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Effects of castration and testosterone in male mice on Schistosoma mansoni
353 TRANSACTIONSOF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. 51. No. 4. July, 1957.
E F F E C T S OF C A S T R A T I O N A N D T E S T O S T E R O N E IN M A L E M I C E ON
SCHISTOSOMA
MANSONI
BY
EDWARD BERG, PH.D.*
(From the Department of Biology, New York State Collegefor Teachers) T h e importance of the reproductive system in lower animal life in reference to the survival and maintenance of species has long been understood. The animal parasites, including Schistosoma mansoni, develop complex genital structures while the other organs remain primitive. T h e necessity for normal development of genital tissue appears essential for the existence of the parasite. There is much to learn regarding host-parasite relations, especially the identity of the vital connecting link between the two. As more information on metabolism is compiled, the application of this mechanism becomes better understood. The present activity on physiological investigations will eventually provide us with the answers. S. mansoni is one of the helminthic parasites being studied in order to provide essential information which might enlighten us about parasitic existence in or on the host. Moore, Vogel, Standen, Bueding and many others have contributed a great deal about the vital connection between host and parasite. Two considerations in this respect are of interest to me. The first concerns the hormonal relationship between host and parasite, whether sexual or otherwise. The second invoives the similarity of enzymatic systems and metabolic cycles of both host and parasite. Schistosomes are the only dioecious parasites of the medically important trematodes, all others being hermaphroditic. However, under strained physical conditions sexual reversal has occurred. Variations in sex ratios and genital structure in different animal hosts have been reported by BUTTNER (1950) and others. Similar responses occurred in different strains of the molluscan intermediate host (VOGEL, 1941 ; STIREWALT, 1951). The introduction of varied sex ratios of schistosomes (STANDEN, 1953b ; MOORE et al., 1954) causes related changes in sexual development of the parasite. It has been demonstrated by ADDIS (1946) that castration Of the rat host results in the alteration of physiological conditions in the tapeworm, Hymenolepis dirninuta. He also found testosterone to be a compensatory agent to the worm when administered to the castrated host. The explanations for these phenomena are not known at the present time. It is, however, apparent that these studies concern two approaches. One involves the possibility of a hormonal relationship by means of a genital secretion from worm to worm. T h e other relationship signifies a hormonal relationship between host and parasite. * I am indebted to the late Professor Ephraim Shorr, Cornell University Medical College, for his assistance on endoerinological aspects ; Dr. Irwin D. J. Bross, of the same institution, for statistical interpretation of the data, Dr. Eloise B. Cram of the National Institutes of Health for infection of the experimental animals and Dr. Louis Freedman of the U.S. Vitamin Corporation for making available the hormone.
354
EFFECTS ON S. mansoni OF CASTRATIONAND TESTOSTERONE IN MOUSE HOST
I t is w i t h t h e s e c o n s i d e r a t i o n s t h a t e x p e r i m e n t s w e r e d e s i g n e d to d e t e r m i n e w h e t h e r sexual h o r m o n e s f r o m t h e h o s t c o u l d cause c h a n g e s in t h e parasite, S. mansoni. MATERIALS AND METHODS O n e h u n d r e d a n d five m a l e a l b i n o m i c e f r o m C a r w o r t h Swiss W e b s t e r stock w e r e d i s t r i b u t e d , at r a n d o m , into six g r o u p s ( T a b l e I). The animals were maintained in white porcelain pans 12 x 8 x 5 inches. Each pan housed 10 or fewer mice. Adequate portions of Purina Laboratory chow were always available. T h e mice were castrated by surgical procedures after intraperitoneal anesthesia with 0.1 rag. of sodium pentobarbitol (Abbott) per g. body weight. A convalescence of 2 weeks ensued. Hormone administration consisted of one subcutaneous injection of an arbitrary 2.5 rag. dose of testosterone in aqueous suspension (Funk) previous to infection with S. mansoni. Post-infection therapy consisted of subsequent similarly equal bi-weekly injections of 1.25 mg. of testosterone. T h e mice were infected with a Puerto Rican strain of N. mansoni to approximately 176 pooled cercaria for 1 hour. T h e cercaria were collected at their most active shedding period (CRAM and FILES, 1946) from snails of the species Australorbis glabratus. T h e method of infection followed the technique of OLIVER and STIREWALT (1952). Faeces examination entailed collection of pooled faeces pellets per pan according to group. Direct smears and the Mathieson and Stoll concentration methods (CRAm and FAUST, 1951) were employed. Ova were first observed 47 days after infection in Group E, followed by the other groups on the 48th day. Necropsies were performed over a 10-day period, starting on the 57th day after infection. T h e mice were selected, a t random, weighed, asphyxiated by ether and dissected. T h e schistosomes were perfused from the liver after the technique of YOLLES et al. (1947). Those trematodes in the mesenteric blood vessels were carefully teased out. T h e y were sexually differentiated and counted. T h e trematodes were fixed, mounted and stained employing Semicon's carmine method (CABLE, 1950). T h e y were mounted on slides and fixed with canada balsam. TABLE I.
