Endocrine Control of Calcium Metabolism in Teleosts

Transcription

1 AMER. ZOOL., 13: (1973). Endocrine Control of Calcium Metabolism in Teleosts PETER K. T. PANG Department of Pharmacology, College of Physicians and Surgeons of Columbia University, New York, New York SYNOPSIS. It is evident that fishes regulate their serum calcium efficiently but that endocrine systems involved may be different from those in tetrapods. A functional parathyroid gland has not yet been demonstrated in fishes. The majority of evidence indicates that calcitonin has little or no effect on fish calcium regulation. Instead, the corpuscles of Stannius and the pituitary gland are necessary for maintaining fish serum calcium levels. In the killifish, Fundulus heteroclitus, the removal of the corpuscles produces hypercalcemia in sea water but not in artificial sea water deficient in calcium. Transplants of the corpuscles or the administration of corpuscle homogenate corrects the increase in calcium. On the other hand, hypophysectomy elicits hypocalcemia under calcium deficient conditions but not in calcium rich sea water. Replacement therapy with pituitary homogenate or hypophysial transplant prevents the fall in calcium. It is postulated that the hypocalcemic corpuscles of Stannius and the hypercalcemic pituitary gland enable the euryhaline killifish to regulate its serum calcium levels in high calcium sea water and low calcium fresh water, respectively. INTRODUCTION The calcium metabolism of fishes is particularly interesting because of their aquatic environment. In sea water or hard, fresh water, the environmental calcium level may be four or more times that of the fish's body fluid while the calcium level in soft fresh water may be one tenth that of fish blood. Fresh water fishes face the constant danger of calcium loss. Therefore, there must be very efficient systems for preventing this loss and for obtaining calcium from the environment. Since the opposite problem exists in sea water, marine or euryhaline fishes must be able to actively excrete the calcium entering from external sources and to limit its rate of entrance. Regardless of the mechanisms actually employed, euryhaline fishes such as eels and killifish are able to maintain their serum calcium levels quite precisely in both fresh and sea water (Chan and Chester Jones, 1968; Pickford et al., 1969; Fenwick The unpublished work described in this review was supported by NIH Grant AM and NSF Grant GB-30598X to Dr. W. H. Sawyer. I am grateful to Dr. Sawyer for reading the manuscript. I also thank Dr. D. H. Copp for the salmon calcitonin, Dr. J. W. Bastian (Armour Pharmaceutical Company) for the porcine calcitonin, Dr. A. S. Ridolfo (Lilly Laboratory) for the parathyroid extract, and NIH for the prolactin. 775 and Forster, 1972). I believe that the aquatic environment renders fish calcium metabolism unique among vertebrates and different from that of the terrestrial tetrapods. Although some tetrapods have an aquatic habitat, most breathe air and have less exchangeable surface area in contact with the surrounding medium than fishes which respire through the gills. The main features of tetrapod calcium metabolism are, essentially, the absorption of calcium from food, the prevention of calcium loss from the body and the ability to mobilize calcium in case of need. To deal with the last problem, tetrapods rely heavily on their calcium storage, the bony tissues. In fishes, the problems of getting rid of excess calcium in sea water or obtaining enough calcium in fresh water are continuous challenges which are closely related to the external medium. In tetrapods, the delicate balance of calcium is under the control of parathyroid hormone and, probably, calcitonin. Disturbances in calcium metabolism are often reflected in serum calcium levels. Thus, parathyroidectomy results in extreme hypocalcemia and, at times, tetanic seizures, while injections of parathyroid hormone correct these changes. On the other hand, in some mammals injection of calcitonin produces

2 776 PETER K. T. PANG hypocalcemia and in some amphibians the removal of the calcitonin source results in an abnormal calcium balance (Robertson, 1970). It seems that parathyroid hormone promotes calcium absorption from the digestive tract and calcium resorption from bone and prevents urinary calcium loss. Calcitonin reduces calcium resorption from storage reservoirs. Probably, it is by the delicate balance of these two systems that precise regulation of calcium metabolism is achieved in tetrapods. Fishes, the most abundant and diversified group of vertebrates, are, as indicated previously, able to regulate their calcium levels. However, their hormonal regulation of this electrolyte is poorly understood and the literature is contradictory and confusing. In recent years, the relation between calcium metabolism and various endocrine systems in fishes has been studied more extensively. However, several problems still remain unsolved. Firstly, it is not known whether a functional parathyroid gland is present. Secondly, whether calcitonin plays a role in fish calcium metabolism is highly controversial. Thirdly, the nature of the calcium regulating endocrine system has not been demonstrated. PARATHYROID GLAND The parathyroid gland has generally been considered absent in fishes (Fleischman, 1947; Hoar, 1951; Pickford, 1953). However, Rasquin and Rosenbloom (1954) suggested that the ultimobranchial gland might have functions analogous to those of the parathyi - oicl gland in this vertebrate group. These authors, studying the effects of complete darkness on the Mexican characin, Astyanax mexicanus, reported skeletal deformation and hypertrophy of the ultimobranchial gland. They concluded that the overactivity of the ultimobranchial body caused excessive skeletal resorption. However, in Funduhis heteroclitus, disturbances in calcium metabolism of fish kept in complete darkness were corrected by the administration of vitamin C (Pang, 1971b). In addition, the ultimobranchial gland of various species has been established as a rich source of calcitonin (Copp et al., 1967; Copp etal., 1968; Pang et al., 1971a). Therefore, it is very unlikely that the ultimobranchial body represents the parathyroid gland in fishes. Nevertheless, it is still possible that a functional parathyroid gland is present and awaits discovery. If this is true, one should be able to demonstrate the effects of parathyroid hormone. Mammalian parathyroid preparations, shown to be effective in nonmammalian tetrapods, have frequently been tested on fishes. Injections of mammalian parathyroid extracts had no consistent hypercalcemic effect on various teleostean fishes (Fleming and Meier, 1960, 1961o>; Clark and Fleming, 1963; Moss, 1963; Oguri and Takada, 1966; Fleming, 1967) and were ineffective in inducing scale growth, resorption, or regeneration (Yamada, 1961) or in changing bone and muscle mineral content (Rampone, cited by Hoar, 1957). Budde (1958) demonstrated an osteological response by Lcbistcs reticulatits to parathyroid extract injections but subsequent investigations in other species of fish failed to show similar changes (Clark and Fleming, 1963; Moss, 1963). Me- Farland (1968) reported that parathyroid tissues had no effect on fish bone resorption in vitro. In the lizard, Anolis carolinensis, injections of parathyroid extract given to intact animals had no statistically significant effect on serum calcium levels. However, when the hormone was injected into parathyroidectomized animals, the hypercalcemic effects of the hormone became evident in these hypocalcemic and tetanic lizards (Clark et al., 1969). Since the experiments on parathyroid hormone administration were performed on intact fishes, the negative findings cannot be viewed as conclusive evidence for the absence of responsiveness to parathyroid hormone. Hypocalcemia and tetanic seizures have recently been successfully induced in the killifish, F. heteroclitus (Pang et al., 19716). This provided an ideal system for testing the hypercalcemic effects of parathyroid hormone. In three separate experiments the same parathyroid extract that was effective in correcting hypocalcemia in para-

