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The Growth of Chicken Lymphoid Organs, Testes, and Adrenals in Relation to the Oxidation State and Concentration of Adrenal and Lymphoid Organ Vitamin C1
VIBRATOR VERSUS NORMAL TURKEYS
ity and/or hatchability. Body weights of the "vibrator" and "normal" poults were not significantly different at either one-day or eight weeks of age. Mortality to eight weeks of age was found to be significantly higher for the "vibrators" than for the "normals." REFERENCES Coleman, T. H., R. K. Ringer, W. J. Mathey, K. G. Rood and C. W. Pope, 1960. Vibrator, a recessive sex-linked mutation in turkeys. J. He-
1463
redity, SI: 158-160. Dixon, W. J., and F. J. Massey, 1957. Introduction to Statistical Analysis. McGraw-Hill Book Co., Inc., New York. Gill, J., 1967. Personal communication. Michigan State University, East Lansing, Mich. Peterson, R. A., R. K. Ringer and T. H. Coleman, 1963. A study of the central nervous system of "vibrator" turkeys. Michigan Agric. Expt. Sta. Quart. Bull. 46: 119-123. Siegel, S., 1956. Non-Parametric Statistics, McGraw-Hill Book Co., Inc., New York.
The Growth of Chicken Lymphoid Organs, Testes, and Adrenals in Relation to the Oxidation State and Concentration of Adrenal and Lymphoid Organ Vitamin C 1 MICHAEL P. DIETER 2 AND R. P. BREITENBACH Department of Zoology, University of Missouri, Columbia, Missouri 65201 (Received for publication February 2, 1968)
T
HE bursa of Fabricius, the thymus, and the spleen are among the more rapidly developing organs in the bird. The growth rate of the bursa has been described in detail by Glick (1956), but those of the chicken's thymus and spleen have not been extensively studied. Because some of the necessary background information for lymphoid tissue growth rates in intact cockerels have never been obtained, the growth of the bursa, thymus, and spleen were followed until age involutions began. The changes in testicular, comb, and adrenal weights, in the growing cockerels were also followed since the steroids secreted by these endocrine organs (testosterone and corticosterone) are known to affect lymphoid organ growth (Dougherty, 19S2). 1 Supported in part by USPH grant Al-05643-01 and 02. 2 Present address: National Institutes of Health, NIAMD-LPB, Room B-1S, Bldg. 2, Bethesda, Md. 20014.
The concentration of vitamin C in the lymphoid organs and adrenals of the different age cockerels was also determined because of the possible participation of this vitamin in developmental processes (Rinaldi, 1951; Backstrom, 1956, 1957). By utilizing a relatively rapid method for determining tissue vitamin C (Hughes, 1956) we were able to obtain estimates for oxidized and reduced, as well as total vitamin C, in the developing lymphoid organs. This type of data may provide more information for the cause of steroid-mediated tissue depletion of vitamin C. The mechanism underlying the tissue depletion response is unknown, but it may depend in part on the particular form of vitamin C present in thes tissues. Martin (1961) has shown that the lipid soluble form of vitamin C, dehydroascorbic acid, traverses the cellular membrane more easily than ascorbic acid. The knowledge of the normal pattern of lymphoid organ growth, vitamin C concen-
1464
M. P. DIETER AND R. P. BREITENBACH
tration and oxidation state in lymphoid tissues at known developmental stages can be used to design hormone treatment experiments, or metabolic experiments in this strain of bird. Further investigations of this nature are being conducted to evaluate the importance of lymphoid tissue vitamin C during organogenesis. MATERIALS AND METHODS One day old Single Comb White Leghorn cockerels of the strain developed by the Poultry Husbandry Department of the University of Missouri, Columbia, Mo. were used in these experiments. They were maintained on a 12:12 light cycle in an air-conditoned animal room (23°C.) and were given Purina chick starter and water, ad libitum. Cage density and social grouping were controlled throughout in order to minimize the effects of social stress. Ten cockerels of 1, 3, 5, 7, 9, 11 and IS weeks of age were utilized to determine the size and growth of the following organs in normal bi^ds: bursa, left thymus, spleen, adrenals, gonads, and comb. Vitamin C concentrations of the lymphoid organs and adrenals were determined. The analyses were performed immediately after necropsy, or the tissues were quick frozen for latter analysis. The birds were killed by administering an over-dose of sodium nembutal into the heart. Vitamin C was analyzed by the method of Hughes (1956). This technique allows one to determine the total, oxidized, and reduced vitamin C in each tissue sample. The sample size for the vitamin determinations varies from four to eighteen as indicated on the subsequent figures. The sample sizes vary for two reasons. First, organs from younger birds (bursa and spleen at one week, and adrenals at one and three weeks) had to be pooled in order to obtain an adequate amount of tissue for analysis. Second, sample sizes from five and seven
week old birds exceed ten because values of control birds from a second experiment were included, in order to obtain the best possible estimate of the true values for birds of those ages. Because of the great change in body weight gain during development, the bursa, thymus, spleen, adrenal, and testis weights are expressed on a percent body weight basis, as well as on an absolute basis. The adrenal and lymphoid organ vitamin C are expressed as mg./lOO gm. tissue weight, whereas the concentration of reduced and oxidized vitamin C are expressed as the ratio of ascorbic/dehydroascorbic acid (ASA/DHA) in mg./lOO gm. tissue weight. RESULTS The maximum rate of growth for the bursa was between three and five weeks while the maximum rate of growth for the thymus was between nine and eleven weeks of age (Fig. 1). Spleen weights increased progressively in one through eleven week old cockerels. The maximum size of all three lymphoid organs occurred in eleven week old cockerels, and by fifteen weeks LYMPHOID ORGAN GROWTH RATES • « gmwt. means+std. e r r o r s o o % wt n=10
1 3 5 7 9 11 15
1 3 5 7 9 11 15
1 3 5 7 9 11 15
AGE IN WEEKS
FIG. 1. Bursa, thymus, and spleen weights of cockerels at different ages. Vertical lines on the curves indicate standard error of the mean.
