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Failure of hog thyrocalcitonin to elicit hypocalcemia in the teleost fish, Fundulus heteroclitus
Comp. Biochem. Physiol., 1967, Vol. 21, pp. 573 to 578. Pergamon Press Ltd. Printed in Great Britain
F A I L U R E OF H O G T H Y R O C A L C I T O N I N TO E L I C I T HYPOCALCEMIA IN T H E T E L E O S T FISH, FUNDULUS HETEROCLITUS P E T E R K. T . P A N G and G R A C E E. P I C K F O R D * The Bingham Laboratory, Department of Biology, Yale University, New Haven, Connecticut, U.S.A.
(Received 19 December 1966) Abstract--1. Hog thyrocalcitonin was administered to three physiologically different groups of male Fundulus heteroclitus at a dose of 0"2 mg (2-4 units) per g wt. of fish. Group 1 were intact fish maintained in salt water and fed; Group 2 were intact fish adapted to fresh water (low calcium) and starved for the final week; Group 3 were hypophysectomized fish maintained in salt water, and fed. 2. Within each group there were no significant differences in serumsoluble calcium between controls and hormone-treated fish. 3. Neither the time to autopsy (1-4 hr) nor the site of the injection (intraperitoneal or intravenous) had any effect. 4. Differences between the three groups could be related to the state of maturation of the testes. INTRODUCTION THE MAMMALIAN thyroid contains a hypocalcemic factor, thyrocalcitonin, that is released in response to high circulating levels of calcium (Foster et al., 1964; Hirsch et al., 1964; Milhaud et al., 1965 ; Talmage et al., 1965 ; G u d m u n d s s o n et al., 1966; M u n s o n et al., 1966). T h e relation of this substance to the parathyroid, with which it was associated by its discoverers (Copp & Cameron, 1961), remains controversial (Copp & Henze, 1964; Care et al., 1966). T o the best of our knowledge, the problem has not been studied in lower vertebrates. An attempt has therefore been made to evaluate the possible response of a fish to injections of mammalian thyrocalcitonin. MATERIALS AND METHODS Experiments were made on intact or hypophysectomized male Fundulus heteroclitus, maintained at ca. 20°C on an 8-hr day. A solution of partially purified hog thyrocalcitonin ( T H V - 6 0 , Munson) was prepared immediately before use by dissolving the required amount in 0.001 N HC1. T h e activity of this preparation assayed on young rats that had been given a calcium-free diet for 1-4 days, was stated by the donor (Dr. Paul L. Munson) to be about 10 units per 0.5-1 rag. W e * Supported by NSF Grant No. GB-0132. 573
574
PETER K. T. PANC AND GRACE E. PICXFORU
employed a high dose, 0.2 mg in 0.01 ml of solvent per g wt. of fish. In view of consistently negative results, lower doses were not investigated. The fish were anesthetized with tricaine methane sulfonate (MS 222, Sandoz). With one exception, noted below, the injections were made into the body cavity. At autopsy, free-flowing blood from lightly anesthetized fish was taken from the cut end of the tail in non-heparinized microhematocrit tubes and allowed to clot. Serum-soluble calcium was estimated on 5-/A samples by an ultramicro modification of the Neutral Fast Red method of Baar (1957). The determinations were made on the same day, without freezing of the serum, except for four samples (two controls and two hormone-treated fish in exp. 2) which were kept frozen at - 2 0 ° C for 24 hr. There was no apparent change that could be attributed to freezing. We employed Pedersen constriction pipettes, Linderstrem-Lang-type magnetic beads for stirring and a Beckman DU spectrophotometer with 50-/A Pyrocell quartz microcells. A standard reference serum (Labtrol, Dade) proved satisfactory and replicate determinations were in the acceptable range (10.28 + 0.10 S.E. mg per cent). EXPERIMENTS AND RESULTS
Experiment 1. Effect of varying the time after injection Eight intact fish, maintained in salt water and fed regularly except on the last day, were given intraperitoneal injections of THV-60 and autopsied after 1, 2 and 4 hr. A ninth fish was given an intravenous injection by way of the cardinal sinus in the posterior wall of the gill chamber. Two controls were not injected, and two received the solvent and were killed after 1 hr. These fish were at the end of the breeding season, but still with relatively large testes. The two control groups gave essentially identical results, and similarly the thyrocalcitonin-injected fish showed no differences that could be correlated with time. The data are therefore combined so that four controls can be compared with the nine fish that received hormonal treatment. The results (Table 1) show that, in this experiment, thyroealcitonin had no effect on the circulating level of serumsoluble calcium. The fish that received an intravenous hormonal injection gave a high reading (3.78 mequiv./1) but there were individuals both in the control and hormone-injected groups that were in this upper limit of the range (3.62 and 3.70 mequiv./l respectively).
