Age and Ageing (1976), 5, 12
FACTORS CONTROLLING GROWTH AND AGE INVOLUTION OF THE RAT THYMUS D. BELLAMY, S. M. HINSULL AND J. G. PHILLIPS* Department of Zoology, University College, Cardiff, and 'Department of Zoology, University of Hull
Thymic development and somatic growth were investigated in three strains of rat. The well established influence of sex and age on somatic growth was confirmed. It was also found that the rate of thymic growth was not influenced by the sex of the animal until four weeks of age, but thereafter, the thymus grew faster in males compared with females of the same strain. Gonadectomy had a marked effect on the thymus, resulting in a higher rate of growth than in normal animaia; but a slower initial rate of involution. The mavimum thymus weights attained in gonadectomized animala were found to be independent of the age at which the animals were gonadectomized or of the sex of the animal Gonadectomy did not influence the point at which involution began. Thymic mitotic indices of gonadectomized animala were found to be higher than those of normal animals The influence of sex hormones on somatic growth and the development of the thymus is discussed, and it is concluded that cell loss is the regulator of thymus growth and involution. INTRODUCTION
Involution of the thymus generally occurs about the time of sexual maturity and this has given rise to ideas that sex hormones are responsible for the initiation and possibly the maintenance of the process of involution (Weaver, 1955). The latter question was investigated by Bellamy (1967), who showed that gonadectomy and injections of sex hormones did not affect the rate of involution although the gonads clearly had some negative feedback control over the total mass of the thymus during the first weeks of life. Whilst this work showed that, in the long term, age involution was independent of the gonads, it did not deal with the questions concerning the timing of involution or with the role of the gonads in maintaining thymus mass during the various stages of development and ageing in the rat. It was with these questions in mind, concerning the timing of involution and the role of the gonads, that the following work was undertaken. Methods Animals Three inbred strains of rats with well established pedigrees were used for the experiments. These were two rWistar derived' animals, respectively the Sheffield strain and a Medical Research Council strain (PVG) and a third group of 'Sprague—Dawley derived' animals from a colony selected by the Medical Research Council (CFY strain). Treatment and experimental methods
Rats were maintained on a commercial pellet diet and water ad lib. and were exposed to 12 h of light per 24 h. They were gonadectomized at 4 weeks, 8 weeks or 15 weeks of age. For histo-
Downloaded from http://ageing.oxfordjournals.org/ at University of California, San Diego on April 10, 2016
Summary
Factors Controlling Growth and Age Involution of the Rat Thymus
13
logical studies, 0.1 nag of colchicine/100 g body weight was administered intraperitoneally 4 h prior to death. Thymic tissue was fixed' in Carnoy, dehydrated, embedded in wax and subsequently stained wilt methyl green-pyronin. RESULTS
300
200
• Normal male • Normal female A Male castrated at 4 weeks T Female ovarx at 4 weeks
100
10
20
30
600
-400 as
O200 to
300
200
100
30 40 Age in weeks
Fig. 1. The effect of gonadectomy on the growth curves of three strains of rat.
Downloaded from http://ageing.oxfordjournals.org/ at University of California, San Diego on April 10, 2016
Effects of gonadectomy on body weight All three strains showed a sexual dimorphism in body weight, males being between 50-100 per cent larger than females at 30 weeks of age. There were, however, differences in the extent of sexual dimorphism between strains as well as differences in the patterns of growth (Fig. 1).
