm-immobilization tests, 01 ml. each of diluted serum, guinea-pig complement, and fresh sperm were combined in glass depression slides and observed during a one-hour period.
OBSERVATIONS
Histologic results
Injections of guinea-pig testis plus adjuvant
Thirty-seven days after the injection of guinea-pig testis (animal No. 58), testicular injury was found corresponding to the type-2 condition (Plate, fig. A). In four guinea-pigs (94-97) permitted to survive 41-47 days after the first of two injections, a variable but increased damage was observed. The extent of damage ranged from impaired testes with masses of exfoliated and necrotic cells in the lumina to a condition with sparse spermatogenic tissue and the absence of material in the lumina. In tubules which still retained some intact cell constituents, sperm-tails without heads were seen in the tubules. The cytoplasm of the primary spermatocytes was highly vacuolated and agranular, and the nuclei were condensed and distorted. In tubules containing few cells, vacuolization of the cytoplasm was so pronounced that cell borders and pycnotic nuclei only were observed. In the most advanced stage of degeneration observed at this time, only Sertoli cells and their processes, with but few isolated spermatogonia and primary spermatocytes, were present. With longer times, 64-169 days, and with a greater number of injections (3-5) the extent of injury was consistently severe (guinea-pigs Nos. 10, 11, 31, 32, 33, 82, 83, and 85). These tubules included a few primary spermatocytes and spermatogonia and a fair population of Sertoli cells whose wavy processes appeared prominently in the lumina. However, even the spermatogonia and Sertoli cells were reduced in number, so that the lumina appeared as vacuoles (Plate, fig. B). In these extreme cases interstitial tissue appeared more prominent than in the normal testis.
Individual variation was encountered in the appearance of tubules within a testis and in testes of animals which received the same treatment. The testes of guinea-pig No. 30, for example, were not so severely damaged as were those of Nos. 31-33, all of which were injected 5 times and carried for 91 days. Moreover, in some instances a more extensive injection schedule was followed by a less severe testicular response. Thus the testes of guinea-pig No. 30 (injected 5 times during 91 days) were not so severely injured as were those of guinea-pig No. 10, which received only three injections during 64 days. Also it was not infrequent that one testicle was more gravely injured than was the other. These differences may reflect a difference in individual sensitivity on the part of the animal as a whole and of the testis. Nevertheless, as appears from the data in Table 1, the total time between injection and sacrifice is important, for guineapigs Nos. 134 and 135 received only one injection each during 114 days, and their testes were maximally affected.
A time factor may also be important in an entirely different respect, namely, in the reversibility of the lesions with subsequent recovery of spermatogenesis. Four guinea-pigs (Nos. 82-85) were injected 5 times with guinea-pig testis during 63 days. On the 70th day animals No. 82 and No. 83 were found to have suffered severe testicular damage. Guinea-pigs No. 85 and No. 84 were permitted to survive for an additional 99 and 133 days respectively. The testes of guinea-pig No. 85 were found to be as profoundly damaged as were those of guinea-pigs Nos. 82 and 83. On the other hand, the degree of damage in guinea-pig No. 84 was considerably less than that in any of the others in this group. Many tubules were sterile, yet several had all stages including secondary spermatocytes, and a few tubules even possessed mature sperm. Whereas recovery may thus be possible after induced aspermatogenesis, the series is too small to justify a conclusion in this regard.
Damaged guinea-pig testis plus adjuvant
Histological examination of the testes of guinea-pigs Nos. 30-33 (Table 1) revealed that the treatment had induced severe depletion of the spermatogenic elements. These damaged testes were then homogenized and emulsified with complete adjuvant in the same manner as the unaffected testes had been prepared in the previous series. Six animals (Nos. 60-65) were then injected on six different occasions with this material over a span of 80-85 days (Table 1). The following observations were made. At sacrifice the testicular weights of all six animals were in the normal or control range, 4 to 6 g. (Table 1), and epididymal sperm were as numerous and as motile as those found in control animals. Extensive histological examination of approximately 200 sections of the testes of all six guinea-pigs disclosed that in no instance had damage beyond the type-1 stage occurred, and this slight impairment was observed in only two animals (Nos. 63 and 65). This effect referred to the occurrence of cells and debris in the lumina of some few tubules, but even in these tubules spermatogenesis had not been arrested. All of these findings (testicular weight, sperm motility and density, and histological data) indicate that the spermatogenic capacity of these animals injected with damaged testis was essentially unaffected by this treatment (Plate, fig. C).