3 CALCIUM METABOLISM IN TELEOSTS 777 TABLE 1. Effects of mammalian parathyroid extract on serum calcium Irvclx of male F. heteroelitus adapted to calcium deficient sea icater. Experiment 1) 5 daily injections Control -+- saline Fluoride treated -f- saline Fluoride treated + PTH 2) 1 injection Hypects. + saline Hj-pects. -f- PTH 3) 5 daily injections Hypects. -f- saline Hypects. + PTH Serum Ca : * (msi/1) 2.95 ± 0.10 (10)* 2.53 ± 0.12 (10)** 2.38 ±0.10 (1 1)" 1.79 ± 0.22 (4) 1.95 ± 0.18 (4) 1.94 ± 0.12 (5) 2.04 ± 0.19 (5) * Data are given as mean ± SB (no. of fish). ** Significantly different from the controls (Student's t test) ; P < (Pajig, unpublished.) thyroidectomized lizards failed to alleviate the hypocalcemia and tetanic seizures of hypophysectomized or sodium fluoride treated F. heteroelitus (Table 1) (Pang, unpublished). Thus, these experiments fail to support the hypothesis that fishes have a functional parathyroid gland. But they do not rule it out. Therefore, further studies are needed. CALCITONIN The discovery of calcitonin as a possible hypocalcemic hormone in mammals (Copp et al., 1962; Hirsch et al., 1964) led to hopeful expectations that this hormone might be important in fish calcium metabolism. Investigations concerning the administration of this hormone to fishes were reviewed by Pang (1971c). In that review, the initial findings by Pang and Pickford (1967) that mammalian calcitonin failed to elicit hypocalcemia in the killifish, F. heteroelitus, were confirmed by subsequent experiments. The absence of effects of mammalian and salmon calcitonins was also shown in other species of fish (catfish and coho salmon). Louw et al. (1967) reported hypocalcemia in catfish treated with a crude mammalian calcitonin preparation. However, these findings have recently been questioned (Kenny, 1972). The hypocalcemic influence of crude mammalian calcitonin preparations or the ultimobranchial body was suggested by work with European and Japanese eels, Anguilla (inguilla and A. japonica, respectively (Chan et al., 1968ft; Chan, 1970). Subsequent investigators failed to confirm the hypocalcemic effects of this hormone in either European or American eels (Hayslett et al., 1971; Dacke, cited by Kenny, 1972). Recently, Orimo et al (1971) isolated calcitonin from Japanese eels and failed to demonstrate a consistent hypocalcemic effect of this purified hormone on A. japonica. In the trout, Salmo gairdneri, maintained in deionized water and treated with thyroxine, hypocalcemia and bone demineralization were evident. Treatment of such fish with calcitonin prevented the bone demineralization but had no effect on the decrease in serum calcium levels (Lopez et al., 1971). Copp et al. (1972) studied the effects of salmon calcitonin on blood and urine calcium metabolism in salmon and failed to observe any effect. Pang (1971c) suggested that calcitonin might be related to osmoregulation. Eel calcitonin has since been shown to decrease serum osmolality, sodium, and chloride in Japanese eels (Orimo et al., 1971, 1972). In a series of experiments with F. heteroelitus, chronic injections of either mammalian or salmon calcitonin appeared to have a hypochloremic effect. However, this effect was evident only in fish receiving fourteen or more injections of calcitonin (Table 2). In one experiment, 21 injections of salmon calcitonin also produced hypercalcemia. Since the experiments were conducted with intact fish, it was suspected that the endogenous calcitonin might exert maximal effects already. The ultimobranchial body, which is the source of calcitonin in fishes, may be under pituitary control (Pang, 1971c). If this is true, the effects of chronic injections of calcitonin would become more pronounced if the fish are hypophysectomized beforehand to reduce the endogenous calcitonin. A pilot experiment with hypophysectomized killifish showed that four injections of calcitonin did indeed produce hypochloremia (Table 3). Dacke et al. (1971) reported an increase in plasma calcitonin levels in fishes injected with a calcium chloride solution. Deftos et