14
D. Bellamy, S. M. HinsuU and J. G. Phillips
Effects of gonadectomy on growth and involution of the thymus There was no significant sex difference between the effects of gonadectomy carried out one or four weeks post partum (Fig. 2ab). The initial growth rate of the thymus in CFY males was about 80 per cent greater than in females. At six weeks of age, there was a sudden loss of weight in both sexes which appeared to proceed in two phases. There was a sharp decline in mass up to about 20 weeks, followed by a slower loss up to 80 weeks. The average rate of weight loss between six and 30 weeks was much greater for males, and this resulted in the male thymus declining to the female weight at about 20 weeks. Thereafter, the thymus lost weight at about the same rate in both sexes (Fig. lab). Gonadectomy at four weeks of age resulted in a prolongation of thymic growth and increased the maximum thymus weight by about 22 per cent in CFY animals. Sudden weight loss was again observed, beginning at nine weeks, which occurred at about the same rate as in normal animals, and proceeded again in two phases. The first phase of rapid weight loss ended at about 40 weeks, when the thymus weights were comparable with control values. Thereafter, gonadectomy did not affect weight loss (Fig. la). The action of gonadectomy in females was to accelerate thymic growth, increasing the maximum thymus weight by about 50 per cent. Weight loss began in the ninth week, as in the gonadectomized males, at about the same organ size. The pattern of weight loss was the same as in the male, with no effect of gonadectomy noticeable after 35 weeks (Fig. 2b). In animals of the Sheffield strain, thymic growth rate was equal to that in CFY
Downloaded from http://ageing.oxfordjournals.org/ at University of California, San Diego on April 10, 2016
In the Sheffield strain, growth rates were similar in both sexes up to about 10 weeks of age, at which time the female weight gain declined. Male growth continued at a high level for another 10 weeks. At 30 weeks, males were about 50 per cent larger than females (Fig. la). Gonadectomy at four weeks reduced the growth rates of males, an effect that was first noticeable at 10 weeks. The operation increased the growth of females, again the effect was first noticeable at 10 weeks of age. In gonadectomized females, the drop in growth rate that appeared in normal animals at 10 weeks was not so marked, although the body weight at 30 weeks was only slightly higher than that of the controls. Over all, the effect of gonadectomy was to reduce sexual dimorphism in body weight by about 50 per cent (Fig. la). Gonadectomy of Sheffield rats at eight and 15 weeks of age again resulted in a decrease in body weight in males and an increase in female weight but no significant difference was observed between the body weights at comparable age groups of animals gonadectomized at four, eight or 15 weeks of age. Despite having growth rates that were about twice as high as in Sheffield rats, CFY animals showed the same pattern of growth and effect of gonadectomy that were observed in the former strain. Sexual dimorphism in body weight was reduced as a result of gonadectomy by about 75 per cent (Fig. 16). The general growth pattern of the males of the PVG strain was different from that of males belonging to the other two strains. There was no significant increase in body weight from 20-65 weeks. Female PVG animals continued to grow, albeit slowly, during this interval, so narrowing the gap in body weight (Fig. lc).
Factors Controlling Growth and Age Involution of the Rat Thymus
15
animals up to about four weeks of age. At this time, in both sexes, the organ growth rate was markedly reduced. A maximum in size was reached at nine weeks, with the female thymus weighing slightly less than that of the male. Females were characterized by a two-phase loss of weight similar to that observed for both sexes in the CFY strain. In contrast, weight loss occurred in the male at a slower, steady rate. At 24 weeks of age, the rates of involution were the same in both sexes (Fig. 3). 1000
800
600 -
Male
400 -
200 -
800 -
eoo -
400 -
200 -
10
15
20 Age in weeks
25
30
Fig. 2. The effect of gonadectomy on thymus growth and involution in CFY rats.
Gonadectomy at four weeks resulted in a continuation in the initial rapid phase of growth beyond the time of marked growth inhibition in normal animals. The timing of the maxima in thymic weight was not altered in either sex, but the peaks in the weight graphs were sharper than in controls; the female maximum was lower than that in males. Involution was faster in males than in females up to 10 weeks of age. Thereafter, both sexes were characterized by the same rate of weight loss, occurring at the same mean thymus size (Fig. 3).