Group .
N u m b e r at start.
Grouping of mike.
Description
N u m b e r at necropsy
A
22
Castrated male mice
20
B
'10
Castrated male mice (sham) plus saline
C
33
Castrated male mice plus testosterone
30
E
20
Uncastrated male mice (control)
19
F
10
Uncastrated male mice (sham) plus saline
9
G
10
Uncastrated male mice plus testosterone
8.
Total
105
8
94
RESULTS Morphological examination revealed no consistent structural differences. The statistical correlations were analysed by the use of the Rank " t " Method (WHITE, 1952) by comparison to the Z2 value at the 0.05 P level with one degree of freedom. Thus any value greater than 3.84 indicated a difference outside the realm of chance. The first experiment tested the effect of castration in male albino mice on 8. mansoni (Table II). The comparison between Group A (castrated) and Group E (uncastratedcontrol) s h o w e d a h i g h l y significant d e c r e a s e in t h e m e a n n u m b e r of m a l e s c h i s t o s o m e s p e r
EDWARD BERG
355
mouse in the castrated group as indicated by an H value of 16.98. Statistical analysis indicated the mean difference between female schistosomes of the two groups to be within the realm of chance, the H value being 0.44. In order to test any mechanical effect of inoculation on uncastrated mice, a comparison was made between Group F (uncastrated-saline) and Group E (uncastrated-control). The results, proved to be within the realm of chance. Analysis for significance gave H values of 1.62 for males and 0.62 for females. The second experiment tested the effect of testosterone in castrated male albino mice on S. mansoni. A comparison between the mean schistosome counts per mouse was made between Group B (castrated-saline) and Group C (castrated-testosterone) (Table II). The results of this comparison are interesting because both sexes of schistosomes decreased in number after testosterone administration (male H value = 4.52 ; female H value = 16. 996). Although the effect of the hormone appeared detrimental to male worms, it was almost lethal to the females, decreasing their number almost threefold. A comparison was made between Group C (castrated-testosterone) and Group G (uncastrated-testosterone) to determine the influencing activity of the hormone as shown in Table II. An H value of 1.89 for the male worms proved statistically insignificant, whereas the females showed a significant 4.22. The third experiment tested the effect of testosterone in uncastrated male mice on S. mansoni (Table II). T h e mean schistosome counts per mouse for Group G (uncastratedtestosterone) and Group F (uncastrated-saline-control) were compared and analysed. Both male and female groups indicated a statistically valid difference with H values of 5.83 and 6.82 respectively. It is clearly evident that the testosterone treated group had fewer male and female schistosomes. TABLE II.