4 778 PETER K. T. PANG TABLE 2. Effects of chronic injections of calcitonin on serum electrolyte levels of intact male F. heteroclitus. Experiment A) Fish adapted to sea water 1) 2 daily injections Saline Calcitonin 2) 4 daily injections Saline Calcitonin 3) 8 daily injections Saline Calcitonin 4) 21 daily injections Saline Calcitonin B) Fish adapted to calcium deficient sea water 1) 4 daily inj ections Saline Calcitonin 2) 14 daily injections Saline Calcitonin 3) 15 daily injections Saline Calcitonin 2.76 ± 0.11 (7) 2.73 ± 0.13 (4) 2.14 ± 0.15 (6) 2.27 ± 0.19 (6) 2.47 ± 0.08 (8) 2.91 ± 0.11 (8)** 2.62 ± 0.20 (9) 2.38 ± 0.07 (9) 2.55 ± 0.13 (4) 2.27 ± 0.09 (4) 2.65 ± 0.05 (7) 2.84 ± 0.13 (6) See Table 1. (Pang and Pang, unpublished.) ± 1.8 (7) ± 1.4 (7) ± 2.9 (7) ± 2.8 (6) ± 1.8 (6) ± 1.8 (6) ± 5.3 (9) ± 2.0 (10) Cl- (mm/1) ± 2.7 (7) ± 2.4 (7) ± 4.3 (7) ± 5.1 (6) ± 3.1 (6) ± 3.2 (6) ± 1.1 (7) ± 2.3 (8)** ±14.9 (6) ± 5.5 (9) ±15.0 (6) ± 4.0 (8)** ± 6.0 (7) ± 3.8 (5)** al. (1972) also described an increase in plasma calcitonin levels in salmon when calcium chloride was added to the surrounding medium. In addition, a fall in circulating hormone was seen in fishes which migrated from sea to fresh water. Nevertheless, the physiological effect of calcitonin on fish calcium metabolism remains obscure. The above discussion indicates that parathyroid hormone and calcitonin, which probably are the two most important endocrine principles in mammalian calcium metabolism, may have little importance in fish calcium regulation. On the other hand, fishes are capable of regulating their serum calcium rather precisely regardless of the calcium challenges they face in their natural habitat. It is, therefore, logical to assume the existence of some other efficient endocrine control. Several endocrine systems were studied in relation to killifish calcium metabolism (Pang, 1970). Of all the systems investigated, two have profound effects on calcium metabolism. They are discussed below with reference to the existing literature. CORPUSCLES OF STANNIUS The first system to be discussed is the corpuscles of Stannius. Fontaine (1964) demonstrated that the surgical removal of this tissue from European eels produced hypercalcemia. Pang (1971a) reviewed subsequent similar findings by various investigators working with European, Japanese, and American eels and goldfish, and reported the hypercalcemic effect of stanniectomy on the killifish, F. heteroclitus. Subsequent un- TABLE 3. Effects of chronic injections of calcitonin on serum chloride levels of hypophysectoviized male F. heteroclitus adapted to sea water. Experiment 4 daily injections Saline Calcitonin See Table 1. (Pang and Pang, unpublished.) Cl- (mm/1) ± 9.3 (5) ±4.7 (6)"

5 CALCIUM METABOLISM IN TELEOSTS 779 TABLE 4. Effects of stanniectomy on serum calcium levels of female F. heteroclitus kept in one-third sea water and fed regular calcium rich food. Experiment 1) 5 days after operation Operated controls Stanniects. 2) 8 days after operation Operated controls Stanniects. 3) 14 days after operation Operated controls Stanniects. See Table 1. (Pang, unpublished.) Ca =t (msi/1) 3.63 ± 0.37 (5) 4.93 ± 0.45 (7)" 2.76 ± 0.24 (5) 5.47 ±0.57 (9)** 2.30 ± 0.12 (7) 3.20 ± 0.16 (5)»* published studies confirmed these findings (Table 4). Therefore, it seems that this gland is important in fish calcium metabolism. However, stanniectomy also elicits changes in other serum electrolytes. It thus becomes important to determine whether the effect on the serum calcium level is specifically related to calcium metabolism or merely a reflection of disturbances in the regulation of other electrolytes or in osmoregulation. Histological examination of this gland in high and low calcium environments should answer this question. When previous investigators conducted histological studies of this gland taken from fish adapted to sea water, which is high in calcium, and to fresh water, which is low in calcium, results indicated that the glands are more active in sea water than in fresh water (Olivereau, 1964; Fontaine and Lopez, 1965; Hanke et al., 1967; Johnson, 1972). This, of course, supports the hypothesis that the corpuscles of Stannius promote hypocalcemia and are directly concerned with calcium metabolism. However, these findings are not decisive since the levels of electrolytes other than calcium, and the osmotic pressure, of fresh water and sea water are very different. Recently, Miss Rochelle Cohen of the Department of Life Sciences, University of Connecticut and I studied the effects of calcium deprivation on the ultrastructure of the corpuscles of Stannius of fish adapted to sea water. Killifish were maintained for four or more weeks in regular sea water or artificial calcium deficient sea water. The calcium levels of these two media were about 40 and 2 mg %, respectively. It is obvious that there is a high calcium challenge in sea water which is reversed in calcium deficient sea water. This may be reflected in the calcium levels of the fish (Pang et al., 19716). Since the stanniectomy experiments showed that these glands have hypocalcemic effects, they should be more active in sea water than they are in calcium deficient sea water. Electron microscopic studies indicate that this is true. The glands from fish in sea water exhibited a high degree of synthetic and secretory activities as indicated by rough endoplasmic reticulum, hyperactive Golgi apparatus, granular depletion, and the presence of lysosome-like bodies. Such indications of activity were not obvious in the glands from fish maintained in calcium deficient sea water. These findings are described in a preliminary report (Cohen et al., 1972). Since the only difference between the two groups of fish was the difference in environmental calcium levels, the difference in glandular activities should be due to calcium and calcium alone. That the corpuscles of Stannius affect calcium metabolism in the teleost fish studied is quite obvious. However, very little is known about the nature of the secretion or the mechanism of action of this gland. It was suggested that the rise in serum calcium after stanniectomy reflected a decrease in urinary calcium loss and that the effect on urine loss could be corrected by infusion of corpuscles of Stannius homogenate or homotransplantation of the tissues (Chan et al., 1969). However, Butler (1969), working with the American eel, Anguilla rostrata, failed to detect a decrease in urinary calcium excretion after the removal of the corpuscles of Stannius and suggested bone as a possible target organ. On the other hand, Lopez (1970) described a decrease in bone resorption after stanniectomy in the European eel. It therefore becomes very important to study kidney function in killifish in relation to the action of the corpuscles of Stannius. I believe that the main function of the corpuscles of Stannius. is to maintain serum