Downloaded from http://ageing.oxfordjournals.org/ at University of California, San Diego on April 10, 2016
Normal rats Gonadx.at 4 weeks Gonadx.at 1 week
D. Bellamy, S. M. Hinsull and J. G. Phillips
16
Gonadectomy of male and female rats of the Sheffield strain at eight or 15 weeks of age resulted in stimulation of thymic growth during the next two weeks. Maximum thymic weights in males were increased by 37 per cent after gonadectomy at eight weeks, whereas gonadectomy at 15 weeks resulted in a 60 per cent increase in thymus weight compared with intact controls. Gonadectomy of females at eight or 15 weeks1 600 Gonaditat 4 weeks
Gonadnat 8 weeks
400
200
Male A Female • Gonadxmale D Gonadx.femal'
100
10
20
30
10
10
20 30 Age in weeks
20
30
Fig. 3. The effect of gonadectomy at four, eight and 15 weeks of age on the growth and involution of thymus in Sheffield rats. 400
Male Female
300
m $ 200 3
100
10
20
30 40 Age In weeks
50
60
Fig. 4. Thymus growth and involution in PVG rate.
of age resulted in weight increases of 108 per cent and 131 per cent respectively compared with control animals. The maximum thymus weights attained by male and female animals gonadectomized at 15 weeks of age were not significantly different from each other nor were they significantly different from the maximum thymus weights attained by male and female rats gonadectomized at eight weeks of age. Thus, involution of
Downloaded from http://ageing.oxfordjournals.org/ at University of California, San Diego on April 10, 2016
*. 300
Factors Controlling Growth and Age Involution of the Rat Thymus
17
the thymus, in all gonadectomized groups was delayed for about one month, when it proceeded at a faster rate than normal for about four weeks. In contrast to the other two strains, there was no significant sexual difference between the rates of thymic growth and involution in animals of the PVG strain. The riming of involution was identical to that in the other strains (Fig. 4).
1-4
1-2
Female gonadx. at 4 weeks Male gonadxat 4 weeks Normal male Normal female
£• o CD
2 §0-8
0-6
10
15
20 26 Age in weeks
30
35
40
Fig. 5. The effect of gonadectomy on mitotic indices of ShefEeld-Wistar rat thypnus. Effect of gonadectomy on mitotic index
The mitotic index dropped by about 50 per cent during the first five weeks of life, with no significant differences between sexes. Gonadectomy at five weeks increased the mean mitotic index slightly but this was only significantly different from control values in the females (Fig. 5). DISCUSSION
The three rat strains used in the present work differed greatly with regard to the rate of growth and the pattern of weight gain. No significant difference was observed in the prepubertal growth rate between male, female and gonadectomized animals. However, postpubertal sexual dimorphism was observed as a characteristic feature, with the males growing faster and reaching a higher body weight than the females. The magnitude of sex-linked weight differences differed between strains. There were also strain differences regarding the extent to which the sexual dimorphism was dependent upon the gonads. Generally, in males, sex hormones appeared to stimulate growth, whereas the opposite seemed to hold for females. The magnitude of the gonadal influence was strain-dependent. These findings on the effect of gonadal status in post-pubertal growth rates are in agreement with the work of Grunt (1964) and Kakolowski, Cox & Valenstein (1968).