Results indicating the number of S. mansoni in male albino mice after collection and sexual differentiation. Parasite sex and mean per mouse
Group (No. of mice)
No. of males
N o . of Mean
females
Mean
i
Castrated (20)
A
Castrated-saline (8)
B
Castrated-testosterone (30)
C
Uncastrated-control (19)
E
Uncastrated saline control (9)
F
Uncastrated-testosterone (8)
G
Total (94)
6
220.00
I I1
256 12.80
11.00
79 00
92 11.50
9.88 222.00 334.00
i
7.40
I
17.58
I 138.00
118
i
3.93 259
I
13.63 112
15.33 73.00
51 9.13
1066.00
12.44 6.33
888
356
EFFECTS ON
S. mansoni OF
CASTRATION AND TESTOSTERONE IN MOUSE HOST
DIscusSlON If a generalized detrimental action is considered in the first experiment, the more fragile female parasites would be expected to undergo the same consequences as the males. Yet only the male population was significantly affected by the action of castration. This may indicate a relationship between the male trematode and the inability of the male mouse to produce secondary sex characteristics. Whether this phenomenon is direct or indirect warrants further consideration. It is interesting to note that upon necropsy, 57 days after infection, only a scant number of trematodes were found in the mesenteric venules of the castrated animals. The majority of parasites were found in the liver. On the other hand, the uncastrated mice carried approximately one-third of the total parasite burden in the mesenteric venules. It has been reported by STANDEN (1949) that under normal conditions the population of S. mansoni in the liver decreased from 53 per cent to 11 per cent. while those of the mesenteric venules increased from 15 per cent. to 77 per cent. between the 35th and 105th day after infectiom - After 50 days, 18 per cent. of the population were located in the mesenteries. The distribution of the parasites in the control mice in this experiment seems to paral!el Standen's results, taking into account the fact that necropsy was performed later. However the castrated experimental animals appeared to contain considerably fewer than 18 per cent. in this area. Another point that STAND~N (1953a) makes is that the migration of the parasites occurs only under optimum conditions. Because the schistosomes in the castrated animals showed delayed migration to the mesenteries, it may be assumed that a necessary developmental factor was lacking. Therefore, the maturation of the parasite may have been inhibited. Several possible criteria may be investigated as the responsible agent for this phenomenon. Since the male mice with excised testes showed a decrease in male schistosomes, it appears that they were affected during or after castration. This may have occurred either as a direct or indirect phenomenon. There are many conditions, such as electrolyte balance, hormone differences, carbohydrate metabolism and others which may change from surgical procedures or infection. Although these changes are usually temporary, a permanent alteration may consequently occur. Whether effects of shock or stress can be of long duration is also important in this respect. The most obvious change after castration is the cessation of normal sexual development. What direct relationship is involved with S. mansoni is not known. ADDIS(1946), however, was able to indicate in male castrated rats infected with H. diminuta, that the parasites were stunted in growth. With testosterone therapy, the tapeworms regained their normal length when compared with uncastrated controls. The second experiment was an attempt to compare the effects on tapeworms with those on schistosomes. Instead of showing morphological changes, however, the worm population was affected. Thus a highly significant decrease in the mean numbers of female worms from the testosterone group occurred. The males appeared affected, but the activity of testosterone is questioned since they had already responded to castration. The dosage of testosterone, although arbitrary, did appear to sustain the sexual behaviour of the mice which corresponds with the results of BEACH and HOLZ-TUCKER (1949). It is, however, possible that the trematodes themselves did not react in the same manner.
EDWARD BERG
357
No explanation can be offered, since a uniform dosage of hormone was employed. Whether an increased or decreased quantity would cause a different effect cannot be stated. However, it is important to remember that variations in testosterone concentration could change the response in the host itself and in this manner alter the host-parasite relationship. The objective, therefore, was to stimulate the castrated mice therapeutically to a more sexually active state. Thus, the effect of testosterone was not indiscriminately tested. An attempt was made to maintain physiological equilibrium, rather than solely to test large doses of testosterone. In this manner any deviation due to excessive activity of the hormone was believed to be checked. Another consideration is that two treatments were actually invoked on the castratedtestosterone group. In combination entirely different effects may result in comparison with individual treatments. The action of testosterone has been described on various subjects in both castrated and uncastrated animals by LEONARD (1952), BARTLETT(1953), and others. However, the responses due to the hormone therapy depend on the dosage employed, the species of animal used and its general physiological state. In the case of castrated male albino mice infected with schistosomiasis, no data concerning the effect of testosterone on the host or the parasite have been reported, to my knowledge. The activity of the hormone on S. rnansoni from an extrinsic source may differ from that of an intrinsic one in these castrated mice. Therefore, from the situation as it appears behaviourwise, a positive effect is possibly directly on the host, but the parasite may exhibit a different response. The true effect of testosterone in this experiment may have been exerted on the female worms rather than on the males. This is demonstrated by the highly significant decrease on the number of female worms in the castrated-testosterone group, whereas the hormone had little effect on the male schistosomes. It is possible, moreover, that by combination with castration, the males were decreased and therefore the females were indirectly affected. It has been reported by MOORE et al. (1954) and STANDEN (1953b) that the female parasites do not develop normally without the presence of mature male schistosomes. It is also indicated (MooRE et al., 1954) that anomalies resulted due to the activity of testosterone proprionate on female S. mansoni. The sex of the experimental mice was not specified. It might be interesting to note whether these anomalies resulted generally, regardless of sex, or whether specific male or female hosts under the testosterone therapy behaved differently. The third experiment seems to confirm that castration in combination with the hormone was a basic factor in the influence of the testosterone, since equal degrees of significance were indicated in both male and female worms in the comparison. From these comparisons concerning testosterone therapy, the following points are apparent : (1) Testosterone therapy affected the female schistosomes in the castrated mice with a highly significant decrease in the n u m b e r collected. T h e male worms did not behave similarly. (2) T h e male parasites were m u c h more inhibited in development by the castration rather t h a n by h o r m o n a l administration. (3) It is also clear that the greatest difference was between the parasites in the uncastrated group and those in the castrated mice on testosterone therapy. (4) T h e fact that the female worms survived more readily in the uncastrated male mice than those in the castrated group tends to indicate that the action of the testosterone is more effective as a detrimental agent w h e n not accompanied by intrinsic male hormones.