Experiment 2. Effect of calcium deficiency It was thought that the negative results in the first experiment might be related to high calcium levels in the sea water and in the diet. A group of fish was therefore adapted to running fresh water wMch, in the New Haven area, is fairly low in calcium (ca. 9 ppm). The fish were maintained for 3 weeks in fresh water to ensure complete adaptation, and, as an additional precaution, they were not fed for 1 week before the experiment. Autopsies were made 2 hr after injection of either solvent or THV-60. The results (Table 1) show no difference between the
575
EFFECT OF THYROCALCITONIN ON F U N D U L U S H E T E R O C L I T U S
two groups, although the absolute levels for serum calcium were (unexpectedly) a little higher than in exp. 1. T h e s e fish had passed into complete sexual regression. TABLE 1 - - S E R U M
CALCIUM LEVELS I N
Fundulus heteroclitus RESULTING FROM
INJECTIONS OF
HOG THYROCALCITONIN
(THV-60, Munson)
Experiment 1
Conditions, date and gonosomatic index (GSI)* Intact, fed in salt water 2-3 Sept. 1966 GSI: 3"11 +0.38
Intact, starved, in fresh water 1 Oct. 1966 GSI: 0-60+0"11
3
Hypophysectomized, fed, in salt water 4-5 Sept. 1966 GSI : 0"34 + 0-03
Treatment None Solvent 1 hr
No. 2 2
Serum calcium (mequiv./l) Mean + S.E. Mean for 4 controls: 3-27 + 0-20
THV-60 1 hr 2hr 4 hr
4 3t 2
Mean for 9 THV-60 injected: 3"35+0-14
Solvent 2 hr
4
Mean : 4"65 + 0"08
THV-60 2 hr
4
Mean: 4"63 + 0"17
Solvent 2 hr
5
Mean: 5"60 + 0"43
THV-60 2hr
5
Mean: 5"18+0"47
* Gonosomatie index (GSI): Gonad weight x 100/body wt. t One fish in this group was given an intravenous injection.
Experiment 3. Effect of hypophysectomy Previous studies have suggested that there may be a disturbance of calcium metabolism in hypophysectomized F. heteroclitus, reflected in a high incidence of carbonate apatite renal calculi (Piekford, 1957). It was therefore decided to employ a group of long-term (7-month) hypophyseetomized fish that had completely recovered in the laboratory during 3.5 months after the termination of another experiment. T h e s e fish all showed the symptoms of complete hypophyseetomy: cessation of growth, pallor, anemia, regressed testes and a 50 per cent incidence of visible renal calculi. T h e y were maintained, of necessity, in salt water since hypophyseetomized fish of this species cannot survive in fresh water (Burden, 1956; Pickford et al., 1965), and were fed regularly until the last day.