Downloaded from http://ageing.oxfordjournals.org/ at University of California, San Diego on April 10, 2016
• o A •
18
D. Bellamy, S. M. HinsuU and J. G. Phillips
Downloaded from http://ageing.oxfordjournals.org/ at University of California, San Diego on April 10, 2016
There was a wide variation in the initial growth rate of the thymus between sexes and strains, which could not be correlated directly with differences in body weight gain. Thymus weight was increased between six and nine weeks of age despite a 50 per cent decrease in the mitotic index during this period. Therefore, there must have been a greater inhibition of cellular loss than mitosis. Involution began at approximately nine weeks of age, but was not accompanied by any change in the rate of decrease in the mitotic index. Previous work has shown that partial thymectomy had no significant influence on the mitotic index (Bellamy & Alkufaishi, 1972). This combined evidence suggests that mitotic jndex is independent of thymus size; also it is unlikely that thymus size is regulated solely through mitosis. In this respect the mass of thymic lymphocytes is possibly controlled mainly by cellular loss; involution being timed by a change in either cell migration (Fichtellius, 1960) or in cellular death (Metcalf, 1966). It has been postulated by Metcalf (1966) that degenerating lymphocytes induce the formation of new cells by the feed-back of breakdown products; however if this is so, during involution this equilibrium must be altered so that cell loss is greater than cell division. Gonadectomy at four weeks of age, depending on strain, results in either an increase in the thymic growth rate or a continuation of the initial growth rate for a longer period. Also the peak of thymus weight could be increased by the operation. Increased thymus weights immediately after gonadectomy at eight or 15 weeks of age demonstrated an influence of sex hormones on the thymus after sexual maturity, when the thymus was normally undergoing involution. The operation resulted in an increase in thymus growth in both sexes thus reversing the normal rate of involution. Maximum thymus weights attained in all four operated groups were not significantly different from each other irrespective of the age when the animals were gonadectomized or the sex of the rats. These findings indicate that sex steroids inhibit thymus growth, either by inhibiting cellular proliferation or by accelerating cellular loss. It is suggested that the degree of inhibition is greater in females than in males since, in intact animals, thymus weights are significantly lower in females than in males, but when this inhibition is released following gonadectomy, a 131 per cent increase in thymus weight was observed in females compared with a 60 per cent increase in thymus weight in males; however both groups attained comparable final thymus weights suggesting that thymic autonomy is maintained at different levels according to the hormonal status of the animal. Similarly, it may be postulated that suppression of thymus mass at 15 weeks of age is greater than at eight weeks of age. Gonadectomy resulted in a higher mitotic index in both male and female animals compared with intact controls, suggesting that the rate of mitosis is inhibited by sex hormones. Although the operation only had a small effect on mitotic index, the thymus is characterized by a high rate of cellular proliferation and this small stimulation of cell division may have been sufficient to produce a large net change in cell mass. The increase in the point of maximum cell mass was probably due to new relationships being established between the rate of mitosis and cellular death. As gonadectomy stimulates growth at all ages it appears that sex hormones have an immediate and direct action on the thymus. There was no clear evidence that the gonads influenced the rate of involution. Indeed, gonadectomy had no effect on the thymus after 35 weeks of age.
Factors Controlling Growth and Age Involution of the Rat Thymus
19
ACKNOWLEDGEMENTS
This project was supported by a research grant from the Tenovus Organization. The expert technical assistance of Miss A. Franklin, Mrs J. Phillips and Mr A. Stevenson is gratefully acknowledged. REFERENCES
D. (1967). Hormonal effects in relation to ageing in mammals. Symp. Soc. Exp. Biol. 21, 427. BELLAMY, D. & ALKUFAISHI, H. (1972). The cellular composition of thymus: a comparison between cortisol-treated and aged C57/BL mice. Age & Ageing 1, 88. FICHTELLIUS, K. E. (1960). Ciba Found. Symp. Haemopoiesis p. 204. GRUNT, J. A. (1964). Endocrinology 75, 446. BELLAMY,
KAKOLOWSKI, J. W., COX, V. C. & VALENSTEIN, E. S. (1968). Psychol. Rep. 22, 547. METCALF, D. (1966). Recent Results in Cancer Research. Berlin and New York: WEAVEH, J. A. (1955). Changes induced in the thymus and lymph nodes of the
Springer-Verlag. rat by the administration of cortisone and sex hormones and by other procedures. J. Pathol. Bacteriol. 69, 133.
Downloaded from http://ageing.oxfordjournals.org/ at University of California, San Diego on April 10, 2016
In summary, the new picture that emerged from the present work is that the normal timing of age involution is set by the gonads. Growth of the thymus may be initiated by gonadectomy during the early stages of age involution, at a rate equal to the preinvolution rate. This indicates the working of a gonad-controlled clock which involves the reversible suppression of a basic process governing the initial size of the lymphocyte population. There appears to be an upper limit to the thymic cell population that is set independently of the gonads. When this new ceiling is reached after post-involution gonadectomy, the cell population is lowered for a second time, and at a faster rate, which appears to be a compensation to reduce an overpopulated gland. Thus, at least four processes govern the mass of thymic lymphocytes: one for rapid growth; a second for gonad-initiated involution; a third for non-gonad-initiated compensatory involution; and a fourth for the steady lowering of the weight ratio of thymus to body, which carries the thymus into old age. If any of these processes act by regulating mitosis of lymphocytes they do so through a very small percentage change in mitotic index which cannot be detected with the present methods.