1465
LYMPHOID ORGAN DEVELOPMENT AND VITAMIN C
there was a marked and significant decrease of bursa and thymus weights when compared to those of eleven week old cockerels. However, when the sizes of these organs are examined on a relative basis (percent body weight), maximum bursa size was achieved between five and seven weeks (Fig. 1). The relative weights of the bursa decreased thereafter and reached a low by fifteen weeks. The thymus and spleen reached a relative maximum weight at five weeks and these weights were maintained until eleven weeks of age. By fifteen weeks the relative weights of the thymus and spleen were significantly lower than those in eleven week old cockerels. The absolute weights of the bursae during the involutionary phase of growth (between eleven and fifteen weeks) varied inversely with the testicular weights. The growth of the thymus and spleen showed much less relationship to testicular size. No relationship was found between the growth rate of the adrenals and those of the lymphoid organs. The growth of the testes and comb was linear from one to nine weeks of age (Fig. 2). An abrupt increase in testicular growth rates (greater than ten-fold increase) occurred between nine and eleven, and again between eleven and fifteen weeks. The slope of the comb growth flattened from nine to eleven weeks, but by fifteen weeks, a ten-fold increase in comb weights had occurred. The adrenals showed consistent growth from one to five weeks, but thereafter adrenal weight increase slowed perceptibly. Examination of the adrenals and testes on a relative basis indicated that adrenals of cockerels eleven weeks and younger were significantly larger than those at fifteen weeks of age; this age or maturation effect also occurred in the testes of young cockerels between one and three weeks of age
ABSOLUTE GROWTH RATES Means+Std. Errors N=10 1
T
1
1
1
1
r
n * ^ 0
i_
j
1
3
5
J
7
9
L
11
15
AGE IN WEEKS FIG. 2. Comb, testes, and adrenal weights of cockerels at different ages. Vertical lines on the curves indicate standard error of the mean.
(Fig. 3). A slow increase in relative testicular weights occurred between three and nine weeks. A greater than tenfold increase in relative testicular mass occurred between nine and eleven, and between eleven and fifteen weeks. The concentration of vitamin C in the lymphoid organs of younger cockerels ranged between 30 and 40 mg./lOO gm. tissue weight (Fig. 4). The concentration of vitamin C in the bursa remained at this level for five weeks, dropped significantly between five and seven weeks, and then increased in a linear fashion through fifteen weeks. The vitamin C concentration of the thymus had significantly decreased below the levels found in younger cockerels by five weeks. The vitamin concentration re-
1466
M. P. DIETER AND R. P. BREITENBACH R E L A T I V E GROWTH RATES means+std, errors n=10 ADRENALS TESTES .60 .11
iX
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1 3 5 7 9 11 15 1 3 5 7 9 1.1 15 AGE
IN WEEKS
FIG. 3. Expression of adrenal and testes weights as a percent of the body weight of cockerels at different ages. Vertical lines above the histograms indicate standard error of the mean.
mained at these low levels through eleven weeks, and then significantly increased between eleven and fifteen weeks. The concentration of vitamin C in the spleen also
fell as the cockerels matured. Significant decreases occurred between three and five weeks, and between five and seven or nine weeks. A significant increase in splenic vitamin C occurred between nine and eleven weeks. The depression of vitamin C concentration in the spleens at fifteen weeks was probably not a valid measure of the vitamin. The splenic filtrates obtained from fifteen week old birds were not clear, and therefore the values obtained are questionable. The oxidation state of vitamin C in the lymphoid organs varied markedly during organogenesis (Fig. 5). There was a linear increase in the ASA/DHA ratio between one and five weeks in the bursa and thymus. The ASA/DHA ratio decreased sharply from five to nine weeks in these organs and reached a level of 3.0 to 4.0 by fifteen weeks. The splenic ASA/DHA ratios were lower throughout the measured time span than those of the bursa or thy-
LYMPHOIDAL VITAMIN C means+std. e r r o r s * sample number RATIOS OF REDUCED TO OXIDIZED VITAMIN C means+std. errors * sample number BURSA
1 3 5 7 9 11 15
THYMUS
1 3 5 7 9 11 15
1 3 5 7 9 11 15
AGE IN WEEKS
FIG. 4. The concentration of bursal, thymic, and splenic vitamin C (ascorbic + dehydroascrobic acid) of cockerels at different ages. Vertical lines on the curves indicate standard error of the mean and the numbers beside them the sample size at each age.