358
EFFECTS ON S .
mansoni
OF CASTRATION AND TESTOSTERONE IN MOUSE HOST
SUMMARY
AND
CONCLUSIONS
A total of 105 albino male mice, 5 weeks old were distributed into six groups to be used in three experiments. Three groups were surgically castrated by excision of the testes at the epididymis level. The others were not surgically treated, One uncastrated and one castrated group received testosterone therapy by subcutaneous inoculation. One uncastrated and one castrated group received physiological saline by subcutaneous inoculation. One uncastrated and one castrated group received no surgical or therapeutic treatment and were used as controls. All animals were infected with approximately 176 pooled cercariae of S. mansoni by percutaneous penetration. The animals were killed from 57 to 67 days after infection and necropsied. The schistosomes were counted and differentiated sexually with the following results : Experiment one tested the effect of castration in male mice on S. mansoni. A significant decrease in the number of male schistosomes resulted in the castrated group as compared with the uncastrated control group. Several statistical comparisons indicated that castration appears to inhibit the development of the male schistosome. Experiment two tested the effect of testosterone in castrated mice on S. mansoni. Both sexes of schistosomes were reduced in number after subcutaneous inoculation with aqueous testosterone suspension. Since castration accounted for a significant decrease in male parasites, the action of the testosterone may be considered as an important influence regarding the decrease of the female schistosomes. Experiment three tested the effect of testosterone in uncastrated mice on S. mansoni. Both sexes were reduced in number. This indicated that testosterone in the dosage utilized exerted some adverse influence on the parasites. REFERENCES ADDIS, C. J., JR. (1946). J. Parasit., 32, 574. BARTLETT,P. D. (1953). Endocrinology, 52, 272. BEACH, F. A. & HOLz-TucI~ER,A. M. (1949). ft. comp. Physiol. Psychol., 42, 433. BUTTNER, A. (1950). Ann. Parasit., 25, 297. CABLE,R. M. (1950). An Illustrated Manual of Parasitology. Minneapolis : Burgess Publishing Co. CRAIG, F. G. & FAUST, E. C. (1951). Clinical Parasitology. Philadelphia : Lea & Febiger. CRAM, E. B. & FILES,V. S. (1946). Amer. J. trop. Med., 26, 715. LEONARD, S. L. (1952). Endocrinology, 51, 293. MOORE, D. V., YOLLES,T. K. & MELENEY,H. E. (1954). J. Parasit., 40, 166. OLIVER, L. & STIREWALT,M. A. (1952). Ibid., 38, 19. STANDEN, O. D. (1949). Ann. trop. Med. Parasit., 43, 268. - (1953a). Ibid., 47, 26. - (1953b). Ibid., 47, 139. STIREWALT,M. A. (1951). J. Parasit., 37, 42. VOGEL,H. (1941). Zbl. Bakt., 148, 78. WHITE, C. (1952). Biometrics, 8, 33. YOLLES,T. K., MOORE,D. V., DEGUISTI,D. L., RIPSOM,C. A. & MELENEY,H. E. (1947). J. Parasit., 33, 419.