576
PETER K . T . PANG AND GRACE E. PICKFORD
As in the two previous experiments, the results (Table 1) show no significant effect of thyrocalcitonin injection. The apparently slightly lower mean level for serum calcium in the THV-60 injected group, as compared with the controls, is not statistically significant. In both the controls and the hormone-injected groups, serum calcium levels were higher and more variable than in intact fish. Visible renal calculi were evident at autopsy in five fish, distributed in both groups, and no doubt sections would have revealed a higher incidence. However, there was no correlation between serum calcium levels and the incidence of visible renal calculi. DISCUSSION The findings reported in this investigation indicate that a partially purified preparation of hog thyrocalcitonin has no effect on serum-soluble calcium levels in the killifish, Fundulus heteroclitus. The hormone was administered to three groups of fish in different physiological conditions. Only males were employed since it is known that serum calcium levels are often high in breeding females of various teleostean species (Fleming & Meier, 1961a; Fleming et al., 1964; Booke, 1964; Phillips et al., 1964) whereas males are little affected or may even show a decline (van Someren, 1937). The first group, in sea water, was in a calcium-rich environment, yet the mean level for serum calcium was lower than in the second group maintained in fresh water under calcium-deficient conditions. There is evidence that teleosts can regulate the level of serum calcium in hypocalcic media, provided sufficient time is allowed for adaptation (Phillips et al., 1960; Montsk6 et al., 1963, 1964). In agreement with this, non-breeding males of F. heteroclitus appear to show no difference in mean serum calcium levels in fresh and in salt water (Pickford & Grant, unpublished). On the other hand, it is possible that the differences between the groups can be correlated with the state of sexual maturity. The fish in the first group were at the end of the breeding season but the testes were still large; the second group had passed into sexual regression. Preliminary observations on other experimental groups (Pickford & Pang, unpublished) suggest that in this species there is an inverse relation between serum calcium and the size of the male gonad. This hypothesis is in agreement with the data presented in Table 1. The effect of hypophysectomy on serum calcium levels in teleostean fishes has been investigated. Fontaine & Callamand (cited by Fontaine, 1956) found that in the eel hypophysectomy results in a decrease in serum calcium. On the other hand, in our experiments, serum calcium is higher in hypophysectomized than in intact males during sexual regression. There was no correlation, however, between the incidence of visible renal calculi and the levels of serum calcium. Further investigations are in progress. It is clear that the differences in the levels of serum calcium in the three groups which we investigated are not due to experimental defects, but can be related to the physiological condition of the animals.
EFFECT OF THYROCALCITONIN ON F U N D U L U S H E T E R O C L I T U S
577
T h e failure to respond to thyrocalcitonin can be explained in two ways: (1) the absence of a thyrocalcitonin m e c h a n i s m in fish, or (2) species specificity of the hormone. I t will be recalled that fishes lack a parathyroid gland and that the only demonstrable effects of exogenous p a r a h o r m o n e on s e r u m calcium levels are in spawning females, or estrogen-treated fish (Fleming & Meier, 1961a, 1961b; Clark & Fleming, 1963). T h e lack of a thyrocalcitonin m e c h a n i s m might be related to the absence of parahormone. I n answer to the second possibility, thyrocalcitonin preparations derived f r o m other sources, including extracts of the teleostean thyroid, m u s t be investigated. A m o n g the m a n y striking effects of radiation thyroidectomy in the rainbow trout, L a Roche et al. (1966) report a statistically insignificant lowering of serum calcium. T h i s would suggest that removal of the thyroid does not remove a hypocalcemic factor. Acknowledgements--We are indebted to Dr. Paul L. Munson, Department of Pharmacology, Medical School, the University of North Carolina, for the preparation employed, and for advice as to the solvent and suitable dosage levels.
REFERENCES BAA~ S. (1957) A micromethod for the estimation of serum calcium. Clin. chim. Acta 2, 567-575. BooKE H. E. (1964) Blood serum protein and calcium levels in yearling brook trout. Progr. Fish-Cult. 26, 107-110. BURDEN C. E. (1956) The failure of hypophysectomized Fundulus heteroclitus to survive in fresh water. Biol. Bull., Woods Hole 110, 8-28. CARE A. D., KEYNES W. M. & DUNCAN T. (1966) An investigation into the parathyroid origin of calcitonin..7. Endocrinol. 34, 299-318. CLARK N. B. & FLEMINGW. R. (1963) The effect of mammalian parathyroid hormone on bone histology and serum calcium levels in Fundulus kamae. Gen. comp. Endocrinol. 3, 461-467. COPP D. H . & CAMERON E. C. (1961) Demonstration of a hypocalcemic factor (calcitonin) in commercial parathyroid extract. Science, N . Y . 134, 2038. CoPP D. H. & HENZE K. G. (1964) Parathyroid origin of calcitonin: Evidence from perfusion of sheep glands. Endocrinology 75, 49-55. FLEMING W. R. & MEIER A. H. (1961a) The effect of mammalian parahormone on the serum calcium levels of Fundulus kansae and Fundulus catenatus. Comp. Biochem. Physiol. 2, 1-7. FLEMING W. R. & MEIER A. H. (1961b) Further studies of the effect of mammalian parathyroid extract on the serum calcium levels of two closely related teleosts. Comp. Biochem. Physiol. 3, 27-29. FLEMINGW. R., STANLEYJ. G. & MEIER A. H. (1964) Seasonal effects of external calcium, estradiol, and A C T H on the serum calcium and sodium levels of Fundulus kansae. Gen. comp. Endocrinol. 4, 61-67. FONTAI~ZE M. (1956) Hormonal control of water and salt-electrolyte metabolism in fish. Mem. Soc. Endocrinol. 5, 69-81. FOSTER G. V., BAGHDIANTZA., KUMARM. A., KUMARE., SOLIMANH. A. & MACINTYREI. (1964) Thyroid origin of calcitonin. Nature, Lond. 202, 1303-1305. GUDMUNDSSON T. V., MACINTYRE I. & SOLIMAN H. A. (1966) The isolation of thyrocalcitonin and a study of its effects in the rat. Proc. R. Soc. B 164, 460-477.
