Camp. Biochem. Physiol. Vol. 72A, No. 4, pp. 709 to 713, 1982 Printed in Great Britain.

EFFECTS OF BLOCKAGE OF EXOGENOUS

0300-9629/82/080709-05$03.00/O 0 1982 Prrgamon Press Ltd

CALCIUM AND PHOSPHORUS ON THE CALCIUM REGULATORY

SYSTEMS IN GOLDFISH

SHIGETAKA YAMANE,* MASAHARU Iciucr-n,t TSLJYOSHI OGAsAwmAt and f(OSHIKAZU NAKAMURAt

*Chemical Biotesting Center, Chemicals Inspection and Testing Institute, 19-14 Chub-Cho, Kurume, Fukuoka 830, Japan and

tFaculty of Fisheries, Hokkaido University, Hakodate, Hokkaido 041, Japan

(Receioed 25 November 1981)

Abstract-l. The effects of calcium and phosphorus deficiency in diet and ambient water on the calcium regulatory systems in goldfish were examined.

2. Hypercalcemia developed after 4 wk though normocalcemia was maintained until 2 wk. 3. Serum inorganic phosphorus was always at a lower level than in controls. 4. The Stannius corouscles were activated and their removal caused hypercalcemia after 9 days of

feeding.

INTRODUCTION

Effects of a low-Ca or a low-P diet on bones, serum concentrations of calcium, inorganic phosphate, PTH and vitamin DS have been studied etiologically in mammals (Freeman & McLean, 1941; Lotz et al., 1968; Baylink et al., 1971; Stauffer et al., 1973; Bruin et al., 1975; Massry, 1978; Brautbar et al., 1979; Rader et al., 1979). In fishes, similar experiments have been carried out to investigate the nutritive require- ments of dietary calcium or phosphorus for growth but the regulatory mechanisms of calcium metabolism under calcium or phosphorus deficient conditions have not been studied.

As to calcium regulatory mechanisms of bony fishes, the parathyroid gland has not been detected yet and the role of vitamin D3 is obscure. On the other hand, the prolactin cells and the Stannius cor- puscles are considered to be implicated in hypercalce- mic (Pang, 1973) and hypocalcemic (Fontaine, 1964) control, respectively. Furthermore, fish calcitonin which is secreted by the ultimobranchial gland and has a hypocalcemic action for mammals appears to be related to ovarian maturation rather than calcium metabolism in fishes (Yamane, 1977, 1980; Yamane & Yamada, 1977). These differences between mammals and fishes may be attributed to the fact that fishes are able to absorb calcium from ambient water across the skin and the gill epithelia as well as from food. Wata- nabe et al. (1980) reported that the body growth and bone mineral content of chum salmon kept in fresh- water containing calcium were not affected by a cal- cium-deficient (CaD) diet, but greatly affected by a phosphorus-deficient (PD) diet in the same ambient water. Therefore, the role played by phosphorus in calcium metabolism in fishes awaits to be studied as it has been in mammals.

Our experiments were undertaken to examine how the blockage of exogenous calcium and phosphorus from food and ambient water affects the metabolism and the regulatory mechanisms of calcium in goldfish.

MATERIALS AND METHODS

Goldfish, Carassius auratus, were obtained from a com- mercial dealer and acclimated in aerated aquaria at a tim- perature between 15 and 25°C. They were fed on commer- cial pellets for carp ad libitum until 2 days before the start of the rearing experiment. One day before the beginning of the rearing experiment, all fish were injected intraperi- toneally with 1 mg tetracycline hydrochloride (Acromycin V, Lederle) in 0.1 ml of 0.9% NaCl per 10 g body weight for time marking on their scales and bones. The control and calcium- and phosphorus-deficient (CaD-PD) diets had the same basal composition (Table 1) but contaitied 0.5% cal- cium-0.8% phosphorus and 0.05% calcium-0.07% phos- phorus, respectively. The mineral mixture of the CaD-PD diet was prepared by omitting sodium phosphate mono- basic and calcium lactate and the loss of sodium was sup- olemented bv addine sodium chloride (Table 2). The con- tents of calcium and phosphorus in the diets were deter- mined by an atomic absorption spectrometer (Hitachi 518) and by the method of Goldenberg & Fernandez (1966), respectively. All fish were sacrificed after rearing experi- ments and their blood, corpuscles of Stannius, ultimobran- chial glands, pituitaries, scales and ribs were collected for chemical or histological examinations.

Experiment 1

Sixty-four animals weighing about 4-11 g were divided into 2 groups. Each group was maintained in a 12 1 aquar- ium containing distilled water which was aerated at 24 & 1°C and renewed at least once a day, and fed either the control or the Ca&PD diet for 4 wk. All fish were fed a diet 3.5% of their body weight each day until sacrifice after 3 days, 14 days and at the end of the experiment.