E F F E C T S OF C A S T R A T I O N A N D T E S T O S T E R O N E IN M A L E M I C E ON
SCHISTOSOMA
MANSONI
BY
EDWARD BERG, PH.D.*
(From the Department of Biology, New York State Collegefor Teachers) T h e importance of the reproductive system in lower animal life in reference to the survival and maintenance of species has long been understood. The animal parasites, including Schistosoma mansoni, develop complex genital structures while the other organs remain primitive. T h e necessity for normal development of genital tissue appears essential for the existence of the parasite. There is much to learn regarding host-parasite relations, especially the identity of the vital connecting link between the two. As more information on metabolism is compiled, the application of this mechanism becomes better understood. The present activity on physiological investigations will eventually provide us with the answers. S. mansoni is one of the helminthic parasites being studied in order to provide essential information which might enlighten us about parasitic existence in or on the host. Moore, Vogel, Standen, Bueding and many others have contributed a great deal about the vital connection between host and parasite. Two considerations in this respect are of interest to me. The first concerns the hormonal relationship between host and parasite, whether sexual or otherwise. The second invoives the similarity of enzymatic systems and metabolic cycles of both host and parasite. Schistosomes are the only dioecious parasites of the medically important trematodes, all others being hermaphroditic. However, under strained physical conditions sexual reversal has occurred. Variations in sex ratios and genital structure in different animal hosts have been reported by BUTTNER (1950) and others. Similar responses occurred in different strains of the molluscan intermediate host (VOGEL, 1941 ; STIREWALT, 1951). The introduction of varied sex ratios of schistosomes (STANDEN, 1953b ; MOORE et al., 1954) causes related changes in sexual development of the parasite. It has been demonstrated by ADDIS (1946) that castration Of the rat host results in the alteration of physiological conditions in the tapeworm, Hymenolepis dirninuta. He also found testosterone to be a compensatory agent to the worm when administered to the castrated host. The explanations for these phenomena are not known at the present time. It is, however, apparent that these studies concern two approaches. One involves the possibility of a hormonal relationship by means of a genital secretion from worm to worm. T h e other relationship signifies a hormonal relationship between host and parasite. * I am indebted to the late Professor Ephraim Shorr, Cornell University Medical College, for his assistance on endoerinological aspects ; Dr. Irwin D. J. Bross, of the same institution, for statistical interpretation of the data, Dr. Eloise B. Cram of the National Institutes of Health for infection of the experimental animals and Dr. Louis Freedman of the U.S. Vitamin Corporation for making available the hormone.
354
EFFECTS ON S. mansoni OF CASTRATIONAND TESTOSTERONE IN MOUSE HOST
I t is w i t h t h e s e c o n s i d e r a t i o n s t h a t e x p e r i m e n t s w e r e d e s i g n e d to d e t e r m i n e w h e t h e r sexual h o r m o n e s f r o m t h e h o s t c o u l d cause c h a n g e s in t h e parasite, S. mansoni. MATERIALS AND METHODS O n e h u n d r e d a n d five m a l e a l b i n o m i c e f r o m C a r w o r t h Swiss W e b s t e r stock w e r e d i s t r i b u t e d , at r a n d o m , into six g r o u p s ( T a b l e I). The animals were maintained in white porcelain pans 12 x 8 x 5 inches. Each pan housed 10 or fewer mice. Adequate portions of Purina Laboratory chow were always available. T h e mice were castrated by surgical procedures after intraperitoneal anesthesia with 0.1 rag. of sodium pentobarbitol (Abbott) per g. body weight. A convalescence of 2 weeks ensued. Hormone administration consisted of one subcutaneous injection of an arbitrary 2.5 rag. dose of testosterone in aqueous suspension (Funk) previous to infection with S. mansoni. Post-infection therapy consisted of subsequent similarly equal bi-weekly injections of 1.25 mg. of testosterone. T h e mice were infected with a Puerto Rican strain of N. mansoni to approximately 176 pooled cercaria for 1 hour. T h e cercaria were collected at their most active shedding period (CRAM and FILES, 1946) from snails of the species Australorbis glabratus. T h e method of infection followed the technique of OLIVER and STIREWALT (1952). Faeces examination entailed collection of pooled faeces pellets per pan according to group. Direct smears and the Mathieson and Stoll concentration methods (CRAm and FAUST, 1951) were employed. Ova were first observed 47 days after infection in Group E, followed by the other groups on the 48th day. Necropsies were performed over a 10-day period, starting on the 57th day after infection. T h e mice were selected, a t random, weighed, asphyxiated by ether and dissected. T h e schistosomes were perfused from the liver after the technique of YOLLES et al. (1947). Those trematodes in the mesenteric blood vessels were carefully teased out. T h e y were sexually differentiated and counted. T h e trematodes were fixed, mounted and stained employing Semicon's carmine method (CABLE, 1950). T h e y were mounted on slides and fixed with canada balsam. TABLE I.