578
PETER K. T. PANG AND GRACE E. PICKFORO
HIRSCH P. F., •OELKEL E. F. & MUNSON P. L. (1964) Thyrocalcitonin: Hypocalcemic hypophosphatemic principle of the thyroid gland. Science, N . Y . 146, 412-413. LAROCHE G., WOODALLA. N., JOHNSON C. L. ~ HALWR J. E. (1966) Thyroid function in the rainbow trout (Salmo gairdnerii Rich) II. Effects of thyroidectomy on the development of young fish. Gen. comp. Endocrinol. 6, 249-266. MILHAUD G., MOUHTAR M. S., BOURICHON J. & PERAULT A. M. (1965) Existence et activit6 de la thyrocalcitonine chez l'homme. C. r. Acad. Sci., Paris 261, 4513-4516. MONTSK6 T., TIG¥I A., LISSA,K K. & DUaACZKVJ. (1964) Calcium regulation of Teleostei. Acta physiol, hung. 24, Suppl. 63. MONTSK6 T., TIGVI A. & THAN Z. (1963) Studies on calcium regulation in Osteichthyes. Acta biol. hung. 13, Suppl. 5, 72. MUNSON P. L., POTTS J. Z., REISFELD R. A., COOPER C. W. ~ VOELKEL E. F. (1966) Further purification of pig thyrocalcitonin. Science, N . Y . 154, 425. PHILLIPS A. M., PODOLIAK H. A., LIVINGSTON D. L., DUMAS R. F. & THOESEN R. W. (1960) Calcium metabolism in brook trout. Cortland Hatchery Rep. 28, 17-36. PHILLIPS A. M., PODOLIAK H. A., POSTON H. A., LIVINGSTON D. L., BOOKE H. E., PYLE E. A. & HAMMER G. L. (1964) T h e nutrition of trout. Cortland Hatchery Rep. 32, 24-35. PICKFORD G. E. (1957) In PICKFORD G. E. & ATZ J. W., The Physiology of the Pituitary Gland of Fishes. pp. 174-176. New York Zoological Society, New York. PICKFORD G. E., ROBERTSON E. E. & SAWYERW. H. (1965) Hypophysectomy, replacement therapy, and the tolerance of the euryhaline killifish, Fundulus heteroditus, to hypotonic media. Gen. comp. Endocrinol. 5, 160-180. SOMEREN V. D. VAN (1937) A preliminary investigation into the causes of scale absorption in salmon (Salmo salar Linn6). Fish Bd Scotland Salmon Fisheries. 1937 (2), 1-12. TALMAGE R. V., NEUENSCHWANDERJ. & KRAINTZ L. (1965) Evidence for the existence of thyrocalcitonin in the rat. Endocrinology 76, 103-107.
F A I L U R E OF H O G T H Y R O C A L C I T O N I N TO E L I C I T HYPOCALCEMIA IN T H E T E L E O S T FISH, FUNDULUS HETEROCLITUS P E T E R K. T . P A N G and G R A C E E. P I C K F O R D * The Bingham Laboratory, Department of Biology, Yale University, New Haven, Connecticut, U.S.A.
