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Age-related variations in the testes of sprague-dawley rats
257
Toxicology Letters, 4 (1979) 257-261 o Elsevier/North-Holland Biomedical Press
AGE-RELATED VARIATIONS IN THE TESTES OF SPRAGUE-DAWLEY RATS
R.W. JAMES and R. HEYWOOD Huntingdon
Research Centre, Huntingdon,
Cam&. PE18 6ES (Great Britain)
(Received June lst, 1979) (Accepted June 5th, 1979)
SUMMARY
Spontaneous variations in testicular weight and histology are reported for 165 Sprague-Dawley rats (CD strain) observed for periods of up to 104 weeks. Few spontaneous changes are expected to occur in rats studied for 13 or 26 weeks. A significant proportion (20%) of ageing rats develop testicular atrophy or inflammatory lesions of testicular blood vessels. Difficulties may arise in the recognition of chemically induced testicular atrophy for studies exceeding 52 weeks’ duration. Spontaneous testicular neoplasia is not an important feature of the Sprague-Dawley rat.
INTRODUCTION
Organ weight analyses and histological examinations are key features of conventional animal safety-evaluation studies. Several authors have published ranges for rat organ weights [ 12,13,15]. The descriptive reproductive pathology of the male rat has been reviewed by King [9]. Heywood and James [6] have discussed the assessment of testicular toxicity in laboratory animals. Although a variety of experimental procedures and spontaneous pathological entities have been described, there do not appear to be any specific reports of the age-related changes encountered for the rat testis. The differentiation between spontaneous pathological changes, exaggerated pharmacological responses and unpredictable lesions forms the interpretative basis of toxicological investigations. This paper reports the findings of a retrospective survey undertaken to establish the range of spontaneous age-related variations found in the testes of laboratory maintained rats. MATERIALS
AND METHODS
Data for this survey were abstracted from the records of chronic toxicity studies undertaken with Sprague-Dawley rats (CD strain*) at the Huntingdon *Charles River Ltd., Manston, U.K., or Wilmington, U.S.A.
258
Research Centre between 1972 and 1975. Details of the rats used as untreated controls were examined. The rats were barrier-maintained and housed in fives in suspended metal cages with wire mesh floors. Room temperature and humidity were maintained at 21” ?r 2°C and 50% f 5%. Lighting was controlled to allow 12 h light (08.00 to 20.00 h) and 12 h dark per day. Spratt’s Laboratory Rodent Diet No. 1 and tap water were freely available. When the experimental periods were completed the rats were killed by carbon dioxide asphyxiation. A full macroscopic post-mortem examination was performed. All the major organs, including the testes, were dissected free of fat and the weights recorded. Tissues were routinely fixed in buffered neutral formalin and after embedding in melted paraffin wax (56”C), 5 pm sections stained with haematoxylin and eosin were examined by light microscopy. The data were classified according to the period for which the rats were maintained. For each period the initial body weight, together with the arithmetic mean, standard deviation and range for terminal body weight and testicular weight were obtained. The examining histopathologist’s comments concerning testicular histology were collated according to the length of study. The lesions were further classified according to the nature and location of the change described. Sufficient data were available to assess the age-related changes occurring in the testes of a sample of 165 Sprague-Dawley (CD strain) rats maintained for periods of 13,26,52 or 104 weeks’ duration. RESULTS
The initial body weights of the rats were all within the range 90.--180 g. The terminal body weight and testicular weight data are given in Table I. The body weights increased according to the duration of the studies; rats maintained for 104 weeks were almost double the weight of those maintained for 13 weeks. The mean testicular weights did not differ appreciably between categories, The standard deviations expressed as percentages of the mean values were 16% and 12% after 104 and 52 weeks respectively, compared with 8% and 4% after 13 or 26 weeks. Thus, the range of testicular weights was widest for rats maintained for 104 weeks. Approx. 13% of the rats mainTABLE I SUMMARY OF BODY WEIGHT AND TESTICULAR WEIGHTS .Study length (weeks) 13 26 52 104
Number of rats
Body weight (g)
Weight of testes (g)
Mean
S.D.