6 780 PETER K. T. PANG calcium levels in high calcium environments, while their role in low calcium environments is minimal. However, previous investigators reported hypercalcemia in stanniectomized fish maintained in fresh water (Fontaine, 1964, 1967; Chester Jones and Henderson, 1965; Chester Jones et al., 1967; Rankin et al., 1967; Chan et al., 1969; Chan, 1970). In some studies where stanniectomy was performed on both freshwater- and seawater-adapted eels, the hypercalcemic response was much greater in seawater-adapted eels than in those adapted to fresh water (Fenwick and Forster, 1972). Fenwick and Forster also showed that hypercalcemia appeared only at 2 weeks after stanniectomy of freshwater eels, while a marked response was seen in seawater eels as early as 10 days after the operation. It is also possible that the fresh water in some of these studies may contain appreciable amounts of calcium. Recent studies on killifish (Pang, unpublished) revealed that the magnitude of the hypercalcemic response to stanniectomy increases with an increase in the environmental calcium challenge. In fish adapted to calcium deficient sea water, a hypercalcemic response was totally absent from the operated animals (Table 5). These studies confirm the hypothesis that the corpuscles of Stannius have a hypocalcemic function in fishes facing a high environmental calcium challenge and that their role in low calcium media is minimal. This information would also define the importance of environmental calcium metabolism. As pointed out in the TABLE 5. Effects of stanniectomy on serum calcium levels of male F. lieteroelitus kept in one-third calcium deficient.sea water and fed low calcium food. Experiment 1) 5 days after operation Operated controls Stanniects. 2) 7 days after operation Operated controls Stanniects. 3) 14 days after operation Operated controls Staiuiiectb. Si'e Table 1. (Pang, unpublished.) Ca" (nim/1) 2.60 ± 0.10 (5) 2.49 ± 0.13 (6) 2.28 ± 0.10 (6) 2.19 ± 0.07 (6) 2.33 ± 0.12 (6) 2.08 ± 0.0G (8) introduction, the relationship of the fish to the aquatic environment makes the problems of calcium regulation in fishes different from those of the terrestrial vetebrates. Such problems are, perhaps, solved with endocrine systems different from those of the tetrapods. The chemical nature of the secretion of the corpuscles of Stannius remains a subject of controversy. Fontaine and Leloup-Hatty (1959), Cedard and Fontaine (1963), Ozon, Fontaine and Cedard (cited by Breuer and Ozon, 1965), Idler and Freeman (1966), Krishnamurthy (1968), and Colombo et al. (1971) claimed to have demonstrated the occurrence or the in vitro synthesis of adrenocorticosteroids in this tissue. However, other investigators failed to do so (Ford, 1959; Phillips and Mulrow, 1959; Roy, 1964; Chester [ones and Henderson, 1965; Chester Jones et al., 1965; Sandor et al., 1966; Arai et al., 1969). The enzymes essential for steroidogenesis are also reported to be absent from this gland (Chieffi and Botte, 1963«,6; Botte etal., 1964; Bara, 1968; Nadkarni and Lapinsky, 1968). From their electron microscopic studies, Oguri (1966), Ogawa (1967), and Tomasulo (1968) observed that the secretion of the gland is proteinaceous in nature. Similar observations were made on the killifish (Cohen and Pang, unpublished). Although a renin-like substance was demonstrated in fish corpuscles of Stannius (Chester (ones and Henderson, 1965; Chester Jones et al., 1966; Sokabe, 1968; Sokabe et al., 1968), they contain relatively little renin compared to the kidney (Sokabe et al., 1970). The physiological importance of renin and angiotensin in the corpuscles would then require further investigation. Fontaine (1964) reported that injections of corpuscles of Stannius extract restored the serum electrolyte levels of stanniectomized eels to normal. Homotransplants of the glands have similar corrective effects on eels (Fenwick and Forster, 1972). In killifish, both treatments are effective (see Table 6). PITUITARY GLAND The other endocrine organ that has a prolound effect on fish calcium metabolism

7 CALCIUM METABOLISM IN TELF.OSTS 781 TABLE 6. Effects of replacement therapy on serum calcium levels of stanniectomized male F. heteroelitus Tccpt in sea icater and fed regular calcium rich food for two weelcs. Groups Ca= 1) Operated controls 3.57 ± 0.08 (8)* 2) Stanniects ± 0.39 (9) 3) Staimiects injections of 3.46 ± 0.08 (9)* Staiuiius corpuscles homogonate 4) Staimiects. -f Staiuiius cor ± 0.17 (8)* puscles transplants See Table 1. * Significantly different from the Stajiniects. (Student's t test) ; P < (Pang, unpublished.) is the pituitary gland. Many investigators have looked into this problem previously. It was pointed out in an earlier publication (Pang et al., 1971) that other investigators failed to establish a distinct relationship between the pituitary gland and fish calcium metabolism. However, our findings in that report show that the pituitary gland regulates calcium metabolism in the killifish, F. heteroclitus, adapted to a low calcium environment. When the pituitary gland was removed surgically from fish adapted to artificial sea water deficient in calcium, the experimental animals exhibited extreme hypocalcemia and tetanic seizures. This was the first time that tetany was induced and correlated with hypocalcemia infishes.a decrease in serum calcium levels after hypophysectomy has been observed in eels adapted to fresh water (Fontaine, 1956; Olivereau and Chartier-Baraduc, 1965; Chan and Chester Jones, 1968; Chan et al., 1968«). However, in those experiments, hypophysectomy also produced hyponatremia and disturbances in osmoregulation. Chan and Chester Jones (1968) believed that the hypocalcemia in the hypophysectomized eels might simply reflect osmoregulatory problems. Since our tetanic fish were maintained in sea water that was only deficient in calcium, no consistent osmotic difficulties were evident, and we therefore suggested that the observed hypocalcemia represented a real defect in calcium metabolism as a result of the removal of the pituitary gland. A subsequent experiment with another species of the genus Fundulus, F. diaphanus, adapted to fresh water supported this hypothesis (Pangetal., 1973«). To substantiate our claim that the pituitary gland has a physiologically hypercalcemic function, replacement therapy was given to hypocalcemic killifish that had been hypophysectomized and maintained in calcium-deficient sea water. In one experiment, either a whole pituitary homogenate was injected daily for 5 days into the experimental fish or the pituitary gland was transplanted under the skin on the side of the body. The transplants survived well. In both cases, normal calcium levels were maintained. Hypophysectomized fish injected with liver homogenate were hypocalcemic. The results are summarized in Table 7. These findings strongly support our hypothesis that the pituitary gland contains a factor or factors essential for the maintenance of serum calcium levels in fish inhabiting environments low in calcium. What is the hypercalcemic hormone (or hormones) in the pituitary gland? To look into this, two approaches have been taken. An attempt has been made to divide the killifish pituitary gland into three different regions, the anterior, median, and posterior parts. Five hundred glands have been divided in such a manner, and homogenates of each part were made. Previous histological studies of killifish pituitary glands showed that the anterior part contains mainly prolactin cells bordered by ACTH cells. The median part is composed of cells producing TSH, STH, and gonadothrophins. The posterior part is equivalent to the pars intermedia of higher vertebrates. The neurophypophysis interdigitates abundantly with the median and posterior regions. It was hoped that replacement therapy with homogenates of the three different parts might reveal the location of the hypercalcemic factor or factors. The results of the experiment are shown in Table 7. Although the homogenates from all three parts were effective in raising serum calcium levels of the hypocalcemic fish, that from the median part was the least effective. Since the three parts were only very roughly divided, contamination of the median part with tissues from the other two parts was