(Received 19 December 1966) Abstract--1. Hog thyrocalcitonin was administered to three physiologically different groups of male Fundulus heteroclitus at a dose of 0"2 mg (2-4 units) per g wt. of fish. Group 1 were intact fish maintained in salt water and fed; Group 2 were intact fish adapted to fresh water (low calcium) and starved for the final week; Group 3 were hypophysectomized fish maintained in salt water, and fed. 2. Within each group there were no significant differences in serumsoluble calcium between controls and hormone-treated fish. 3. Neither the time to autopsy (1-4 hr) nor the site of the injection (intraperitoneal or intravenous) had any effect. 4. Differences between the three groups could be related to the state of maturation of the testes. INTRODUCTION THE MAMMALIAN thyroid contains a hypocalcemic factor, thyrocalcitonin, that is released in response to high circulating levels of calcium (Foster et al., 1964; Hirsch et al., 1964; Milhaud et al., 1965 ; Talmage et al., 1965 ; G u d m u n d s s o n et al., 1966; M u n s o n et al., 1966). T h e relation of this substance to the parathyroid, with which it was associated by its discoverers (Copp & Cameron, 1961), remains controversial (Copp & Henze, 1964; Care et al., 1966). T o the best of our knowledge, the problem has not been studied in lower vertebrates. An attempt has therefore been made to evaluate the possible response of a fish to injections of mammalian thyrocalcitonin. MATERIALS AND METHODS Experiments were made on intact or hypophysectomized male Fundulus heteroclitus, maintained at ca. 20°C on an 8-hr day. A solution of partially purified hog thyrocalcitonin ( T H V - 6 0 , Munson) was prepared immediately before use by dissolving the required amount in 0.001 N HC1. T h e activity of this preparation assayed on young rats that had been given a calcium-free diet for 1-4 days, was stated by the donor (Dr. Paul L. Munson) to be about 10 units per 0.5-1 rag. W e * Supported by NSF Grant No. GB-0132. 573
574
PETER K. T. PANC AND GRACE E. PICXFORU
employed a high dose, 0.2 mg in 0.01 ml of solvent per g wt. of fish. In view of consistently negative results, lower doses were not investigated. The fish were anesthetized with tricaine methane sulfonate (MS 222, Sandoz). With one exception, noted below, the injections were made into the body cavity. At autopsy, free-flowing blood from lightly anesthetized fish was taken from the cut end of the tail in non-heparinized microhematocrit tubes and allowed to clot. Serum-soluble calcium was estimated on 5-/A samples by an ultramicro modification of the Neutral Fast Red method of Baar (1957). The determinations were made on the same day, without freezing of the serum, except for four samples (two controls and two hormone-treated fish in exp. 2) which were kept frozen at - 2 0 ° C for 24 hr. There was no apparent change that could be attributed to freezing. We employed Pedersen constriction pipettes, Linderstrem-Lang-type magnetic beads for stirring and a Beckman DU spectrophotometer with 50-/A Pyrocell quartz microcells. A standard reference serum (Labtrol, Dade) proved satisfactory and replicate determinations were in the acceptable range (10.28 + 0.10 S.E. mg per cent). EXPERIMENTS AND RESULTS
Experiment 1. Effect of varying the time after injection Eight intact fish, maintained in salt water and fed regularly except on the last day, were given intraperitoneal injections of THV-60 and autopsied after 1, 2 and 4 hr. A ninth fish was given an intravenous injection by way of the cardinal sinus in the posterior wall of the gill chamber. Two controls were not injected, and two received the solvent and were killed after 1 hr. These fish were at the end of the breeding season, but still with relatively large testes. The two control groups gave essentially identical results, and similarly the thyrocalcitonin-injected fish showed no differences that could be correlated with time. The data are therefore combined so that four controls can be compared with the nine fish that received hormonal treatment. The results (Table 1) show that, in this experiment, thyroealcitonin had no effect on the circulating level of serumsoluble calcium. The fish that received an intravenous hormonal injection gave a high reading (3.78 mequiv./1) but there were individuals both in the control and hormone-injected groups that were in this upper limit of the range (3.62 and 3.70 mequiv./l respectively).