Group .
N u m b e r at start.
Grouping of mike.
Description
N u m b e r at necropsy
A
22
Castrated male mice
20
B
'10
Castrated male mice (sham) plus saline
C
33
Castrated male mice plus testosterone
30
E
20
Uncastrated male mice (control)
19
F
10
Uncastrated male mice (sham) plus saline
9
G
10
Uncastrated male mice plus testosterone
8.
Total
105
8
94
RESULTS Morphological examination revealed no consistent structural differences. The statistical correlations were analysed by the use of the Rank " t " Method (WHITE, 1952) by comparison to the Z2 value at the 0.05 P level with one degree of freedom. Thus any value greater than 3.84 indicated a difference outside the realm of chance. The first experiment tested the effect of castration in male albino mice on 8. mansoni (Table II). The comparison between Group A (castrated) and Group E (uncastratedcontrol) s h o w e d a h i g h l y significant d e c r e a s e in t h e m e a n n u m b e r of m a l e s c h i s t o s o m e s p e r
EDWARD BERG
355
mouse in the castrated group as indicated by an H value of 16.98. Statistical analysis indicated the mean difference between female schistosomes of the two groups to be within the realm of chance, the H value being 0.44. In order to test any mechanical effect of inoculation on uncastrated mice, a comparison was made between Group F (uncastrated-saline) and Group E (uncastrated-control). The results, proved to be within the realm of chance. Analysis for significance gave H values of 1.62 for males and 0.62 for females. The second experiment tested the effect of testosterone in castrated male albino mice on S. mansoni. A comparison between the mean schistosome counts per mouse was made between Group B (castrated-saline) and Group C (castrated-testosterone) (Table II). The results of this comparison are interesting because both sexes of schistosomes decreased in number after testosterone administration (male H value = 4.52 ; female H value = 16. 996). Although the effect of the hormone appeared detrimental to male worms, it was almost lethal to the females, decreasing their number almost threefold. A comparison was made between Group C (castrated-testosterone) and Group G (uncastrated-testosterone) to determine the influencing activity of the hormone as shown in Table II. An H value of 1.89 for the male worms proved statistically insignificant, whereas the females showed a significant 4.22. The third experiment tested the effect of testosterone in uncastrated male mice on S. mansoni (Table II). T h e mean schistosome counts per mouse for Group G (uncastratedtestosterone) and Group F (uncastrated-saline-control) were compared and analysed. Both male and female groups indicated a statistically valid difference with H values of 5.83 and 6.82 respectively. It is clearly evident that the testosterone treated group had fewer male and female schistosomes. TABLE II.