Mean
S.D.
range
39 59 15 52
533 652 798 904
* 53 f 17 f 127 f 52
5.0 5.1 4.9 5.0
f + f *
4.3-6.6 4.3-6.1 3.8-5.9 2.7-6.3
0.4 0.2 0.6 0.8
259
tained for 52 or 104 weeks were found to have testicular weights below the lower limit recorded in the remaining categories. The most notable histological findings (Table II) were the increased incidence of atrophied germinal epithelium (19%) and of inflammatory lesions involving testicular blood vessels (17%) among rats kept for 104 weeks. It was unusual for both lesions to occur in the same testis. Atrophy of the germinal epithelium was almost exclusively diffuse in nature, but unilateral and bilatTABLE II SUMMARY OF HISTOPATHOLOGICAL
FINDINGS
Study length
13 weeks
26 weeks
52 weeks
104 weeks
Number examined Number with : Interstitial cell adenoma Interstitial cell hyperplasia Incomplete spermatogenesis Atrophic germinal epithelium (a) unilateral (b) bilateral Spermatocoele granuloma Epididymitis Arteritic changes
39
59
15
52
-
-
-
-
-
-
-
-
-
1
1
-
1
-
-
1 -
1 1
6 5 1 9
eral cases were found at similar frequencies. One case of unilateral atrophy was reported among the 15 rats examined after 52 weeks. Another rat examined after 52 weeks showed arrest of spermatogenesis at the primary spermatocyte stage. Neoplastic or hyperplastic lesions were rarely identified (2%) and then only among the oldest rats. A single spermatocoele granuloma was found among rats kept for 104 weeks. Less than 3% of the rats maintained for 13 or 26 weeks showed any remarkable pathology. The only changes recorded for these rats were minor inflammatory cell infiltrations in the epididymides. DISCUSSION
The results of this survey suggest that spontaneous v~ations in testicular weight and histology are rarely encountered in Sprague-Dawley rats maintained for 13- or 26-week observation periods. Rats maintained for longer periods tend to show an increasing incidence of testicular atrophy and correspondingly reduced testicular weights. The changes in pituitary gonadotrophin secretion and androgen production observed in ageing male rats [ 51 support the hypothesis that this is a senescent phenomenon. Ribelin [lo] , has suggested that atrophy of the rat testis is a useful index of chemical toxicity. However, age-related atrophy must be considered in studies exceeding 12 months’ duration.
Spontaneous testicular tumours are rarely reported for Sprague-Dawley rats [3,11,14] . Bullock and Curtis [ 11 observed a possible seminoma in a single aged rat of unspecified strain, In this series one interstitial (Leydig) cell adenoma was reported. Leydig cell tumours frequently occur in aged Rochester [ 21 and Fischer rats [ 71. A high incidence of spontaneous testicular tumours would not be expected to occur during life-span studies with the Sprague-Dawley rat. Yang [16] has discussed the inflammatory changes observed in the arteries of ageing Spra~e-Dawley rats, which resemble human poly~teritis nodosa. A 14.6% incidence was recorded in rats aged 541-797 days. Lesions were present in mesentery, pancreas, kidneys, stomach, intestine and testes. Muscular arteries were more frequently involved than arterioles. The incidence of the lesions did not differ between control or experimental rats. The aetiological factors remain obscure, but autoimmune hypersensitivity reactions may be involved. Spermatocoele granulomata occur following extravasation of spermatozoa into the epididymal stroma [ 41. James et al. [ 81 have reported the production of epididymal sperm granulomata in rats given the antifertility compound 1-amino-3-chloro-2-propanol. Such lesions would be expected to arise spontaneously only on isolated occasions. REFERENCES 1 F.R. Bullock and M.R. Curtis, Spontaneous tumours of the rat, J. Cane. Res., 15 (1930) 1-115. 2 R.C. Crain, Spontaneous tumours in the Rochester strain of the Wistar rat, Am. J. Pathol., 34 (1958) 311-335. 