Experiment 2. Effect of calcium deficiency It was thought that the negative results in the first experiment might be related to high calcium levels in the sea water and in the diet. A group of fish was therefore adapted to running fresh water wMch, in the New Haven area, is fairly low in calcium (ca. 9 ppm). The fish were maintained for 3 weeks in fresh water to ensure complete adaptation, and, as an additional precaution, they were not fed for 1 week before the experiment. Autopsies were made 2 hr after injection of either solvent or THV-60. The results (Table 1) show no difference between the
575
EFFECT OF THYROCALCITONIN ON F U N D U L U S H E T E R O C L I T U S
two groups, although the absolute levels for serum calcium were (unexpectedly) a little higher than in exp. 1. T h e s e fish had passed into complete sexual regression. TABLE 1 - - S E R U M
CALCIUM LEVELS I N
Fundulus heteroclitus RESULTING FROM
INJECTIONS OF
HOG THYROCALCITONIN
(THV-60, Munson)
Experiment 1
Conditions, date and gonosomatic index (GSI)* Intact, fed in salt water 2-3 Sept. 1966 GSI: 3"11 +0.38
Intact, starved, in fresh water 1 Oct. 1966 GSI: 0-60+0"11
3
Hypophysectomized, fed, in salt water 4-5 Sept. 1966 GSI : 0"34 + 0-03
Treatment None Solvent 1 hr
No. 2 2
Serum calcium (mequiv./l) Mean + S.E. Mean for 4 controls: 3-27 + 0-20
THV-60 1 hr 2hr 4 hr
4 3t 2
Mean for 9 THV-60 injected: 3"35+0-14
Solvent 2 hr
4
Mean : 4"65 + 0"08
THV-60 2 hr
4
Mean: 4"63 + 0"17
Solvent 2 hr
5
Mean: 5"60 + 0"43
THV-60 2hr
5
Mean: 5"18+0"47
* Gonosomatie index (GSI): Gonad weight x 100/body wt. t One fish in this group was given an intravenous injection.
Experiment 3. Effect of hypophysectomy Previous studies have suggested that there may be a disturbance of calcium metabolism in hypophysectomized F. heteroclitus, reflected in a high incidence of carbonate apatite renal calculi (Piekford, 1957). It was therefore decided to employ a group of long-term (7-month) hypophyseetomized fish that had completely recovered in the laboratory during 3.5 months after the termination of another experiment. T h e s e fish all showed the symptoms of complete hypophyseetomy: cessation of growth, pallor, anemia, regressed testes and a 50 per cent incidence of visible renal calculi. T h e y were maintained, of necessity, in salt water since hypophyseetomized fish of this species cannot survive in fresh water (Burden, 1956; Pickford et al., 1965), and were fed regularly until the last day.
576
PETER K . T . PANG AND GRACE E. PICKFORD
As in the two previous experiments, the results (Table 1) show no significant effect of thyrocalcitonin injection. The apparently slightly lower mean level for serum calcium in the THV-60 injected group, as compared with the controls, is not statistically significant. In both the controls and the hormone-injected groups, serum calcium levels were higher and more variable than in intact fish. Visible renal calculi were evident at autopsy in five fish, distributed in both groups, and no doubt sections would have revealed a higher incidence. However, there was no correlation between serum calcium levels and the incidence of visible renal calculi. DISCUSSION The findings reported in this investigation indicate that a partially purified preparation of hog thyrocalcitonin has no effect on serum-soluble calcium levels in the killifish, Fundulus heteroclitus. The hormone was administered to three groups of fish in different physiological conditions. Only males were employed since it is known that serum calcium levels are often high in breeding females of various teleostean species (Fleming & Meier, 1961a; Fleming et al., 1964; Booke, 1964; Phillips et al., 1964) whereas males are little affected or may even show a decline (van Someren, 1937). The first group, in sea water, was in a calcium-rich environment, yet the mean level for serum calcium was lower than in the second group maintained in fresh water under calcium-deficient conditions. There is evidence that teleosts can regulate the level of serum calcium in hypocalcic media, provided sufficient time is allowed for adaptation (Phillips et al., 1960; Montsk6 et al., 1963, 1964). In agreement with this, non-breeding males of F. heteroclitus appear to show no difference in mean serum calcium levels in fresh and in salt water (Pickford & Grant, unpublished). On the other hand, it is possible that the differences between the groups can be correlated with the state of sexual maturity. The fish in the first group were at the end of the breeding season but the testes were still large; the second group had passed into sexual regression. Preliminary observations on other experimental groups (Pickford & Pang, unpublished) suggest that in this species there is an inverse relation between serum calcium and the size of the male gonad. This hypothesis is in agreement with the data presented in Table 1. The effect of hypophysectomy on serum calcium levels in teleostean fishes has been investigated. Fontaine & Callamand (cited by Fontaine, 1956) found that in the eel hypophysectomy results in a decrease in serum calcium. On the other hand, in our experiments, serum calcium is higher in hypophysectomized than in intact males during sexual regression. There was no correlation, however, between the incidence of visible renal calculi and the levels of serum calcium. Further investigations are in progress. It is clear that the differences in the levels of serum calcium in the three groups which we investigated are not due to experimental defects, but can be related to the physiological condition of the animals.