Results indicating the number of S. mansoni in male albino mice after collection and sexual differentiation. Parasite sex and mean per mouse
Group (No. of mice)
No. of males
N o . of Mean
females
Mean
i
Castrated (20)
A
Castrated-saline (8)
B
Castrated-testosterone (30)
C
Uncastrated-control (19)
E
Uncastrated saline control (9)
F
Uncastrated-testosterone (8)
G
Total (94)
6
220.00
I I1
256 12.80
11.00
79 00
92 11.50
9.88 222.00 334.00
i
7.40
I
17.58
I 138.00
118
i
3.93 259
I
13.63 112
15.33 73.00
51 9.13
1066.00
12.44 6.33
888
356
EFFECTS ON
S. mansoni OF
CASTRATION AND TESTOSTERONE IN MOUSE HOST
DIscusSlON If a generalized detrimental action is considered in the first experiment, the more fragile female parasites would be expected to undergo the same consequences as the males. Yet only the male population was significantly affected by the action of castration. This may indicate a relationship between the male trematode and the inability of the male mouse to produce secondary sex characteristics. Whether this phenomenon is direct or indirect warrants further consideration. It is interesting to note that upon necropsy, 57 days after infection, only a scant number of trematodes were found in the mesenteric venules of the castrated animals. The majority of parasites were found in the liver. On the other hand, the uncastrated mice carried approximately one-third of the total parasite burden in the mesenteric venules. It has been reported by STANDEN (1949) that under normal conditions the population of S. mansoni in the liver decreased from 53 per cent to 11 per cent. while those of the mesenteric venules increased from 15 per cent. to 77 per cent. between the 35th and 105th day after infectiom - After 50 days, 18 per cent. of the population were located in the mesenteries. The distribution of the parasites in the control mice in this experiment seems to paral!el Standen's results, taking into account the fact that necropsy was performed later. However the castrated experimental animals appeared to contain considerably fewer than 18 per cent. in this area. Another point that STAND~N (1953a) makes is that the migration of the parasites occurs only under optimum conditions. Because the schistosomes in the castrated animals showed delayed migration to the mesenteries, it may be assumed that a necessary developmental factor was lacking. Therefore, the maturation of the parasite may have been inhibited. Several possible criteria may be investigated as the responsible agent for this phenomenon. Since the male mice with excised testes showed a decrease in male schistosomes, it appears that they were affected during or after castration. This may have occurred either as a direct or indirect phenomenon. There are many conditions, such as electrolyte balance, hormone differences, carbohydrate metabolism and others which may change from surgical procedures or infection. Although these changes are usually temporary, a permanent alteration may consequently occur. Whether effects of shock or stress can be of long duration is also important in this respect. The most obvious change after castration is the cessation of normal sexual development. What direct relationship is involved with S. mansoni is not known. ADDIS(1946), however, was able to indicate in male castrated rats infected with H. diminuta, that the parasites were stunted in growth. With testosterone therapy, the tapeworms regained their normal length when compared with uncastrated controls. The second experiment was an attempt to compare the effects on tapeworms with those on schistosomes. Instead of showing morphological changes, however, the worm population was affected. Thus a highly significant decrease in the mean numbers of female worms from the testosterone group occurred. The males appeared affected, but the activity of testosterone is questioned since they had already responded to castration. The dosage of testosterone, although arbitrary, did appear to sustain the sexual behaviour of the mice which corresponds with the results of BEACH and HOLZ-TUCKER (1949). It is, however, possible that the trematodes themselves did not react in the same manner.
EDWARD BERG
357
No explanation can be offered, since a uniform dosage of hormone was employed. Whether an increased or decreased quantity would cause a different effect cannot be stated. However, it is important to remember that variations in testosterone concentration could change the response in the host itself and in this manner alter the host-parasite relationship. The objective, therefore, was to stimulate the castrated mice therapeutically to a more sexually active state. Thus, the effect of testosterone was not indiscriminately tested. An attempt was made to maintain physiological equilibrium, rather than solely to test large doses of testosterone. In this manner any deviation due to excessive activity of the hormone was believed to be checked. Another consideration is that two treatments were actually invoked on the castratedtestosterone group. In combination entirely different effects may result in comparison with individual treatments. The action of testosterone has been described on various subjects in both castrated and uncastrated animals by LEONARD (1952), BARTLETT(1953), and others. However, the responses due to the hormone therapy depend on the dosage employed, the species of animal used and its general physiological state. In the case of castrated male albino mice infected with schistosomiasis, no data concerning the effect of testosterone on the host or the parasite have been reported, to my knowledge. The activity of the hormone on S. rnansoni from an extrinsic source may differ from that of an intrinsic one in these castrated mice. Therefore, from the situation as it appears behaviourwise, a positive effect is possibly directly on the host, but the parasite may exhibit a different response. The true effect of testosterone in this experiment may have been exerted on the female worms rather than on the males. This is demonstrated by the highly significant decrease on the number of female worms in the castrated-testosterone group, whereas the hormone had little effect on the male schistosomes. It is possible, moreover, that by combination with castration, the males were decreased and therefore the females were indirectly affected. It has been reported by MOORE et al. (1954) and STANDEN (1953b) that the female parasites do not develop normally without the presence of mature male schistosomes. It is also indicated (MooRE et al., 1954) that anomalies resulted due to the activity of testosterone proprionate on female S. mansoni. The sex of the experimental mice was not specified. It might be interesting to note whether these anomalies resulted generally, regardless of sex, or whether specific male or female hosts under the testosterone therapy behaved differently. The third experiment seems to confirm that castration in combination with the hormone was a basic factor in the influence of the testosterone, since equal degrees of significance were indicated in both male and female worms in the comparison. From these comparisons concerning testosterone therapy, the following points are apparent : (1) Testosterone therapy affected the female schistosomes in the castrated mice with a highly significant decrease in the n u m b e r collected. T h e male worms did not behave similarly. (2) T h e male parasites were m u c h more inhibited in development by the castration rather t h a n by h o r m o n a l administration. (3) It is also clear that the greatest difference was between the parasites in the uncastrated group and those in the castrated mice on testosterone therapy. (4) T h e fact that the female worms survived more readily in the uncastrated male mice than those in the castrated group tends to indicate that the action of the testosterone is more effective as a detrimental agent w h e n not accompanied by intrinsic male hormones.