FACTORS CONTROLLING GROWTH AND AGE INVOLUTION OF THE RAT THYMUS D. BELLAMY, S. M. HINSULL AND J. G. PHILLIPS* Department of Zoology, University College, Cardiff, and 'Department of Zoology, University of Hull
Thymic development and somatic growth were investigated in three strains of rat. The well established influence of sex and age on somatic growth was confirmed. It was also found that the rate of thymic growth was not influenced by the sex of the animal until four weeks of age, but thereafter, the thymus grew faster in males compared with females of the same strain. Gonadectomy had a marked effect on the thymus, resulting in a higher rate of growth than in normal animaia; but a slower initial rate of involution. The mavimum thymus weights attained in gonadectomized animala were found to be independent of the age at which the animals were gonadectomized or of the sex of the animal Gonadectomy did not influence the point at which involution began. Thymic mitotic indices of gonadectomized animala were found to be higher than those of normal animals The influence of sex hormones on somatic growth and the development of the thymus is discussed, and it is concluded that cell loss is the regulator of thymus growth and involution. INTRODUCTION
Involution of the thymus generally occurs about the time of sexual maturity and this has given rise to ideas that sex hormones are responsible for the initiation and possibly the maintenance of the process of involution (Weaver, 1955). The latter question was investigated by Bellamy (1967), who showed that gonadectomy and injections of sex hormones did not affect the rate of involution although the gonads clearly had some negative feedback control over the total mass of the thymus during the first weeks of life. Whilst this work showed that, in the long term, age involution was independent of the gonads, it did not deal with the questions concerning the timing of involution or with the role of the gonads in maintaining thymus mass during the various stages of development and ageing in the rat. It was with these questions in mind, concerning the timing of involution and the role of the gonads, that the following work was undertaken. Methods Animals Three inbred strains of rats with well established pedigrees were used for the experiments. These were two rWistar derived' animals, respectively the Sheffield strain and a Medical Research Council strain (PVG) and a third group of 'Sprague—Dawley derived' animals from a colony selected by the Medical Research Council (CFY strain). Treatment and experimental methods
Rats were maintained on a commercial pellet diet and water ad lib. and were exposed to 12 h of light per 24 h. They were gonadectomized at 4 weeks, 8 weeks or 15 weeks of age. For histo-
Downloaded from http://ageing.oxfordjournals.org/ at University of California, San Diego on April 10, 2016
Summary
Factors Controlling Growth and Age Involution of the Rat Thymus
13
logical studies, 0.1 nag of colchicine/100 g body weight was administered intraperitoneally 4 h prior to death. Thymic tissue was fixed' in Carnoy, dehydrated, embedded in wax and subsequently stained wilt methyl green-pyronin. RESULTS
300
200
• Normal male • Normal female A Male castrated at 4 weeks T Female ovarx at 4 weeks
100
10
20
30
600
-400 as
O200 to
300
200
100
30 40 Age in weeks
Fig. 1. The effect of gonadectomy on the growth curves of three strains of rat.
Downloaded from http://ageing.oxfordjournals.org/ at University of California, San Diego on April 10, 2016
Effects of gonadectomy on body weight All three strains showed a sexual dimorphism in body weight, males being between 50-100 per cent larger than females at 30 weeks of age. There were, however, differences in the extent of sexual dimorphism between strains as well as differences in the patterns of growth (Fig. 1).