1 3 5 7 9 11 15
1 3 5 7 9 11 15
1 3 5 7 9 11 15
AGE IN WEEKS
FIG. 5. The ratio of reduced to oxidized vitamin C (ASA/DHA) in the bursa, thymus, and spleen of cockerels at different ages. Vertical lines on the curves indicate standard error of the mean and the numbers beside them the sample size at each age.
LYMPHOID ORGAN DEVELOPMENT AND VITAMIN C
mus (more of the splenic vitamin was in the oxidized state). A moderate decline in the ratio of splenic ASA/DHA occurred from one through seven weeks; the low ratios persisted through eleven weeks. The increase depicted at fifteen weeks is probably incorrect. Unlike the aliquots used in the other determinations, these were darkly colored after the initial filtration procedure. Thus the transmittance values obtained were not accurate for estimating the concentration of vitamin C in the spleens of fifteen week old cockerels. From one to seven weeks the concentration of adrenal vitamin C was about 100% greater than in adrenals of nine to fifteen week old cockerels (Fig. 6). The levels were about 200 mg./lOO gm. adrenal tissue in 1, 3, 5, and 7 week old cockerels; at 9, 11, and 15 weeks the adrenal vitamin C concentration was approximately 100 mg./lOO gm. adrenal tissue. In view of the suggested relationship between vitamin C in the bursa and adrenal (Perek and Eilat, ADRENAL VITAMIN C DURING EARLY A G E Means+Std. Errors *Sample Number
200 O O O
\ CD 2
100
3 5 7 9 11 A G E IN W E E K S
15
FIG. 6. The concentration of adrenal vitamin C (ascorbic + dehydroascorbic acid) of cockerels at different ages. Vertical lines on the curve indicate standard error of the mean and the numbers beside them the sample size at each age.
1467
1960), it is interesting to note that bursal vitamin C fell between five and seven weeks (Fig. 3), and then rose sharply as the adrenal vitamin C concentration fell. The ASA/DHA ratio in adrenals of different age cockerels showed no consistent changes and were not included. DISCUSSION
The maximum weight of the lymphoid organs (11 weeks) was attained shortly after the testicular weights showed a sharp increase (9 weeks); adrenal weight increases had reached a plateau during this period. The beginning of age involution (between 11 and IS weeks) coincided with a further logarithmic increase in testicular mass. The gravimetric changes in the adrenals, testes, and comb suggest that endogenous testosterone levels increase just before the beginning of age involution and may act in conjunction with basal levels of corticosterone secretion. It is possible that corticosteroids may affect the lymphoid tissue of younger birds so that a later testosterone-induced involutionary response may occur (permissive effect of corticoids), or the critical circulating levels of corticoid/androgen in older birds may initiate a sequence of events causing involution (synergistic effect of corticoids and androgens). Dorfman and Dorfman (1962) demonstrated that testosterone would intensify or potentiate the thymolytic effect of corticoids when injected into ovariectomized, adrenalectomized mice. Further evidence for these hypotheses is based on the age-dependent dichotomy in the secretory rate of avian adrenals and testes. This gravimetric data and especially the avian adrenocortical secretory rate studies of Nagra et al. (1960, 1963), deRoos (1961), Breitenbach (1962), Greenman (1962), show that young chickens secrete adrenal corticosterone at a rate comparable to that of older chickens. Unfortunately, only a
1468
M. P. DIETER AND R. P. BEEITENBACH
few quantitative measurements of testoste- tion occurred after five weeks. Perhaps the rone secretion have been made in the bird changes in vitamin C concentration and in (Connell et al., 1965). However, the avian the ratio of ASA/DHA in the developing testicular secretion rate, as judged by comb/ lymphoid organs reflect intracellular testes correlations with age, continues to changes in the state of lymphoid organ inincrease through the period of lymphoid termediary metabolism. However, other reorgan involution (see Figure 2 and 3; sults (Backstrom, 1956, 1957; Price, Nalbandov, 1964). Until further research 1966) indicate vitamin C, rather than has been conducted, neither hypothesis being an inert storage product in the lym(permissive effect of corticoids vs. syner- phoid organs, may be of physiological imgistic effect of corticoids and androgens) portance during organogenesis. can be disproved. On the basis of endocrine SUMMARY gland growth and secretion rates, maturation of the avian hypothalamic-hypophysMaximum weights of the bursa, thymus, eal-adrenal control system appears to occur and spleen in White Leghorn cockerels much earlier than maturation of the gonadal were attained at eleven weeks of age; by control system. fifteen weeks, age involution had begun. The marked differences in the tissue con- Testicular and comb weights increased centrations of vitamin C in various age maximally after nine weeks, but no further cockerels also suggests hormonal involve- increase in adrenal weights occurred during ment. Previous investigators have demon- this period. The relationship between the strated that depletion of bursal ascorbic changing mass of lymphoid organs and that acid (Perek and Eilat, 1960) or splenic as- of the gonads and testes suggests that corcorbic acid (Lahiri and Lloyd, 1962), oc- ticoids and androgens may act synergisticurred in chicks or rats following ACTH cally to induce lymphoid organ involution. administration. Our results showed that viMarket fluctuations in the concentration tamin depletion in the lymphoid organs oc- and oxidation state of tissue vitamin C occurred as adrenal growth slowed, but prior curred during development. By five weeks to the increase in testes or comb weights. of age, the concentration of vitamin C in Adrenal vitamin C depletion occurred even the thymus and spleen had decreased thirty later than the depletion of lymphoid organ to fifty percent below earlier levels. By vitamin C—adrenal vitamin C depletion seven weeks a similar response had ocmay have been due to ACTH release, since curred in the bursa. Thereafter the concenagonistic behavior is probably heightened tration of vitamin C in the bursa rose with the onset of pubertal development. steadily, but in the thymus and spleen it The evidence accumulated for decreased. The ratio of reduced to oxidized ASA/DHA ratios did not indicate tissue vi- vitamin C in the bursa and thymus intamin C depletion occurred solely because creased sharply through five weeks, and of oxidation of ascorbic acid and loss of then fell abruptly so that fifteen to twenlipid soluble dehydroascorbic acid. The ty-five percent of the vitamin C was in the ASA/DHA ratios showed similar changes oxidized state by fifteen weeks. The ratio in the bursa, and in the thymus or spleen, of reduced to oxidized vitamin C was conbut the concentration of bursal vitamin C tinually lower in the spleen than in the increased in older cockerels; the oxidation bursa or thymus; the spleens of older cockstate of adrenal vitamin C showed no con- erels (seven to eleven weeks) contained sistent changes but adrenal vitamin deple- more oxidized than reduced vitamin C.