3 J, Gillmann, C. Gilbert and I. Spence, Phaeochromocytoma in the rat, pathogenesis and collateral reactions and its relation to comparable tumours in man, Cancer, 6 (1953) 494-511. 4 F.J. Glassy and F.K. Mostofi, Spermatic granulomas of the epididymis, Am. J. Clin. Pathol., 26 (1956) 1303-1313. 5 C.D. Gray, Changes in the levels of luteinising hormone and testosterone in the circulation of ageing male rats, J. Endocr., 76 (1978) 551-552. 6 R. Heywood and R.W. James, Assessment of testicular toxicity in laboratory animals, Environ. Hlth. Persp., 24 (1978) 73-80. 7 B.B. Jacobs and R.A. Huseby, Neoplasms occurring in aged Fischer rats with special reference to testicular, uterine and thyroid turnours, J. Nat. Cancer Inst., 39 (1967) 303-30s. 8 R.W. James, R. Heywood, J. Colley and B. Hunter, The oral toxicity of 1-amino-3chloro-2.propanol hydrochloride (CL 88236) in rats, Toxicology, 11 (1978) 235-243. 9 N.W. King, The Reproductive Tract, in K. Benirschke, F.M. Garner and T.C. Jones (Eds.), Pathology of Laboratory Animals, Vol. I, Springer-Verlag, New York, 1978, pp. 554-580. 10 WE. Ribelin, Atrophy of rat testis as index of chemical toxicity, Arch. Pathol., 75 (1963) 229-235. 11 A.B. Russfield, Pathology of the endocrine glands, ovary and testis of rats and mice, in E. Cotchin and F.J.C. Roe (Eds.), Pathology of Laboratory Rats and Mice, BlackweI1, Oxford, 1967, pp. 391-465.
261 12 K. Schtirer, The effects of chronic underfeeding on organ weights of rats, Toxicology, 7 (1977) 45-56. 13 M.T. Stevens, The value of relative organ weights, Toxicology, 5 (1975) 311-318. 14 S.W. Thompson, R.A. Huseby, M.A. Fox, CL. Davis and R.D. Hunt, Spontaneous tumours in the Sprague-Dawley rat, J. Natl. Cancer Inst., 27 (1961) 1037-1057. 15 G. Trieb, G. Pappritz and L. Liitzens, Allometric analysis of organ weights: I, Rats, Toxicol. Appl. Pharmacol., 35 (1976) 531-542. 16 Y.H. Yang, Polyarteritis nodosa in laboratory rats, Lab. Invest., 14 (1965) 81-38.
Toxicology Letters, 4 (1979) 257-261 o Elsevier/North-Holland Biomedical Press
AGE-RELATED VARIATIONS IN THE TESTES OF SPRAGUE-DAWLEY RATS
R.W. JAMES and R. HEYWOOD Huntingdon
Research Centre, Huntingdon,
Cam&. PE18 6ES (Great Britain)
(Received June lst, 1979) (Accepted June 5th, 1979)
SUMMARY
Spontaneous variations in testicular weight and histology are reported for 165 Sprague-Dawley rats (CD strain) observed for periods of up to 104 weeks. Few spontaneous changes are expected to occur in rats studied for 13 or 26 weeks. A significant proportion (20%) of ageing rats develop testicular atrophy or inflammatory lesions of testicular blood vessels. Difficulties may arise in the recognition of chemically induced testicular atrophy for studies exceeding 52 weeks’ duration. Spontaneous testicular neoplasia is not an important feature of the Sprague-Dawley rat.
INTRODUCTION
Organ weight analyses and histological examinations are key features of conventional animal safety-evaluation studies. Several authors have published ranges for rat organ weights [ 12,13,15]. The descriptive reproductive pathology of the male rat has been reviewed by King [9]. Heywood and James [6] have discussed the assessment of testicular toxicity in laboratory animals. Although a variety of experimental procedures and spontaneous pathological entities have been described, there do not appear to be any specific reports of the age-related changes encountered for the rat testis. The differentiation between spontaneous pathological changes, exaggerated pharmacological responses and unpredictable lesions forms the interpretative basis of toxicological investigations. This paper reports the findings of a retrospective survey undertaken to establish the range of spontaneous age-related variations found in the testes of laboratory maintained rats. MATERIALS
AND METHODS
Data for this survey were abstracted from the records of chronic toxicity studies undertaken with Sprague-Dawley rats (CD strain*) at the Huntingdon *Charles River Ltd., Manston, U.K., or Wilmington, U.S.A.