EFFECT OF THYROCALCITONIN ON F U N D U L U S H E T E R O C L I T U S
577
T h e failure to respond to thyrocalcitonin can be explained in two ways: (1) the absence of a thyrocalcitonin m e c h a n i s m in fish, or (2) species specificity of the hormone. I t will be recalled that fishes lack a parathyroid gland and that the only demonstrable effects of exogenous p a r a h o r m o n e on s e r u m calcium levels are in spawning females, or estrogen-treated fish (Fleming & Meier, 1961a, 1961b; Clark & Fleming, 1963). T h e lack of a thyrocalcitonin m e c h a n i s m might be related to the absence of parahormone. I n answer to the second possibility, thyrocalcitonin preparations derived f r o m other sources, including extracts of the teleostean thyroid, m u s t be investigated. A m o n g the m a n y striking effects of radiation thyroidectomy in the rainbow trout, L a Roche et al. (1966) report a statistically insignificant lowering of serum calcium. T h i s would suggest that removal of the thyroid does not remove a hypocalcemic factor. Acknowledgements--We are indebted to Dr. Paul L. Munson, Department of Pharmacology, Medical School, the University of North Carolina, for the preparation employed, and for advice as to the solvent and suitable dosage levels.
REFERENCES BAA~ S. (1957) A micromethod for the estimation of serum calcium. Clin. chim. Acta 2, 567-575. BooKE H. E. (1964) Blood serum protein and calcium levels in yearling brook trout. Progr. Fish-Cult. 26, 107-110. BURDEN C. E. (1956) The failure of hypophysectomized Fundulus heteroclitus to survive in fresh water. Biol. Bull., Woods Hole 110, 8-28. CARE A. D., KEYNES W. M. & DUNCAN T. (1966) An investigation into the parathyroid origin of calcitonin..7. Endocrinol. 34, 299-318. CLARK N. B. & FLEMINGW. R. (1963) The effect of mammalian parathyroid hormone on bone histology and serum calcium levels in Fundulus kamae. Gen. comp. Endocrinol. 3, 461-467. COPP D. H . & CAMERON E. C. (1961) Demonstration of a hypocalcemic factor (calcitonin) in commercial parathyroid extract. Science, N . Y . 134, 2038. CoPP D. H. & HENZE K. G. (1964) Parathyroid origin of calcitonin: Evidence from perfusion of sheep glands. Endocrinology 75, 49-55. FLEMING W. R. & MEIER A. H. (1961a) The effect of mammalian parahormone on the serum calcium levels of Fundulus kansae and Fundulus catenatus. Comp. Biochem. Physiol. 2, 1-7. FLEMING W. R. & MEIER A. H. (1961b) Further studies of the effect of mammalian parathyroid extract on the serum calcium levels of two closely related teleosts. Comp. Biochem. Physiol. 3, 27-29. FLEMINGW. R., STANLEYJ. G. & MEIER A. H. (1964) Seasonal effects of external calcium, estradiol, and A C T H on the serum calcium and sodium levels of Fundulus kansae. Gen. comp. Endocrinol. 4, 61-67. FONTAI~ZE M. (1956) Hormonal control of water and salt-electrolyte metabolism in fish. Mem. Soc. Endocrinol. 5, 69-81. FOSTER G. V., BAGHDIANTZA., KUMARM. A., KUMARE., SOLIMANH. A. & MACINTYREI. (1964) Thyroid origin of calcitonin. Nature, Lond. 202, 1303-1305. GUDMUNDSSON T. V., MACINTYRE I. & SOLIMAN H. A. (1966) The isolation of thyrocalcitonin and a study of its effects in the rat. Proc. R. Soc. B 164, 460-477.
578
PETER K. T. PANG AND GRACE E. PICKFORO
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