LYMPHOID ORGAN DEVELOPMENT AND VITAMIN C
The concentration of adrenal vitamin C was fifty percent lower in cockerels seven weeks of age and older than in younger cockerels. No consistent changes occurred in the oxidation state of adrenal vitamin C. The loss of tissue vitamin C was not solely related to the state of vitamin oxidation. The marked changes in the tissue concentration and oxidation state of vitamin C during lymphoid organogenesis suggests that it has a dynamic function for lymphoidal vitamin C. REFERENCES Backstrom, S., 1956. The total content of ascorbic acid in the developing sea urchin egg. Exptl. Cell. Res. 1 1 : 322-326. Backstrom, S., 1957. Content and distribution of ascorbic acid in sea urchin embryos of different developmental trends. Exptl. Cell Res. 13: 333-340. Breitenbach, R. P., 1962. The effect of ACTH on adrenocortical secretion and ascorbic acid depletion in normal and testosterone treated cockerels. Poultry Sci. 4 1 : 1318-1324. Connell, G. M., C. H. Connell and K. B. Eik-Nes, 1965. Testosterone biosynthesis by the twoday-old chick testes in vitro. Amer. Zool. 5 : 218. deRoos, R., 1961. The corticoids of the avian adrenal gland. Gen. Comp. Endocrinol. 1: 494-512. Dorfman, R. I., and A. S. Dorfman, 1962. Thymolytic activity of corticoids in combination with other steroids. Endocrinology, 7 1 : 271-276.
1469
Dougherty, T. F., 1952. Effect of hormones on lymphatic tissue. Physiol. Rev. 32: 379-401. Glick, B., 1956. Normal growth of the bursa of Fabricius in chickens. Poultry Sci. 35:844-851. Greenman, D. L., 1962. The avian pituitary-adrenal axis. Ph.D. Dissertation, Purdue University, Lafayette, Indiana, 132 pp. Hughes, R. E., 1956. The use of homocystein in the estimation of dehydroascorbic acid. Biochem. J. 64: 203-208. Lahiri, S., and B. B. Lloyd, 1962. The effect of stress and corticotrophin on the concentrations of vitamin C in blood and tissues of the rat. Biochem. J. 84:478-483. Martin, G. R., 1961. Studies on the tissue distribution of ascorbic acid. Ann. New York Acad. Sci. 92: 141-147. Nagra, C. L., G. J. Baum and R. K. Meyer. 1960. Corticosterone levels in adrenal effluent blood of some gallinaceous birds. Proc. Soc. Exptl. Biol. Med. 105: 68-70. Nagra, C. L., J. G. Birnie, G. J. Baum and R. K. Meyer. 1963. The role of the pituitary in regulating steroid secretion by the avian adrenal. Gen. Comp. Endocrinol. 3 : 274-280. Nalbandov, A. V., 1964. Reproductive Physiology: Comparative Physiology of Domestic Animals, Laboratory Animals, and Man. Second Edition, W. H. Freeman & Co., San Francisco & London. 316 pp. Perek, M., and A. Eilat, 1960. The bursa of Fabricius and adrenal ascorbic acid depletion following ACTH injections in chicks. J. Endocrinology, 20: 251-255. Price, C. E., 1966. Ascorbate stimulation of RNA synthesis. Nature, 212: 1481. Rinaldi, L. M., 1951. Report 15. Strangeways Research Lab., Cambridge.
NEWS AND NOTES (continued from page 144?) C. F. Brodersen, formerly in New York and Detroit as an independent consultant to medical centers in public information and fund-raising efforts, has been appointed Director of Public Information at the American Veterinary Medical Association. Mr. Brodersen majored in the life sciences as a post-war student at the Massachusetts Institute of Technology, where he received bachelor's and master's degrees. During World War II he served in the Pacific
Theater in the Army Ordnance Department and was discharged as a Captain in 1946. Soon after graduation from M.I.T., he became Assistant Director for Penicillin and Streptomycin Development at Schenley Laboratories. Later he was Manager of Vitamin Products for the International Division of Merck & Co. for whom he travelled extensively in Peru, Chile, Argentina, and Brazil. He also introduced flour enrichment in Chile in 1951, the first such program in South America.