8 782 PETER K. T. PANG inevitable. This would easily explain the significant corrective effect of the homogenate of the median part. Since the anterior and posterior parts were equally effective, it was suspected that during the collection of tissues, there might inadvertently have been some interchanging of the two parts. On the other hand, it is known that the molecular structure of mammalian MSH and ACTH are very similar. It is then possible that the ACTH from the anterior part and the MSH from the posterior part mimic each other at high dosage levels. It is evident from the over-correction of the serum calcium levels that the dose of the TABLE 7. Effects of replacement therapy on serum calcium levels of hypophysectomized male F. heteroclitus adapted to calcium deficient sea water and fed low calcium food. Groups Ca 2 * (mm/1) 1) Operated controls 2.68 ± 0.14 (5) 2) Hypeets ± 0.09 (5)** 3) Hypeets. + liverhomogenate 1.94 ± 0.12 (8)** 4) Hypeets. + whole pituitary 2.47 ± 0.11 (10) homogenate 5) Hypeets. + homogenate of 2.73 ± 0.21 (9) anterior part of pituitary gland 6) Hypeets. + homogenate of 2.49 ± 0.21 (9) middle part of pituitary gland 7) Hypeets. + homogenate of 2.88 ± 0.20 (9) posterior part of pituitary gland 8) Hypeets. + pituitary 2.43 ± 0.11 (10) transplant See Table 1. (Pang et al., ) injection was too high. The serum calcium levels of fish receiving homogenates from these two parts of the pituitary gland were higher than the mock operated controls. To pursue this further, ACTH or mammalian MSH was given to the hypocalcemic fish in another experiment. In addition, cortisol and ovine prolactin were also tested, since ACTH should stimulate cortisol release, and prolactin should be abundant in the homogenate of the anterior part of the pituitary gland. The results are summarized in Table 8. It is interesting to note that although ACTH, cortisol, and prolactin were all effective in correcting the hypocalcemia, MSH was ineffective. The experiment was TABLE 8. Effects of pituitary hormones on serum calcium levels of hypophysectomized male F. heteroclitus adapted to calcium deficient sea water and fed low calcium food. (Experiment 1.) Groups Ca 2 1) Operated controls + saline 2.36 ± 0.15 (5)* 2) Hypeets. + saline 1.75 ± 0.13 (5) 3) Hypeets. + cortisol, 2.5 Ag/g 2.18 ± 0.06 (10)* 4) Hypects. + ACTH, 0.05 IU/g 2.50 ± 0.27 (10)* 5) Hypeets. + prolactin, 5 ^g/g 2.49 ± 0.14 (5)* 6) Hypeets. + MSH, 2.5 /jg/g 1.97 ± 0.O8 (10) See Table 1. * Significantly different from group 2 (Student's t test) ; P < (Pang et al., ) subsequently repeated with lower doses of ACTH and prolactin and a higher dose of MSH. In that experiment, the lower doses of ACTH and the high dose of MSH had no effect while prolactin, even at the lowest dose, one-tenth of that used in the previous experiment, was effective (Table 9). The evidence collected to date is far from conclusive, but it does suggest that prolactin, and probably also ACTH, are involved in the regulation of serum calcium levels of killifish maintained in a low calcium environment (Pang et al., 19736). The hypercalcemic effect of prolactin has been observed in eels (Olivereau and Olivereau, 1970). It is possible that ACTH and prolactin act together in hypercalcemic regulation. The synergistic effect of these two hormones on calcium regulation has been demonstrated in eels (Chan et al., 1968a). The participation of prolactin in hypercalcemic regulation fits the current views on TABLE 9. Effects of pituitary hormones on serum calcium levels of male F. heteroclitus adapted to calcium deficient sea water and fed low calcium food. (Experiment 2.) Groups Ca a+ 1) Operated controls + saline 2) Hypeets. -f saline 3) Hypeets. + AOTH, 0.02 IU/g 4) Hypects. + ACTH, IU/g 5) Hypeets. -+- prolactin, 2/Jg/g 6) Hypects. + prolaetin, j g g 05 / 7) Hypects.+1ISH. See Table 8. (Pang et al., ) 2.87 ±0.08 (9)» 2.25 ± 0.06 (10) 2.34 ± 0.06 (10) 2.23 ± 0.05 (9) 2.58 ± 0.57 (8)* 2.64 ±0.05 (10)" 2.16 ± 0.07 (7)