258
Research Centre between 1972 and 1975. Details of the rats used as untreated controls were examined. The rats were barrier-maintained and housed in fives in suspended metal cages with wire mesh floors. Room temperature and humidity were maintained at 21” ?r 2°C and 50% f 5%. Lighting was controlled to allow 12 h light (08.00 to 20.00 h) and 12 h dark per day. Spratt’s Laboratory Rodent Diet No. 1 and tap water were freely available. When the experimental periods were completed the rats were killed by carbon dioxide asphyxiation. A full macroscopic post-mortem examination was performed. All the major organs, including the testes, were dissected free of fat and the weights recorded. Tissues were routinely fixed in buffered neutral formalin and after embedding in melted paraffin wax (56”C), 5 pm sections stained with haematoxylin and eosin were examined by light microscopy. The data were classified according to the period for which the rats were maintained. For each period the initial body weight, together with the arithmetic mean, standard deviation and range for terminal body weight and testicular weight were obtained. The examining histopathologist’s comments concerning testicular histology were collated according to the length of study. The lesions were further classified according to the nature and location of the change described. Sufficient data were available to assess the age-related changes occurring in the testes of a sample of 165 Sprague-Dawley (CD strain) rats maintained for periods of 13,26,52 or 104 weeks’ duration. RESULTS
The initial body weights of the rats were all within the range 90.--180 g. The terminal body weight and testicular weight data are given in Table I. The body weights increased according to the duration of the studies; rats maintained for 104 weeks were almost double the weight of those maintained for 13 weeks. The mean testicular weights did not differ appreciably between categories, The standard deviations expressed as percentages of the mean values were 16% and 12% after 104 and 52 weeks respectively, compared with 8% and 4% after 13 or 26 weeks. Thus, the range of testicular weights was widest for rats maintained for 104 weeks. Approx. 13% of the rats mainTABLE I SUMMARY OF BODY WEIGHT AND TESTICULAR WEIGHTS .Study length (weeks) 13 26 52 104
Number of rats
Body weight (g)
Weight of testes (g)
Mean
S.D.
Mean
S.D.
range
39 59 15 52
533 652 798 904
* 53 f 17 f 127 f 52
5.0 5.1 4.9 5.0
f + f *
4.3-6.6 4.3-6.1 3.8-5.9 2.7-6.3
0.4 0.2 0.6 0.8
259
tained for 52 or 104 weeks were found to have testicular weights below the lower limit recorded in the remaining categories. The most notable histological findings (Table II) were the increased incidence of atrophied germinal epithelium (19%) and of inflammatory lesions involving testicular blood vessels (17%) among rats kept for 104 weeks. It was unusual for both lesions to occur in the same testis. Atrophy of the germinal epithelium was almost exclusively diffuse in nature, but unilateral and bilatTABLE II SUMMARY OF HISTOPATHOLOGICAL
FINDINGS
Study length
13 weeks
26 weeks
52 weeks
104 weeks
Number examined Number with : Interstitial cell adenoma Interstitial cell hyperplasia Incomplete spermatogenesis Atrophic germinal epithelium (a) unilateral (b) bilateral Spermatocoele granuloma Epididymitis Arteritic changes
39
59
15
52
-
-
-
-
-
-
-
-
-
1
1
-
1
-
-
1 -
1 1
6 5 1 9
eral cases were found at similar frequencies. One case of unilateral atrophy was reported among the 15 rats examined after 52 weeks. Another rat examined after 52 weeks showed arrest of spermatogenesis at the primary spermatocyte stage. Neoplastic or hyperplastic lesions were rarely identified (2%) and then only among the oldest rats. A single spermatocoele granuloma was found among rats kept for 104 weeks. Less than 3% of the rats maintained for 13 or 26 weeks showed any remarkable pathology. The only changes recorded for these rats were minor inflammatory cell infiltrations in the epididymides. DISCUSSION
The results of this survey suggest that spontaneous v~ations in testicular weight and histology are rarely encountered in Sprague-Dawley rats maintained for 13- or 26-week observation periods. Rats maintained for longer periods tend to show an increasing incidence of testicular atrophy and correspondingly reduced testicular weights. The changes in pituitary gonadotrophin secretion and androgen production observed in ageing male rats [ 51 support the hypothesis that this is a senescent phenomenon. Ribelin [lo] , has suggested that atrophy of the rat testis is a useful index of chemical toxicity. However, age-related atrophy must be considered in studies exceeding 12 months’ duration.