(continued on page 1487)
ity and/or hatchability. Body weights of the "vibrator" and "normal" poults were not significantly different at either one-day or eight weeks of age. Mortality to eight weeks of age was found to be significantly higher for the "vibrators" than for the "normals." REFERENCES Coleman, T. H., R. K. Ringer, W. J. Mathey, K. G. Rood and C. W. Pope, 1960. Vibrator, a recessive sex-linked mutation in turkeys. J. He-
1463
redity, SI: 158-160. Dixon, W. J., and F. J. Massey, 1957. Introduction to Statistical Analysis. McGraw-Hill Book Co., Inc., New York. Gill, J., 1967. Personal communication. Michigan State University, East Lansing, Mich. Peterson, R. A., R. K. Ringer and T. H. Coleman, 1963. A study of the central nervous system of "vibrator" turkeys. Michigan Agric. Expt. Sta. Quart. Bull. 46: 119-123. Siegel, S., 1956. Non-Parametric Statistics, McGraw-Hill Book Co., Inc., New York.
The Growth of Chicken Lymphoid Organs, Testes, and Adrenals in Relation to the Oxidation State and Concentration of Adrenal and Lymphoid Organ Vitamin C 1 MICHAEL P. DIETER 2 AND R. P. BREITENBACH Department of Zoology, University of Missouri, Columbia, Missouri 65201 (Received for publication February 2, 1968)
T
HE bursa of Fabricius, the thymus, and the spleen are among the more rapidly developing organs in the bird. The growth rate of the bursa has been described in detail by Glick (1956), but those of the chicken's thymus and spleen have not been extensively studied. Because some of the necessary background information for lymphoid tissue growth rates in intact cockerels have never been obtained, the growth of the bursa, thymus, and spleen were followed until age involutions began. The changes in testicular, comb, and adrenal weights, in the growing cockerels were also followed since the steroids secreted by these endocrine organs (testosterone and corticosterone) are known to affect lymphoid organ growth (Dougherty, 19S2). 1 Supported in part by USPH grant Al-05643-01 and 02. 2 Present address: National Institutes of Health, NIAMD-LPB, Room B-1S, Bldg. 2, Bethesda, Md. 20014.
The concentration of vitamin C in the lymphoid organs and adrenals of the different age cockerels was also determined because of the possible participation of this vitamin in developmental processes (Rinaldi, 1951; Backstrom, 1956, 1957). By utilizing a relatively rapid method for determining tissue vitamin C (Hughes, 1956) we were able to obtain estimates for oxidized and reduced, as well as total vitamin C, in the developing lymphoid organs. This type of data may provide more information for the cause of steroid-mediated tissue depletion of vitamin C. The mechanism underlying the tissue depletion response is unknown, but it may depend in part on the particular form of vitamin C present in thes tissues. Martin (1961) has shown that the lipid soluble form of vitamin C, dehydroascorbic acid, traverses the cellular membrane more easily than ascorbic acid. The knowledge of the normal pattern of lymphoid organ growth, vitamin C concen-
1464
M. P. DIETER AND R. P. BREITENBACH
tration and oxidation state in lymphoid tissues at known developmental stages can be used to design hormone treatment experiments, or metabolic experiments in this strain of bird. Further investigations of this nature are being conducted to evaluate the importance of lymphoid tissue vitamin C during organogenesis. MATERIALS AND METHODS One day old Single Comb White Leghorn cockerels of the strain developed by the Poultry Husbandry Department of the University of Missouri, Columbia, Mo. were used in these experiments. They were maintained on a 12:12 light cycle in an air-conditoned animal room (23°C.) and were given Purina chick starter and water, ad libitum. Cage density and social grouping were controlled throughout in order to minimize the effects of social stress. Ten cockerels of 1, 3, 5, 7, 9, 11 and IS weeks of age were utilized to determine the size and growth of the following organs in normal bi^ds: bursa, left thymus, spleen, adrenals, gonads, and comb. Vitamin C concentrations of the lymphoid organs and adrenals were determined. The analyses were performed immediately after necropsy, or the tissues were quick frozen for latter analysis. The birds were killed by administering an over-dose of sodium nembutal into the heart. Vitamin C was analyzed by the method of Hughes (1956). This technique allows one to determine the total, oxidized, and reduced vitamin C in each tissue sample. The sample size for the vitamin determinations varies from four to eighteen as indicated on the subsequent figures. The sample sizes vary for two reasons. First, organs from younger birds (bursa and spleen at one week, and adrenals at one and three weeks) had to be pooled in order to obtain an adequate amount of tissue for analysis. Second, sample sizes from five and seven
week old birds exceed ten because values of control birds from a second experiment were included, in order to obtain the best possible estimate of the true values for birds of those ages. Because of the great change in body weight gain during development, the bursa, thymus, spleen, adrenal, and testis weights are expressed on a percent body weight basis, as well as on an absolute basis. The adrenal and lymphoid organ vitamin C are expressed as mg./lOO gm. tissue weight, whereas the concentration of reduced and oxidized vitamin C are expressed as the ratio of ascorbic/dehydroascorbic acid (ASA/DHA) in mg./lOO gm. tissue weight. RESULTS The maximum rate of growth for the bursa was between three and five weeks while the maximum rate of growth for the thymus was between nine and eleven weeks of age (Fig. 1). Spleen weights increased progressively in one through eleven week old cockerels. The maximum size of all three lymphoid organs occurred in eleven week old cockerels, and by fifteen weeks LYMPHOID ORGAN GROWTH RATES • « gmwt. means+std. e r r o r s o o % wt n=10
1 3 5 7 9 11 15
1 3 5 7 9 11 15
1 3 5 7 9 11 15
AGE IN WEEKS
FIG. 1. Bursa, thymus, and spleen weights of cockerels at different ages. Vertical lines on the curves indicate standard error of the mean.