9 CALCIUM METABOLISM IN TELEOSTS 783 the physiological function of this hormone in fishes. It has been well established that prolactin is important in osmoregulation in the freshwater fishes. In nature, the only low calcium environment that a fish would encounter is fresh water. That the hormone for osmoregulation also regulates calcium would then be understandable. At present these are simply speculations and many more experiments are required to clarify the identity of the calcium regulating hormone from the pituitary gland. We should note that the hypercalcemic function of the pituitary gland is important only in environments that are low in calcium. If the fish are hypophysectomized in sea water which is rich in calcium, no change in serum calcium levels would be seen. Also, the hypocalcemia in hypophysectomized fish maintained in calcium deficient sea water can be corrected by returning the fish to regular sea water (Pang et al., 19716). Obviously, then, the hormone or hormones would be secreted when the fish are maintained in a low calcium environment. Therefore, histological studies of this gland in high and low calcium sea water would help us identify the hormone or hormones in the pituitary gland. Such studies are in progress and the results will become available in the near future. OTHER ENDOCRINE ORGANS Other endocrine organs, including the gonads, and the thyroid, interrenal, and pineal glands, have been implicated in fish calcium metabolism. These studies will not be discussed in detail. Instead, pertinent literature will be tabulated for the convenience of the reader and some important features and contradictions will be discussed in the text. It is be lieved that apart from the ovaries these systems contribute little to fish calcium metabolism. Gonads Table 10 summarizes the reported studies on the gonads and calcium metabolism. These studies clearly indicate that female fish have higher serum calcium levels during ovarian maturation, probably due to increased estrogen secretion. The administration of estradiol increases fish serum calcium levels. This increase is associated with a rise in protein bound calcium and not in ionic calcium (Bailey, 1957a; Chan and Chester Jones, 1968; Urist and Sehjeide, 1961). It is believed that the protein is incorporated into the yolk and stored for future embryonic development. Most studies on male fish failed to show a distinct relation between testicular maturation and serum calcium levels (see table 10). However, a significant decrease in serum calcium levels was observed in male Salmo salar spawning in fresh water (van Someren, 1937; Fontaine et al., 1969). On the other hand, hypercalcemia occurred in mature male Gadus morhna in sea water (Woodhead and Woodhead, 1964; Woodhead, 1968; Woodhead and Plack, 1968). Since fresh water is low in calcium and sea water is high in this ion, it is possible that these two species failed to regulate their calcium in relation to the environmental challenge during sexual maturation. The most logical approach to this problem is to observe the effects of male sex hormones on fish calcium levels. Testosterone propionate did not affect serum calcium levels in male goldfish (Bailey, 1957a), but Peterson and Shehadeh (1971), citing the data from one fish, claimed that methyltestosterone increased serum calcium in a male mullet (Mugil cephalus). Administration of crude fish gonadotropins induced testicular hydration in male goldfish, but serum calcium was not changed (Grant et al., 1969). Many reports show that female sex hormones have a hypercalcemic effect on male fish (see Table 10), but unless a high level of the female sex hormone is demonstrated in reproductively active male fishes, hypercalcemic effects of estrogen on these organisms have little or no meaning in the context of their normal physiology. Male killifish reproduce annually in the laboratory even under constant periods of light and dark and constant temperature (Pickford, unpublished). When serum calcium levels in killifish are measured in association with testicular maturation, no correlation is seen between these levels and testicular size (Fig. 1). In the same figure, the serum calcium levels are given for cas-

10 TABLE 10. Jiclationship betwien the gonads and calcium metabolism in fishes. Authors Mii-sclier (1897) Hcssetal. (1928) van SomeiTii (1937) Bailey (3957a) Bailey (1957b) Garrod and Newell (1958) (cited by Woodhead, 1968) Sano (1960) Fleming and Meier (1961a) Ho and Vanstone (1961) Trist and Schjcide (1961) Clark and Fleming (1963) Booke (1964) Fleming et al. (1964) Fontaine et al. (1964) Woodhead and Woodhead (1964) ORiiri and Takada (1966) Chan and Chester Jones (1968) Woodhead (1968) Woodhead and Plack (1968) Fontaine et al. (1969) Stanley (1969) Woodhead (1969a) Woodhead (1969ft) Peterson and Sheliaded (1971) Species Medium Conclusions Cod and puffer Sdltno salar Carass-ins auraius C. auraius Tilapia csculcnta Salmo gairdnerii Fundulus Tcansae, F. eatenatus Oncorhynchns nertca Paralabrax claihratits F. leansae Salvelinns fontinalis F. Tcansae F. Tcansae Anguilla anguilla Gadits morhua Cliana argus A. anguilla G. morhua G. morhua S. salar Alosa psrudoharengus Scyliorhinus canicnla G. morhua Mngil cephalus SW rw salt spring SW SW SW SW SW Serum calcium levels were elevated during egg laying season. Females had higher serum calcium levels than males. These could be correlated with gonadal maturation. Males maintained constant, serum calcium levels until just, before spa.wni.ng; females showed increase during maturation. At spawning, decrease seen, in both sexes. One injection of estradiol increased total and non-ultrafiltrable calcium levels for 20 days. Testosterone propionate had no effect. Same as above. Females showed higher serum calcium levels when sexually mature; males did not. Claimed higher serum calcium levels in summer, but no sex differentiation. Estradiol induced hypercalcemia in males and females. Estradiol benzoato elevated serum calcium levels of both sexes. Serum calcium in the form of calcium proreina.te was increased by estrone. Estradiol increased serum total calcium. Histology and x-ray of bone showed no changes. Females had higher serum calcium levels during spawning; males did not. Males showed no seasonal cycle in sorum calcium levels but females exhibited hypercalcemia. in summer. Estradiol increased serum calcium level in females. Tho rise was greater if there was calcium in the water. Females had higher serum calcium levels when sexually mature. Hypercalcemia. coincided with gonadal maturation in both sexes. Estradiol elevated serum calcium levels but sexes of fish were not separated. Estrogen treatment increased total and non-ultrafiltrable calcium levels. Gonadal maturation raised plasma calcium levels in both sexes. Same as above. Both sexes showed decrease in serum ca.lcium levels during spawning. Claimed hypercalcemia in breeding males but not females. Estradiol benzoate increased plasma, total calcium in females. Estradiol benzoate increased plasma, calcium in both sexes. Methyltestosterone increased serum calcium level in an experiment with one male mullet. ft 7a -A a