14
D. Bellamy, S. M. HinsuU and J. G. Phillips
Effects of gonadectomy on growth and involution of the thymus There was no significant sex difference between the effects of gonadectomy carried out one or four weeks post partum (Fig. 2ab). The initial growth rate of the thymus in CFY males was about 80 per cent greater than in females. At six weeks of age, there was a sudden loss of weight in both sexes which appeared to proceed in two phases. There was a sharp decline in mass up to about 20 weeks, followed by a slower loss up to 80 weeks. The average rate of weight loss between six and 30 weeks was much greater for males, and this resulted in the male thymus declining to the female weight at about 20 weeks. Thereafter, the thymus lost weight at about the same rate in both sexes (Fig. lab). Gonadectomy at four weeks of age resulted in a prolongation of thymic growth and increased the maximum thymus weight by about 22 per cent in CFY animals. Sudden weight loss was again observed, beginning at nine weeks, which occurred at about the same rate as in normal animals, and proceeded again in two phases. The first phase of rapid weight loss ended at about 40 weeks, when the thymus weights were comparable with control values. Thereafter, gonadectomy did not affect weight loss (Fig. la). The action of gonadectomy in females was to accelerate thymic growth, increasing the maximum thymus weight by about 50 per cent. Weight loss began in the ninth week, as in the gonadectomized males, at about the same organ size. The pattern of weight loss was the same as in the male, with no effect of gonadectomy noticeable after 35 weeks (Fig. 2b). In animals of the Sheffield strain, thymic growth rate was equal to that in CFY
Downloaded from http://ageing.oxfordjournals.org/ at University of California, San Diego on April 10, 2016
In the Sheffield strain, growth rates were similar in both sexes up to about 10 weeks of age, at which time the female weight gain declined. Male growth continued at a high level for another 10 weeks. At 30 weeks, males were about 50 per cent larger than females (Fig. la). Gonadectomy at four weeks reduced the growth rates of males, an effect that was first noticeable at 10 weeks. The operation increased the growth of females, again the effect was first noticeable at 10 weeks of age. In gonadectomized females, the drop in growth rate that appeared in normal animals at 10 weeks was not so marked, although the body weight at 30 weeks was only slightly higher than that of the controls. Over all, the effect of gonadectomy was to reduce sexual dimorphism in body weight by about 50 per cent (Fig. la). Gonadectomy of Sheffield rats at eight and 15 weeks of age again resulted in a decrease in body weight in males and an increase in female weight but no significant difference was observed between the body weights at comparable age groups of animals gonadectomized at four, eight or 15 weeks of age. Despite having growth rates that were about twice as high as in Sheffield rats, CFY animals showed the same pattern of growth and effect of gonadectomy that were observed in the former strain. Sexual dimorphism in body weight was reduced as a result of gonadectomy by about 75 per cent (Fig. 16). The general growth pattern of the males of the PVG strain was different from that of males belonging to the other two strains. There was no significant increase in body weight from 20-65 weeks. Female PVG animals continued to grow, albeit slowly, during this interval, so narrowing the gap in body weight (Fig. lc).
Factors Controlling Growth and Age Involution of the Rat Thymus
15
animals up to about four weeks of age. At this time, in both sexes, the organ growth rate was markedly reduced. A maximum in size was reached at nine weeks, with the female thymus weighing slightly less than that of the male. Females were characterized by a two-phase loss of weight similar to that observed for both sexes in the CFY strain. In contrast, weight loss occurred in the male at a slower, steady rate. At 24 weeks of age, the rates of involution were the same in both sexes (Fig. 3). 1000
800
600 -
Male
400 -
200 -
800 -
eoo -
400 -
200 -
10
15
20 Age in weeks
25
30
Fig. 2. The effect of gonadectomy on thymus growth and involution in CFY rats.
Gonadectomy at four weeks resulted in a continuation in the initial rapid phase of growth beyond the time of marked growth inhibition in normal animals. The timing of the maxima in thymic weight was not altered in either sex, but the peaks in the weight graphs were sharper than in controls; the female maximum was lower than that in males. Involution was faster in males than in females up to 10 weeks of age. Thereafter, both sexes were characterized by the same rate of weight loss, occurring at the same mean thymus size (Fig. 3).