1465
LYMPHOID ORGAN DEVELOPMENT AND VITAMIN C
there was a marked and significant decrease of bursa and thymus weights when compared to those of eleven week old cockerels. However, when the sizes of these organs are examined on a relative basis (percent body weight), maximum bursa size was achieved between five and seven weeks (Fig. 1). The relative weights of the bursa decreased thereafter and reached a low by fifteen weeks. The thymus and spleen reached a relative maximum weight at five weeks and these weights were maintained until eleven weeks of age. By fifteen weeks the relative weights of the thymus and spleen were significantly lower than those in eleven week old cockerels. The absolute weights of the bursae during the involutionary phase of growth (between eleven and fifteen weeks) varied inversely with the testicular weights. The growth of the thymus and spleen showed much less relationship to testicular size. No relationship was found between the growth rate of the adrenals and those of the lymphoid organs. The growth of the testes and comb was linear from one to nine weeks of age (Fig. 2). An abrupt increase in testicular growth rates (greater than ten-fold increase) occurred between nine and eleven, and again between eleven and fifteen weeks. The slope of the comb growth flattened from nine to eleven weeks, but by fifteen weeks, a ten-fold increase in comb weights had occurred. The adrenals showed consistent growth from one to five weeks, but thereafter adrenal weight increase slowed perceptibly. Examination of the adrenals and testes on a relative basis indicated that adrenals of cockerels eleven weeks and younger were significantly larger than those at fifteen weeks of age; this age or maturation effect also occurred in the testes of young cockerels between one and three weeks of age
ABSOLUTE GROWTH RATES Means+Std. Errors N=10 1
T
1
1
1
1
r
n * ^ 0
i_
j
1
3
5
J
7
9
L
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15
AGE IN WEEKS FIG. 2. Comb, testes, and adrenal weights of cockerels at different ages. Vertical lines on the curves indicate standard error of the mean.
(Fig. 3). A slow increase in relative testicular weights occurred between three and nine weeks. A greater than tenfold increase in relative testicular mass occurred between nine and eleven, and between eleven and fifteen weeks. The concentration of vitamin C in the lymphoid organs of younger cockerels ranged between 30 and 40 mg./lOO gm. tissue weight (Fig. 4). The concentration of vitamin C in the bursa remained at this level for five weeks, dropped significantly between five and seven weeks, and then increased in a linear fashion through fifteen weeks. The vitamin C concentration of the thymus had significantly decreased below the levels found in younger cockerels by five weeks. The vitamin concentration re-
1466
M. P. DIETER AND R. P. BREITENBACH R E L A T I V E GROWTH RATES means+std, errors n=10 ADRENALS TESTES .60 .11
iX
n
W .06 UJ
^
.04
X
> Q
O .02 CD
S2
0
nnnnnnn
n
i=.
•v.
:» LJ II
i/i
1 3 5 7 9 11 15 1 3 5 7 9 1.1 15 AGE
IN WEEKS
FIG. 3. Expression of adrenal and testes weights as a percent of the body weight of cockerels at different ages. Vertical lines above the histograms indicate standard error of the mean.
mained at these low levels through eleven weeks, and then significantly increased between eleven and fifteen weeks. The concentration of vitamin C in the spleen also
fell as the cockerels matured. Significant decreases occurred between three and five weeks, and between five and seven or nine weeks. A significant increase in splenic vitamin C occurred between nine and eleven weeks. The depression of vitamin C concentration in the spleens at fifteen weeks was probably not a valid measure of the vitamin. The splenic filtrates obtained from fifteen week old birds were not clear, and therefore the values obtained are questionable. The oxidation state of vitamin C in the lymphoid organs varied markedly during organogenesis (Fig. 5). There was a linear increase in the ASA/DHA ratio between one and five weeks in the bursa and thymus. The ASA/DHA ratio decreased sharply from five to nine weeks in these organs and reached a level of 3.0 to 4.0 by fifteen weeks. The splenic ASA/DHA ratios were lower throughout the measured time span than those of the bursa or thy-
LYMPHOIDAL VITAMIN C means+std. e r r o r s * sample number RATIOS OF REDUCED TO OXIDIZED VITAMIN C means+std. errors * sample number BURSA
1 3 5 7 9 11 15
THYMUS
1 3 5 7 9 11 15
1 3 5 7 9 11 15
AGE IN WEEKS
FIG. 4. The concentration of bursal, thymic, and splenic vitamin C (ascorbic + dehydroascrobic acid) of cockerels at different ages. Vertical lines on the curves indicate standard error of the mean and the numbers beside them the sample size at each age.
1 3 5 7 9 11 15
1 3 5 7 9 11 15
1 3 5 7 9 11 15
AGE IN WEEKS
FIG. 5. The ratio of reduced to oxidized vitamin C (ASA/DHA) in the bursa, thymus, and spleen of cockerels at different ages. Vertical lines on the curves indicate standard error of the mean and the numbers beside them the sample size at each age.