Injections of guinea-pig brain plus adjuvant
Four guinea-pigs (Nos. 20-23) were injected with homologous brain emulsified in complete adjuvant. Four series of inoculations were given during 94 days (Table 1). One of the animals (No. 23) lost weight, and for that reason its data are not included. The testicle weights of the other three guinea-pigs at sacrifice were as follows: 1·6, 2·8, and 4·0 g. for Nos. 20-22 respectively. Histological examination disclosed that the epididymides of guinea-pig No. 20 contained much debris, which included fragmented sperm and spermatogenic tissue. The degree of injury in the testes of this animal was variable but generally was severe; damage ranged from that in tubules, in which only spermatogonia and Sertoli cells could be seen, to a condition in a few tubules where secondary spermatocytes were encountered. The abundance of tubules which contained no stages beyond the primary spermatocyte, and the necrobiotic appearance of these cells, indicated a type-3 reaction (Plate, fig. D). A variable but generally less severe response than that of guineapig No. 20 was found in the testes of animals No. 21 and No. 22. The injury observed in the former case was predominantly of type-2 wherein few or no mature sperm were in the tubules, spermatids were lacking, and the kind as well as the amount of debris in the lumina indicated sloughing of the primary spermatocytes. In the testes of guinea-pig No. 22 little evidence of mature sperm or spermatids could be found, but the depletion of spermatocytes was less severe than that observed in the other animals in this series.
Injections of heterologous testis plus adjuvant
Two guinea-pigs, Nos. 66 and 67, were injected 5 times with rooster testis during 75 and 125 days respectively. The lesions in the spermatogenic tissue were moderate, namely, types-2 and 1 respectively. Again, the injury was not uniform throughout the testes but varied from tubule to tubule. A similar destructive effect was observed in two animals which received five injections of human ejaculate during 75 and 124 days respectively. The injection of rat testis 4 times during 62 days (guinea-pig No. 81), however, produced no damage. Two or three injections of rooster testis, one injection of bull testis, and two injections of human ejaculate into six guineapigs (Nos. 69,73,952,955,973, and 978) led to varying degrees of damage which ranged from a mild effect to severe destruction. In animal No. 73 one testicle was affected maximally and the other had a type-3 response (Plate, figs. E, F).
Two guinea-pigs responded to injections of monkey testis. Animal No. 122 received two injections and after 61 days had a type-3 injury; No. 124, after 97 days, showed a type-1 condition.
Control injections
In Table 1 it can be seen that guinea-pigs which received four or five injections of complete adjuvant alone or in combination with histidine, hyaluronic acid, or galacturonic acid over a period of 67 to 107 days had testes that were indistinguishable from those of untreated animals selected at random from the colony. Freund et al. (1953) have previously shown that certain other organ preparations (liver and kidney) plus adjuvant had no effect on the testes.
Saline homogenates of guinea-pig testis
Two guinea-pigs (Nos. 56 and 57) were injected with saline preparations of guinea-pig testis not combined with adjuvant. The material was given intravenously, intraperitoneally, and subcutaneously. The testes of these animals were found to have suffered a slight injury (type-1); cellular debris and exfoliated cells were present in some of the lumina, but the majority of tubules were unaffected.
Accessory organs of reproduction
The seminal vesicles and prostatic glands of the treated animals were not different from those of the controls. At autopsy these glands were observed to be normal in size and to contain abundant secretion. Histological examination confirmed the fact that these organs of the treated guinea-pigs were indistinguishable from those of untreated animals.
Serological observations
Complement-fixing antibody titers were found when the sera of guinea-pigs injected with homologous testis were incubated with guinea-pig sperm. No complement-fixing antibodies, on the other hand, were detected in the sera from guinea-pigs which received adjuvant alone. It was later found in the studies with rabbits that high titers of antisperm complement-fixing antibodies were evoked by injections of homologous sperm, even though no lesions were observed in the testes of these animals. Consequently the relationship between complement-fixing antibodies and testicular damage must be viewed with caution.
The sera of guinea-pigs sensitized with homologous testis plus adjuvant were found to immobilize 50 per cent, of fresh sperm within one-half hour. This inhibition of motility was frequently accompanied by head-to-head, tail-to-tail, and indiscriminate agglutination. The results obtained with precipitin, agglutination, and ring tests were inconsistent, and therefore these methods had to be abandoned.