Downloaded from http://ageing.oxfordjournals.org/ at University of California, San Diego on April 10, 2016
Normal rats Gonadx.at 4 weeks Gonadx.at 1 week
D. Bellamy, S. M. Hinsull and J. G. Phillips
16
Gonadectomy of male and female rats of the Sheffield strain at eight or 15 weeks of age resulted in stimulation of thymic growth during the next two weeks. Maximum thymic weights in males were increased by 37 per cent after gonadectomy at eight weeks, whereas gonadectomy at 15 weeks resulted in a 60 per cent increase in thymus weight compared with intact controls. Gonadectomy of females at eight or 15 weeks1 600 Gonaditat 4 weeks
Gonadnat 8 weeks
400
200
Male A Female • Gonadxmale D Gonadx.femal'
100
10
20
30
10
10
20 30 Age in weeks
20
30
Fig. 3. The effect of gonadectomy at four, eight and 15 weeks of age on the growth and involution of thymus in Sheffield rats. 400
Male Female
300
m $ 200 3
100
10
20
30 40 Age In weeks
50
60
Fig. 4. Thymus growth and involution in PVG rate.
of age resulted in weight increases of 108 per cent and 131 per cent respectively compared with control animals. The maximum thymus weights attained by male and female animals gonadectomized at 15 weeks of age were not significantly different from each other nor were they significantly different from the maximum thymus weights attained by male and female rats gonadectomized at eight weeks of age. Thus, involution of
Downloaded from http://ageing.oxfordjournals.org/ at University of California, San Diego on April 10, 2016
*. 300
Factors Controlling Growth and Age Involution of the Rat Thymus
17
the thymus, in all gonadectomized groups was delayed for about one month, when it proceeded at a faster rate than normal for about four weeks. In contrast to the other two strains, there was no significant sexual difference between the rates of thymic growth and involution in animals of the PVG strain. The riming of involution was identical to that in the other strains (Fig. 4).
1-4
1-2
Female gonadx. at 4 weeks Male gonadxat 4 weeks Normal male Normal female
£• o CD
2 §0-8
0-6
10
15
20 26 Age in weeks
30
35
40
Fig. 5. The effect of gonadectomy on mitotic indices of ShefEeld-Wistar rat thypnus. Effect of gonadectomy on mitotic index
The mitotic index dropped by about 50 per cent during the first five weeks of life, with no significant differences between sexes. Gonadectomy at five weeks increased the mean mitotic index slightly but this was only significantly different from control values in the females (Fig. 5). DISCUSSION
The three rat strains used in the present work differed greatly with regard to the rate of growth and the pattern of weight gain. No significant difference was observed in the prepubertal growth rate between male, female and gonadectomized animals. However, postpubertal sexual dimorphism was observed as a characteristic feature, with the males growing faster and reaching a higher body weight than the females. The magnitude of sex-linked weight differences differed between strains. There were also strain differences regarding the extent to which the sexual dimorphism was dependent upon the gonads. Generally, in males, sex hormones appeared to stimulate growth, whereas the opposite seemed to hold for females. The magnitude of the gonadal influence was strain-dependent. These findings on the effect of gonadal status in post-pubertal growth rates are in agreement with the work of Grunt (1964) and Kakolowski, Cox & Valenstein (1968).