LYMPHOID ORGAN DEVELOPMENT AND VITAMIN C
mus (more of the splenic vitamin was in the oxidized state). A moderate decline in the ratio of splenic ASA/DHA occurred from one through seven weeks; the low ratios persisted through eleven weeks. The increase depicted at fifteen weeks is probably incorrect. Unlike the aliquots used in the other determinations, these were darkly colored after the initial filtration procedure. Thus the transmittance values obtained were not accurate for estimating the concentration of vitamin C in the spleens of fifteen week old cockerels. From one to seven weeks the concentration of adrenal vitamin C was about 100% greater than in adrenals of nine to fifteen week old cockerels (Fig. 6). The levels were about 200 mg./lOO gm. adrenal tissue in 1, 3, 5, and 7 week old cockerels; at 9, 11, and 15 weeks the adrenal vitamin C concentration was approximately 100 mg./lOO gm. adrenal tissue. In view of the suggested relationship between vitamin C in the bursa and adrenal (Perek and Eilat, ADRENAL VITAMIN C DURING EARLY A G E Means+Std. Errors *Sample Number
200 O O O
\ CD 2
100
3 5 7 9 11 A G E IN W E E K S
15
FIG. 6. The concentration of adrenal vitamin C (ascorbic + dehydroascorbic acid) of cockerels at different ages. Vertical lines on the curve indicate standard error of the mean and the numbers beside them the sample size at each age.
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1960), it is interesting to note that bursal vitamin C fell between five and seven weeks (Fig. 3), and then rose sharply as the adrenal vitamin C concentration fell. The ASA/DHA ratio in adrenals of different age cockerels showed no consistent changes and were not included. DISCUSSION
The maximum weight of the lymphoid organs (11 weeks) was attained shortly after the testicular weights showed a sharp increase (9 weeks); adrenal weight increases had reached a plateau during this period. The beginning of age involution (between 11 and IS weeks) coincided with a further logarithmic increase in testicular mass. The gravimetric changes in the adrenals, testes, and comb suggest that endogenous testosterone levels increase just before the beginning of age involution and may act in conjunction with basal levels of corticosterone secretion. It is possible that corticosteroids may affect the lymphoid tissue of younger birds so that a later testosterone-induced involutionary response may occur (permissive effect of corticoids), or the critical circulating levels of corticoid/androgen in older birds may initiate a sequence of events causing involution (synergistic effect of corticoids and androgens). Dorfman and Dorfman (1962) demonstrated that testosterone would intensify or potentiate the thymolytic effect of corticoids when injected into ovariectomized, adrenalectomized mice. Further evidence for these hypotheses is based on the age-dependent dichotomy in the secretory rate of avian adrenals and testes. This gravimetric data and especially the avian adrenocortical secretory rate studies of Nagra et al. (1960, 1963), deRoos (1961), Breitenbach (1962), Greenman (1962), show that young chickens secrete adrenal corticosterone at a rate comparable to that of older chickens. Unfortunately, only a
1468
M. P. DIETER AND R. P. BEEITENBACH
few quantitative measurements of testoste- tion occurred after five weeks. Perhaps the rone secretion have been made in the bird changes in vitamin C concentration and in (Connell et al., 1965). However, the avian the ratio of ASA/DHA in the developing testicular secretion rate, as judged by comb/ lymphoid organs reflect intracellular testes correlations with age, continues to changes in the state of lymphoid organ inincrease through the period of lymphoid termediary metabolism. However, other reorgan involution (see Figure 2 and 3; sults (Backstrom, 1956, 1957; Price, Nalbandov, 1964). Until further research 1966) indicate vitamin C, rather than has been conducted, neither hypothesis being an inert storage product in the lym(permissive effect of corticoids vs. syner- phoid organs, may be of physiological imgistic effect of corticoids and androgens) portance during organogenesis. can be disproved. On the basis of endocrine SUMMARY gland growth and secretion rates, maturation of the avian hypothalamic-hypophysMaximum weights of the bursa, thymus, eal-adrenal control system appears to occur and spleen in White Leghorn cockerels much earlier than maturation of the gonadal were attained at eleven weeks of age; by control system. fifteen weeks, age involution had begun. The marked differences in the tissue con- Testicular and comb weights increased centrations of vitamin C in various age maximally after nine weeks, but no further cockerels also suggests hormonal involve- increase in adrenal weights occurred during ment. Previous investigators have demon- this period. The relationship between the strated that depletion of bursal ascorbic changing mass of lymphoid organs and that acid (Perek and Eilat, 1960) or splenic as- of the gonads and testes suggests that corcorbic acid (Lahiri and Lloyd, 1962), oc- ticoids and androgens may act synergisticurred in chicks or rats following ACTH cally to induce lymphoid organ involution. administration. Our results showed that viMarket fluctuations in the concentration tamin depletion in the lymphoid organs oc- and oxidation state of tissue vitamin C occurred as adrenal growth slowed, but prior curred during development. By five weeks to the increase in testes or comb weights. of age, the concentration of vitamin C in Adrenal vitamin C depletion occurred even the thymus and spleen had decreased thirty later than the depletion of lymphoid organ to fifty percent below earlier levels. By vitamin C—adrenal vitamin C depletion seven weeks a similar response had ocmay have been due to ACTH release, since curred in the bursa. Thereafter the concenagonistic behavior is probably heightened tration of vitamin C in the bursa rose with the onset of pubertal development. steadily, but in the thymus and spleen it The evidence accumulated for decreased. The ratio of reduced to oxidized ASA/DHA ratios did not indicate tissue vi- vitamin C in the bursa and thymus intamin C depletion occurred solely because creased sharply through five weeks, and of oxidation of ascorbic acid and loss of then fell abruptly so that fifteen to twenlipid soluble dehydroascorbic acid. The ty-five percent of the vitamin C was in the ASA/DHA ratios showed similar changes oxidized state by fifteen weeks. The ratio in the bursa, and in the thymus or spleen, of reduced to oxidized vitamin C was conbut the concentration of bursal vitamin C tinually lower in the spleen than in the increased in older cockerels; the oxidation bursa or thymus; the spleens of older cockstate of adrenal vitamin C showed no con- erels (seven to eleven weeks) contained sistent changes but adrenal vitamin deple- more oxidized than reduced vitamin C.