DISCUSSION
One of the goals of the current investigation was to determine whether the antispermatogenic factor is a property of the spermatogenic tissue or is found also in the nongerminal portions of the testis as well. The answer is provided by the results obtained on the animals that were injected with testis in which only remnants of germinal tissue remained as a result of previous immunization with homologous testicular extracts. The lack of damage, despite six separate injections during 80 to 85 days, points clearly to the fact that the antispermatogenic factor resides in the gametogenic tissue. Another important fact is demonstrated by these experiments in which damaged testis was injected, namely, that androgenic substance present in the testes cannot be responsible for the lesions. The androgenic activity of the damaged testis was not different from that of undamaged testis as judged by the size and histological condition of the seminal vesicles and prostatic glands. The injection of damaged testis had no significant effect on the testes of the recipient animals.
Another aim of the present work was to determine whether heterologous testicular homogenates and homologous brain tissue could induce injury in the spermatogenic tissue. The results obtained with rooster or monkey testis, human ejaculate, or homologous brain extract indicate that these materials do induce injury. The fact that the lesions so obtained are not so severe or as uniform as are those evoked by homologous testicular tissue may be a reflection of the fact that interspecific cross-reactions are rarely so strong immunologically as are intraspecific ones (Mudd & Mudd, 1929; Henle, 1938). Brain-testicle interactions are also weaker than are those between the same organs. These points have been made clear by Lewis (1934) and Henle (1938), who referred to such interactions as species and organ ‘selectivity’ rather than the more rigid species and organ ‘specificity’. Freund et al. (1953) failed to induce aspermatogenesis in the guineapig after injecting bull sperm or testis from the rabbit, hamster, or sheep. They concluded that the reaction was species specific. It may be that the heterologous testicular materials employed by these investigators were less closely related serologically to the guinea-pig than are those tissues obtained from the monkey and man. The same workers also recorded only mild testicular lesions following injections of homologous brain preparation, and they concluded that the mechanism of damage may be unrelated to that induced by testis, since loss of body-weight was observed in these animals. In the current work, however, testicular lesions induced by brain homogenate were observed in healthy, growing animals. Thus, the probability of brain-testicle interaction is strongly indicated.
With regard to the third objective of these studies, the available evidence supports the view that the testicular damage results from an immune response. Lewis (1934) demonstrated that antisera against the testis of a given species reacted most strongly with the testes of individuals of the same species; crossreactions were weaker between such antisera and the testes of animals from closely related species; and no serological reactions occurred with the testes of distantly related animals. These principles should be applicable to aspermatogenesis if the process by which it is produced is an immune one. The evidence indicates that this is so. In the current work guinea-pig testis was more effective in causing damage in guinea-pig testicles than was monkey and human testicular material, which, however, did give positive reactions. In the work of Freund et al. (1953), aspermatogenesis was unobtainable by testicular material from bull, rabbit, hamster, or sheep. The results with brain injections also support the view that aspermatogenesis is induced by an immune reaction. Henle (1938) provided evidence that brain and testicle have a common antigen. In the present studies brain injections did cause testicular damage. The most likely interpretation is that the injury was due to an immune response. The work of Lewis (1941) also supports this interpretation, for he revealed that brain and testicle reacted with each other but did not react with liver, kidney, heart, lung, or spleen. Consequently if aspermatogenesis is induced by an immune mechanism, only testis or brain material should cause this effect. This appears to be true, for Freund et al. (1953) have shown that liver and kidney were unable to damage guinea-pig testes.
Further and compelling evidence for an immune reaction is presented in the current work. If testicular material damages selectively only the germinal elements of the testes and not the nongerminal portions, then the antigenic material should be found only in the spermatogenic tissue. This report points clearly to this selectivity, since guinea-pigs’ testes damaged by treatment with homologous testis and then injected into other recipients induced no significant injury.
The mild damage induced in the testes of guinea-pigs No. 66 and No. 67 by rooster testis is a curious result, since cross-reactions between two such distantly related forms is unusual. This finding implies some common antigenicity shared by rooster and guinea-pig testes. The problem is being investigated further.