358
EFFECTS ON S .
mansoni
OF CASTRATION AND TESTOSTERONE IN MOUSE HOST
SUMMARY
AND
CONCLUSIONS
A total of 105 albino male mice, 5 weeks old were distributed into six groups to be used in three experiments. Three groups were surgically castrated by excision of the testes at the epididymis level. The others were not surgically treated, One uncastrated and one castrated group received testosterone therapy by subcutaneous inoculation. One uncastrated and one castrated group received physiological saline by subcutaneous inoculation. One uncastrated and one castrated group received no surgical or therapeutic treatment and were used as controls. All animals were infected with approximately 176 pooled cercariae of S. mansoni by percutaneous penetration. The animals were killed from 57 to 67 days after infection and necropsied. The schistosomes were counted and differentiated sexually with the following results : Experiment one tested the effect of castration in male mice on S. mansoni. A significant decrease in the number of male schistosomes resulted in the castrated group as compared with the uncastrated control group. Several statistical comparisons indicated that castration appears to inhibit the development of the male schistosome. Experiment two tested the effect of testosterone in castrated mice on S. mansoni. Both sexes of schistosomes were reduced in number after subcutaneous inoculation with aqueous testosterone suspension. Since castration accounted for a significant decrease in male parasites, the action of the testosterone may be considered as an important influence regarding the decrease of the female schistosomes. Experiment three tested the effect of testosterone in uncastrated mice on S. mansoni. Both sexes were reduced in number. This indicated that testosterone in the dosage utilized exerted some adverse influence on the parasites. REFERENCES ADDIS, C. J., JR. (1946). J. Parasit., 32, 574. BARTLETT,P. D. (1953). Endocrinology, 52, 272. BEACH, F. A. & HOLz-TucI~ER,A. M. (1949). ft. comp. Physiol. Psychol., 42, 433. BUTTNER, A. (1950). Ann. Parasit., 25, 297. CABLE,R. M. (1950). An Illustrated Manual of Parasitology. Minneapolis : Burgess Publishing Co. CRAIG, F. G. & FAUST, E. C. (1951). Clinical Parasitology. Philadelphia : Lea & Febiger. CRAM, E. B. & FILES,V. S. (1946). Amer. J. trop. Med., 26, 715. LEONARD, S. L. (1952). Endocrinology, 51, 293. MOORE, D. V., YOLLES,T. K. & MELENEY,H. E. (1954). J. Parasit., 40, 166. OLIVER, L. & STIREWALT,M. A. (1952). Ibid., 38, 19. STANDEN, O. D. (1949). Ann. trop. Med. Parasit., 43, 268. - (1953a). Ibid., 47, 26. - (1953b). Ibid., 47, 139. STIREWALT,M. A. (1951). J. Parasit., 37, 42. VOGEL,H. (1941). Zbl. Bakt., 148, 78. WHITE, C. (1952). Biometrics, 8, 33. YOLLES,T. K., MOORE,D. V., DEGUISTI,D. L., RIPSOM,C. A. & MELENEY,H. E. (1947). J. Parasit., 33, 419.