LYMPHOID ORGAN DEVELOPMENT AND VITAMIN C
The concentration of adrenal vitamin C was fifty percent lower in cockerels seven weeks of age and older than in younger cockerels. No consistent changes occurred in the oxidation state of adrenal vitamin C. The loss of tissue vitamin C was not solely related to the state of vitamin oxidation. The marked changes in the tissue concentration and oxidation state of vitamin C during lymphoid organogenesis suggests that it has a dynamic function for lymphoidal vitamin C. REFERENCES Backstrom, S., 1956. The total content of ascorbic acid in the developing sea urchin egg. Exptl. Cell. Res. 1 1 : 322-326. Backstrom, S., 1957. Content and distribution of ascorbic acid in sea urchin embryos of different developmental trends. Exptl. Cell Res. 13: 333-340. Breitenbach, R. P., 1962. The effect of ACTH on adrenocortical secretion and ascorbic acid depletion in normal and testosterone treated cockerels. Poultry Sci. 4 1 : 1318-1324. Connell, G. M., C. H. Connell and K. B. Eik-Nes, 1965. Testosterone biosynthesis by the twoday-old chick testes in vitro. Amer. Zool. 5 : 218. deRoos, R., 1961. The corticoids of the avian adrenal gland. Gen. Comp. Endocrinol. 1: 494-512. Dorfman, R. I., and A. S. Dorfman, 1962. Thymolytic activity of corticoids in combination with other steroids. Endocrinology, 7 1 : 271-276.
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Dougherty, T. F., 1952. Effect of hormones on lymphatic tissue. Physiol. Rev. 32: 379-401. Glick, B., 1956. Normal growth of the bursa of Fabricius in chickens. Poultry Sci. 35:844-851. Greenman, D. L., 1962. The avian pituitary-adrenal axis. Ph.D. Dissertation, Purdue University, Lafayette, Indiana, 132 pp. Hughes, R. E., 1956. The use of homocystein in the estimation of dehydroascorbic acid. Biochem. J. 64: 203-208. Lahiri, S., and B. B. Lloyd, 1962. The effect of stress and corticotrophin on the concentrations of vitamin C in blood and tissues of the rat. Biochem. J. 84:478-483. Martin, G. R., 1961. Studies on the tissue distribution of ascorbic acid. Ann. New York Acad. Sci. 92: 141-147. Nagra, C. L., G. J. Baum and R. K. Meyer. 1960. Corticosterone levels in adrenal effluent blood of some gallinaceous birds. Proc. Soc. Exptl. Biol. Med. 105: 68-70. Nagra, C. L., J. G. Birnie, G. J. Baum and R. K. Meyer. 1963. The role of the pituitary in regulating steroid secretion by the avian adrenal. Gen. Comp. Endocrinol. 3 : 274-280. Nalbandov, A. V., 1964. Reproductive Physiology: Comparative Physiology of Domestic Animals, Laboratory Animals, and Man. Second Edition, W. H. Freeman & Co., San Francisco & London. 316 pp. Perek, M., and A. Eilat, 1960. The bursa of Fabricius and adrenal ascorbic acid depletion following ACTH injections in chicks. J. Endocrinology, 20: 251-255. Price, C. E., 1966. Ascorbate stimulation of RNA synthesis. Nature, 212: 1481. Rinaldi, L. M., 1951. Report 15. Strangeways Research Lab., Cambridge.
NEWS AND NOTES (continued from page 144?) C. F. Brodersen, formerly in New York and Detroit as an independent consultant to medical centers in public information and fund-raising efforts, has been appointed Director of Public Information at the American Veterinary Medical Association. Mr. Brodersen majored in the life sciences as a post-war student at the Massachusetts Institute of Technology, where he received bachelor's and master's degrees. During World War II he served in the Pacific
Theater in the Army Ordnance Department and was discharged as a Captain in 1946. Soon after graduation from M.I.T., he became Assistant Director for Penicillin and Streptomycin Development at Schenley Laboratories. Later he was Manager of Vitamin Products for the International Division of Merck & Co. for whom he travelled extensively in Peru, Chile, Argentina, and Brazil. He also introduced flour enrichment in Chile in 1951, the first such program in South America.
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