The principal objection against an immune mechanism as the causative factor in aspermatogenesis was raised by Freund et al. (1953). These workers injected a ‘mitochondrial’ fraction obtained from guinea-pig testes and found that, while severe damage was present in the testes of the treated guinea-pigs, there were no significant complement-fixing antibody titers in the sera of these animals. It is entirely likely, however, that complement-fixing antibody titers do not reveal the true nature of the immune response. It is to be recalled that injections of homologous testes into rabbits caused a significant elevation in complementfixing antibody titers, but no damage was seen in the testes of these animals. Other methods of measuring antibody levels in guinea-pigs injected with testis were no more revealing than was the complement-fixation method. The clues to the demonstration of the immune mechanism responsible for destruction of the spermatogenic tissue may be that the response is an allergic one. In such cases, there is an apparent lack of correlation between circulating antibody titer and hypersensitivity (Ehrenkranz & Waksman, 1956). However, induction of aspermatogenesis by means of transfer of cells must be explored as a possibility before a strong position can be taken with regard to the mechanism involved.
SUMMARY
In confirmation of the work of Freund et al. (1953), it was found that intra-cutaneous injection of an emulsion of guinea-pig testis and complete adjuvant resulted in damage to the spermatogenic tissue of recipient guinea-pigs. The degree of damage varied from mild to severe. In the moderately injured testicles sperm were absent, spermatogenesis was disrupted, and exfoliated germinal cells were found in the lumina of the tubules; in severe injury most germinal elements, including spermatocytes and spermatogonia, were severely depleted, and frequently only Sertoli cells and few spermatogonia remained. The degree of damage is a function of time as well as of sensitivity of the individual animal. The suggestion is made, further, that partial recovery of spermatogenesis may occur if the damage is not too severe and sufficient time is allowed to lapse after injection.
The antispermatogenic factor resides in the spermatogenic tissue and not in the nongametogenic portions of the testes.
Androgen present in the testicular homogenates is not responsible for the lesions in the guinea-pig’s testes.
The seminal vesicles and prostatic glands of all recipient guinea-pigs remain normal, which clearly demonstrates that the treatment has no effect on androgenic activity.
Testicular tissue from roosters and monkeys, human semen, and guineapig brain, each incorporated into adjuvant, also induced spermatogenic lesions. These materials were, however, less efficacious in evoking as uniform and severe a lesion as consistently resulted from injections of guinea-pig testis. These findings are explainable on an immune basis, since reports in the literature concerning interspecific cross-reactivity indicate the weaker nature of such interactions when compared with intraspecific reactivity. Reports also indicate that brain and testicle share a common antigen. The precise relation of the immune response to the induction of aspermatogenesis remains to be revealed.
ACKNOWLEDGEMENTS
The Difco Laboratories and Mr. H. W. Schoenlein were most generous in making available the supplies of adjuvant used in these studies.
The technical assistance of Mr. William Duncan in the preparation of the tissues for histological examination is gratefully acknowledged.
This study was supported in part with funds made available by the Population Council, Inc., and The Rockefeller Institute for Medical Research.
REFERENCES
EXPLANATION OF PLATE
Reproduced at × 100
Fig. A. Animal No. 58. Mild damage in seminiferous tubules 37 days after first of two injections of homologous testis. Note luminal debris and vacuolated spermatocytes.
Fig. B. Animal No. 33. Severe damage 91 days after first of five injections of homologous testis. Only spermatogonia and Sertoli cells remain.
Fig. C. Animal No. 60. No damage in testis 80 days after first of six injections of damaged homologous testicles. This gonad is indistinguishable from normal, control testes.
Fig. D. Animal No. 20. Injury induced by homologous brain. Seminiferous tubules lack stages beyond primary spermatocyte. Animal sacrificed 94 days after first of four injections.
Figs. E, F. Animal No. 73. Seminiferous tubules 75 days after first of five injections of heterologous testicular material (human ejaculate, rooster, and bull testes). Damage in left gonad is maximal and in right testis is less severe.
Fig. A. Animal No. 58. Mild damage in seminiferous tubules 37 days after first of two injections of homologous testis. Note luminal debris and vacuolated spermatocytes.
Fig. B. Animal No. 33. Severe damage 91 days after first of five injections of homologous testis. Only spermatogonia and Sertoli cells remain.
Fig. C. Animal No. 60. No damage in testis 80 days after first of six injections of damaged homologous testicles. This gonad is indistinguishable from normal, control testes.
Fig. D. Animal No. 20. Injury induced by homologous brain. Seminiferous tubules lack stages beyond primary spermatocyte. Animal sacrificed 94 days after first of four injections.
Figs. E, F. Animal No. 73. Seminiferous tubules 75 days after first of five injections of heterologous testicular material (human ejaculate, rooster, and bull testes). Damage in left gonad is maximal and in right testis is less severe.