Spontaneous testicular tumours are rarely reported for Sprague-Dawley rats [3,11,14] . Bullock and Curtis [ 11 observed a possible seminoma in a single aged rat of unspecified strain, In this series one interstitial (Leydig) cell adenoma was reported. Leydig cell tumours frequently occur in aged Rochester [ 21 and Fischer rats [ 71. A high incidence of spontaneous testicular tumours would not be expected to occur during life-span studies with the Sprague-Dawley rat. Yang [16] has discussed the inflammatory changes observed in the arteries of ageing Spra~e-Dawley rats, which resemble human poly~teritis nodosa. A 14.6% incidence was recorded in rats aged 541-797 days. Lesions were present in mesentery, pancreas, kidneys, stomach, intestine and testes. Muscular arteries were more frequently involved than arterioles. The incidence of the lesions did not differ between control or experimental rats. The aetiological factors remain obscure, but autoimmune hypersensitivity reactions may be involved. Spermatocoele granulomata occur following extravasation of spermatozoa into the epididymal stroma [ 41. James et al. [ 81 have reported the production of epididymal sperm granulomata in rats given the antifertility compound 1-amino-3-chloro-2-propanol. Such lesions would be expected to arise spontaneously only on isolated occasions. REFERENCES 1 F.R. Bullock and M.R. Curtis, Spontaneous tumours of the rat, J. Cane. Res., 15 (1930) 1-115. 2 R.C. Crain, Spontaneous tumours in the Rochester strain of the Wistar rat, Am. J. Pathol., 34 (1958) 311-335. 3 J, Gillmann, C. Gilbert and I. Spence, Phaeochromocytoma in the rat, pathogenesis and collateral reactions and its relation to comparable tumours in man, Cancer, 6 (1953) 494-511. 4 F.J. Glassy and F.K. Mostofi, Spermatic granulomas of the epididymis, Am. J. Clin. Pathol., 26 (1956) 1303-1313. 5 C.D. Gray, Changes in the levels of luteinising hormone and testosterone in the circulation of ageing male rats, J. Endocr., 76 (1978) 551-552. 6 R. Heywood and R.W. James, Assessment of testicular toxicity in laboratory animals, Environ. Hlth. Persp., 24 (1978) 73-80. 7 B.B. Jacobs and R.A. Huseby, Neoplasms occurring in aged Fischer rats with special reference to testicular, uterine and thyroid turnours, J. Nat. Cancer Inst., 39 (1967) 303-30s. 8 R.W. James, R. Heywood, J. Colley and B. Hunter, The oral toxicity of 1-amino-3chloro-2.propanol hydrochloride (CL 88236) in rats, Toxicology, 11 (1978) 235-243. 9 N.W. King, The Reproductive Tract, in K. Benirschke, F.M. Garner and T.C. Jones (Eds.), Pathology of Laboratory Animals, Vol. I, Springer-Verlag, New York, 1978, pp. 554-580. 10 WE. Ribelin, Atrophy of rat testis as index of chemical toxicity, Arch. Pathol., 75 (1963) 229-235. 11 A.B. Russfield, Pathology of the endocrine glands, ovary and testis of rats and mice, in E. Cotchin and F.J.C. Roe (Eds.), Pathology of Laboratory Rats and Mice, BlackweI1, Oxford, 1967, pp. 391-465.
261 12 K. Schtirer, The effects of chronic underfeeding on organ weights of rats, Toxicology, 7 (1977) 45-56. 13 M.T. Stevens, The value of relative organ weights, Toxicology, 5 (1975) 311-318. 14 S.W. Thompson, R.A. Huseby, M.A. Fox, CL. Davis and R.D. Hunt, Spontaneous tumours in the Sprague-Dawley rat, J. Natl. Cancer Inst., 27 (1961) 1037-1057. 15 G. Trieb, G. Pappritz and L. Liitzens, Allometric analysis of organ weights: I, Rats, Toxicol. Appl. Pharmacol., 35 (1976) 531-542. 16 Y.H. Yang, Polyarteritis nodosa in laboratory rats, Lab. Invest., 14 (1965) 81-38.