Downloaded from http://ageing.oxfordjournals.org/ at University of California, San Diego on April 10, 2016
• o A •
18
D. Bellamy, S. M. HinsuU and J. G. Phillips
Downloaded from http://ageing.oxfordjournals.org/ at University of California, San Diego on April 10, 2016
There was a wide variation in the initial growth rate of the thymus between sexes and strains, which could not be correlated directly with differences in body weight gain. Thymus weight was increased between six and nine weeks of age despite a 50 per cent decrease in the mitotic index during this period. Therefore, there must have been a greater inhibition of cellular loss than mitosis. Involution began at approximately nine weeks of age, but was not accompanied by any change in the rate of decrease in the mitotic index. Previous work has shown that partial thymectomy had no significant influence on the mitotic index (Bellamy & Alkufaishi, 1972). This combined evidence suggests that mitotic jndex is independent of thymus size; also it is unlikely that thymus size is regulated solely through mitosis. In this respect the mass of thymic lymphocytes is possibly controlled mainly by cellular loss; involution being timed by a change in either cell migration (Fichtellius, 1960) or in cellular death (Metcalf, 1966). It has been postulated by Metcalf (1966) that degenerating lymphocytes induce the formation of new cells by the feed-back of breakdown products; however if this is so, during involution this equilibrium must be altered so that cell loss is greater than cell division. Gonadectomy at four weeks of age, depending on strain, results in either an increase in the thymic growth rate or a continuation of the initial growth rate for a longer period. Also the peak of thymus weight could be increased by the operation. Increased thymus weights immediately after gonadectomy at eight or 15 weeks of age demonstrated an influence of sex hormones on the thymus after sexual maturity, when the thymus was normally undergoing involution. The operation resulted in an increase in thymus growth in both sexes thus reversing the normal rate of involution. Maximum thymus weights attained in all four operated groups were not significantly different from each other irrespective of the age when the animals were gonadectomized or the sex of the rats. These findings indicate that sex steroids inhibit thymus growth, either by inhibiting cellular proliferation or by accelerating cellular loss. It is suggested that the degree of inhibition is greater in females than in males since, in intact animals, thymus weights are significantly lower in females than in males, but when this inhibition is released following gonadectomy, a 131 per cent increase in thymus weight was observed in females compared with a 60 per cent increase in thymus weight in males; however both groups attained comparable final thymus weights suggesting that thymic autonomy is maintained at different levels according to the hormonal status of the animal. Similarly, it may be postulated that suppression of thymus mass at 15 weeks of age is greater than at eight weeks of age. Gonadectomy resulted in a higher mitotic index in both male and female animals compared with intact controls, suggesting that the rate of mitosis is inhibited by sex hormones. Although the operation only had a small effect on mitotic index, the thymus is characterized by a high rate of cellular proliferation and this small stimulation of cell division may have been sufficient to produce a large net change in cell mass. The increase in the point of maximum cell mass was probably due to new relationships being established between the rate of mitosis and cellular death. As gonadectomy stimulates growth at all ages it appears that sex hormones have an immediate and direct action on the thymus. There was no clear evidence that the gonads influenced the rate of involution. Indeed, gonadectomy had no effect on the thymus after 35 weeks of age.
Factors Controlling Growth and Age Involution of the Rat Thymus
19
ACKNOWLEDGEMENTS
This project was supported by a research grant from the Tenovus Organization. The expert technical assistance of Miss A. Franklin, Mrs J. Phillips and Mr A. Stevenson is gratefully acknowledged. REFERENCES
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Downloaded from http://ageing.oxfordjournals.org/ at University of California, San Diego on April 10, 2016
In summary, the new picture that emerged from the present work is that the normal timing of age involution is set by the gonads. Growth of the thymus may be initiated by gonadectomy during the early stages of age involution, at a rate equal to the preinvolution rate. This indicates the working of a gonad-controlled clock which involves the reversible suppression of a basic process governing the initial size of the lymphocyte population. There appears to be an upper limit to the thymic cell population that is set independently of the gonads. When this new ceiling is reached after post-involution gonadectomy, the cell population is lowered for a second time, and at a faster rate, which appears to be a compensation to reduce an overpopulated gland. Thus, at least four processes govern the mass of thymic lymphocytes: one for rapid growth; a second for gonad-initiated involution; a third for non-gonad-initiated compensatory involution; and a fourth for the steady lowering of the weight ratio of thymus to body, which carries the thymus into old age. If any of these processes act by regulating mitosis of lymphocytes they do so through a very small percentage change in mitotic index which cannot be detected with the present methods.