11 CALCIUM METABOLISM IN TELEOSTS 785 III 2 s.2 ts o O I if! Ill; i 2 's 2 = -SO a v o ill c; u c o.3 o o f, is' c~i 'S ^. a Si CaoH o -js «6? 1 b yl&."«"=? "3 j? < «I so 3 =. s r' 8 s r S o CO - o f Jill fisstjk 8? o FIG. 1. Testicular activities and serum total calcium levels in killifish, F. heteroclitus, maintained in sea water. trated males and operated controls (Pang, unpublished). Again, no difference is seen. These studies support the hypothesis that calcium metabolism is not affected by testicular development. This is understandable since, compared to the females, mobilization of calcium into the gonads is much less drastic in male fish. Thyroid gland Previous investigations of the thyroid gland and fish calcium metabolism are summarized in Table 11. Most studies indicated that thyroxine prevented estrogen induced hypercalcemia. When rainbow trout were immersed in a solution of thyroxine and tri-idothyronine, hypocalcemia was observed (Chartier-Baraduc, 1968). However, radiothyroidectomized trout had normal serum calcium levels (La Roche et al., 1966). Ball and Ensor (1967) reported hypercalcemia in freshwater- or dilute seawater-adapted Poecillia latipinna, injected with thyrotropin. This problem has been investigated in male F. heteroclitus (Table 12) (Pang unpublished). In three separate experiments, food containing thyroid powder produced hypocalcemia. Ten injections of thyroxine or three injections of TSH had a similar effect. However, in other experiments, ten or twenty injections of thyroxine or the administration of thyroxine and tri-iodothyronine by injection or in the external media had no significant effect. Such contra-

12 786 PETER K. T. PANG TABLE 12. Effects of thyroid substances on serum calcium levels of male hillifish, F. heteroclitus. Serum Ca 2 * (mm/1) Experiment Controls Experimentals 1) Thyroid feeding, F.W. 2) Thyroid feeding, S.W. 3) Thyroxine (T4), 10 inj., F.W. 4) T4, 20 inj., F.W. 5) T4, 10 inj., hypect, S.W. 6) Thyroid feeding, F.W. 7) TSH, 3 inj., F.W. 8) T3 and T4, in F.W. 9) T3 andt4, 3 inj., F.W. See Table 1. (Pang, unpublished.) dictions even within the same species o fish makes it impossible to draw any conclusion concerning the relation between thyroid function and fish calcium metabolism. Probably, thyroid hormones possess a hypocalcemic effect, demonstrable only under some special physiological conditions which have yet to be identified. Interrenal gland In the section concerning the pituitary gland, the possible participation of the pituitary-interrenal axis in conjunction with prolactin in the regulation of serum calcium levels in low calcium environments was discussed. Literature on interrenal hormones and fish calcium metabolism is presented in Table 13, but no consistent picture can be seen in all these studies. In a series of studies on eels (Chan et al., 1967; Chan and Chester Jones, 1968), it was reported that interrenalectomy caused a fall in plasma calcium in freshwater silver eels and a rise in that of sea water silver eels, but not in yellow eels maintained in either media. Treatment with cortisol, aldosterone, and adrenocorticotropin (ACTH) did not have consistent effects either. Butler et al. (1969) detected a decrease in plasma calcium in one of three (16) (7) (8) (10) i (6) t (7) (6) (8) (7) ** (16) ** (7) i- 0.09** (8) t (H) t (9) ** (7) i- 0.09** (8) * (8) t (8) groups of interrenalectomized freshwater eels. Fleming, et al., (1964) reported hypocalcemia in winter but not summer male and female F. kansae held in deionized water and treated with ACTH. In Channa argus, a freshwater fish cortisol had no effect on serum or urine calcium levels (Oguri and Takada, 1966). Injections of cortisol given to hypophysectomized male or female F. heteroclitus in sea water had no effect on total or dialytic calcium levels (Pickford et al., 1970). In another experiment, treatment of seawateradapted male killifish with metopirone produced no significant effects on serum calcium levels (Table 14) (Pang, unpublished). As discussed above in the section concerning the pituitary gland, a synergistic effect of prolactin and ACTH was suggested by Chan et al. (1968a). Therefore, if the pituitaryinterrenal axis is involved in calcium metabolism, it would probably be for the maintenance of calcium levels in a low calcium environment. Pineal gland Skeletal deformities have been demonstrated in pinealectomized guppies and small salmon (Pflugfelder, 1953, 1967). Un-

13 CALCIUM METABOLISM IN TELEOSTS 787 fortunately, serum calcium levels were not studied in those experiments. However, in view of the skeletal changes, it was suspected that the pineal gland might be involved in fish calcium metabolism. Weisbart and Fenwick (1968) failed to show any change in serum calcium levels in pinealectomized goldfish maintained in fresh water. Similar negative findings were obtained with F. heteroclitus pinealectomized in sea water (Table 15) (Pang, unpublished). Therefore; a pineal effect on fish calcium regulation seems rather unlikely. CONCLUSION Our studies with F. heteroclitus, using to date more than 2,500 fish, have been very rewarding. These studies have shown us that, at least in killifish, the endocrine control of calcium metabolism is very different from that of tetrapods. Parathyroid hormone and calcitonin appear to have little or no physiological significance in the calcium metabolism of this species of fish. Of all the endocrine systems studied, the pituitary gland and the corpuscles of Stannius are most consistently demonstrated to be involved; the former being hypercalcemic and the latter hypocalcemic. It is necessary to reiterate that the Stannius corpuscles are important in environments high in calcium but probably not in low calcium environments, and the opposite is true for the pituitary gland. The possession of these two systems enables a euryhaline fish like F. heteroclitus to regulate its calcium in sea water as well as in fresh water. The adjustment of their activities under different environmental calcium challenges helps the fish to maintain a stable serum calcium level. As discussed above, the fact that the corpuscles of Stannius are dispensable in environments poor in calcium and the pituitary gland in environments rich in calcium, strongly supports my idea that these two systems are physiologically meaningful and that the exchange with the environment is an integral part of calcium, metabolism in killifish. It is impossible to generalize for all fishes from our data since killifish have acellular 3 i I > ' o ft 03.3 a ^ 2~ ag is a o ft. 6 9 * S3^ 2^ i oi 3 «S Is goo -5 a 8 3 O " A Sip: S 8 O 3 O oo? Q ills a S «3 a a so S S 3 o o T3 1966) ci a tan O 6 s o 1-5 nd Cl *> e 3 o o _o 3 6 M

14 788 PETER K. T. PANG TABLE 14. Effects of meiopironr treatment on xerum. calcium levin of clitus, kepi in sea( water. Group Controls Metopirone treated See Table 1. (Pang, unpublished.) bone. It has been suggested that acellular bone, in contrast to cellular bone, is physiologically dead and that its calcium cannot be resorbed for regulation (Moss, 1962; Simmons, 1971). Thus, the calcium metabolism of fishes with cellular bone may be different. Nevertheless, work with eels, a fish TABLE 15. Effects of pinealeciomy on serum calcium levels of male, killifish, F. heteroelitus, kept in sea water. G roup Ca 2+ Mock-operated controls 2.82 ± 0.22 (8) Pinenlects ± 0.32 (4) See Table 1. (Pang, unpublished.) with cellular bone, has revealed the presence of hypercalcemia after stanniectomy and hypocalcemia following hypophysectomy, and it is possible that fishes with either cellular or acellular bone may have similar endocrine regulation of calcium metabolism. It seems obvious that fishes regulate their calcium in relation to the environmental FIG. 2. Evolution of endocrine control of calcium metabolism in vertebrates. B: bone; Ca: calcium; C.S.: coipusclcs of Slannkis; CTX: calcitonin; F.W.: fresh water; I: intestine; K: kidney; Pit.: pituitary gland; 1'IH: paratlmoid hormone; S.W.: stu water. Totiil C:\r* (lllm/1) ± 0,.15 (5) (5) Bldlf killifish, F. hetero- Dialytic Ca 2 * (nim/1) 0.71 H- 0.,12 (5) 0.73,18(5) ± 0, calcium challenge. When tetrapods moved onto land from the aquatic habitat, they also left the continuous calcium challenge. The importance of the bony tissues as calcium reservoirs then becomes evident and with it, a different set of endocrine controls for calcium metabolism comes into play (Fig. 2). If this hypothesis is true, this would be one of the important adjustments vertebrates made when they moved from water to land during evolution. REFERENCES Arai, R., H. Tajima, and B. I. Tainaoki In vitro transformation of steroids by the head kidney, the body kidney and the corpuscles of Stannius of the rainbow trout. Gen. Comp. Entlocrinol. 12: Bailey, R. E. 1957a. The effect of estradiol on serum calcium, phosphorus and protein of goldfish. J. Exp. Zool. 136: Bailey, R. E. 1957b. Effect of thyroxine and estradiol on the serum calcium, phosphorus and protein of goldfish. Anat. Rec. 128: Ball, J. N., and D. M. Ensor Specific action of prolactin on plasma sodium levels in hypophysectomized Poecilia latipinna (Teleostei). Gen. Comp. Endocrinol. 8: Bara, G Histochemical study of 3/3 and 3 a, 11/3 and 17/3 hydroxysteroid dehydrogenases in the adrenocortical tissue and the corpuscles of Stannius of Fundulus heteroelitus. Gen. Comp. Endocrinol. 10: Booke, H. E Blood scrum protein and calcium levels in yearling brook trout. Prog. Fish-Cult. 26: Botte, V., C. Buonanno, and G. Chiefli Osservazioni sulla istofisiologia deh'interrenale e dei corpuscoli di Stannius di alcuni Teleostei. Boll. Zool. 31: Breuer, H., and R. Ozon Metabolisme des hormones steroides androgencs et oestrogenes chez les vertebrates inferieurs. Arch. Anat. Microsc. 54: Budde, Sr. >f. L The effects of parathyroid extract upon the leleost fish Lebistes reticulatus. Growth 22: Butler, D. G Corpuscles of Stannius and renal physiology in the eel (Anguilla rostrata). J. Fish. RLS. Board Can. 2fi:639-fu4.

15 CALCIUM METABOLISM IN TELEOSTS 789 Butler, D. G., W. C. Clarke, E. M. Donaldson, and R. W. Langford Surgical adrenalectomy of a teleost fish (Anguilla rostrata Le Sueur): effect on plasma cortisol and tissue electrolyte and carbohydrate concentrations. Gen. Comp. Endocrinol. 12: C&lard, L., and M. Fontaine Sur la presence de steroides sexuels dans les corpuscles de Stannius du salmon atlantique {Salmo salar L.). C. R. Hebd. Seances Acad. Sci. (Paris) 257: Chan, D. K. O Endocrine regulation o calcium and inorganic phosphate balance in freshwater adapted teleost fish, Anguilla anguilla and A. japonica, p Proc. 3rd Int. Congr. Endocrinol. Excerpta Med. Int. Cong. Ser. No Chan, D. K. O., and I. Chester Jones Regulation and distribution of plasma calcium and inorganic phosphate in the European eel {Anguilla anguilla L.). J. Endocrinol. 32: Chan, D. K. O., I. Chester Jones, I. W. Henderson, and J. C. 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