Experiment 2

Thirty males weighing about 614 g were maintained in one third seawater for one week to facilitate surgical re- moval of the Stannius corpuscles (Ogawa, 1963). After stanniectomy or sham-operation, the fish were transferred to one fourth seawater separately in a small compartment, and maintained without feeding for 4 days. Four days after the operation all fish were acclimated to freshwater with- out feeding, and injected with tetracycline hydrochloride. Five days after the operation both the operated and sham-

709

7 IO SHIWTAKA YAMAM rt trl

Table I. Basal composition of control and CaD-PD diets branchial glands and Stannius corpuscles) or with azan

fed to goldfish (pituitaries).

Ingredient Per cent

Egg albumin* 40 r-Starch 20 Dextrin 24 Soybean oilt 3 Cod liver oil 2 Vitamin mixture3 2 Mineral mixture 9

The scales and rtbs were taken from a defined portion of the abdomen and immersed in glycerol. The tetracycline labels of the glycerinated spectmens were observed ut toto by a fluorescence microscope to determine the growth dur- ing the experimental period. Subsequently. the same speci- mens were stained by von Kossa’s method to observe the degree of mineralization. The growth of the scales and ribs was represented as average values of apposition measured at three points per sample.

* Denatured by boiling with ethyl alcohol and extracted with diethyl ether.

The total serum calcium and inorganic phosphate con- centration were determined by the same methods as de- scribed for diets.

t Contained 40 mg of vitamin E per 100 g of total diet. : Mixture of thiamine-HCI, 6; riboflavin, 20; pyridoxine-

HCI, 4; nicotinic acid, 80; calcium pantothenate, 28; inositol, 400; biotin, 0.6; folic acid, 1.5; p-aminobenzoic acid, 40; cholin chloride, 800; vitamin B,2. 0.009; ascorbic acid, 200; a-tocopherol, 40; manadione, 4 (each in mg/lOO g of total diet).

RESULTS

Experiment 1

operated fish were divided into two groups, transferred to distilled water and fed the control or the CaD-PD diet 3.5?,;, of body weight a day for 9 days. Other procedures were the same as described in Experiment 1.

The stanniectomy was performed after anesthetizing the fish with MS-222. An inverted V-shaped incision was made on the abdomen and the corpuscles of Stannius, located posteriorly to the kidney, were carefully removed with fine watchmaker’s forceps, working under a binocular micro- scope. The incised abdomen was sewn up after the oper- ation The sham-operation involved the same procedures except for the actual removal of the corpuscles of Stannius. Total or partial removal of the Stannius corpuscles was checked by histological inspection of adjacent tissues after the rearing experiment.

All fish looked healthy and behaved normally dur- ing the experimental period. The body weight of the control diet group increased 28% after 2 wk and 93% after 4 wk in distilled water, whereas the CaD-PD diet group showed increases of 16 and 53% respect- ively. The growth of the scales and ribs in the CaDPD diet fish was not as good as that of the control diet fish (Fig. 1). In the control diet fish, newly grown portions of both scales and ribs were fully mineralized except for the narrow marginal osteoids but these portions in the CaDPD diet fish were under mineralized (Fig. 2). However, scale ridges were found to be normal in all fish.

Blood samples were obtained by caudal section without using anesthetics and centrifuged at 3000 rpm for 20 min. The separated sera were stored at -40°C until chemical analyses were performed.

The fish fed on the CaD-PD diet maintained their serum calcium level in the normal range until 2 wk, but developed hypercalcemia after 4 wk (Fig. 3). In contrast, the CaD-PD diet lowered the serum in- organic phosphate level rapidly, and fish on this diet developed severe hypophosphatemia after 4 wk. The Stannius corpuscles showed histological features of increased activity after 3 days of CaD-PD diet in half

The corpuscles of Stannius and the ultimobranchial glands with surrounding tissues were fixed with Bouin’s fluid. The pituitaries with surrounding bones were fixed with Bouin-Holland fluid and then decalcified with a 10% EDTA solution. The fixed tissues were dehydrated in etha- nol and embedded in paraffin. Paraffin sections were stained with Delafield’s hematoxylin and eosin (ultimo-

C : control diet

E : CaD-PD diet 0 osteoid zone LZI calcified zcne

Scale

Table 2. Composition of the mineral mixture

Ingredient CaDPD

Control diet

NaCl 1.87 7.62 MgSO, -7Hz0 6.75 6.75 NaH2P0,-2H20 39.16 FeC6H507-xH20 2.97 2.97 CaCH,CH(OH)COO,-5H,O 43.66 AlCI,--6H,O 0.018 0.018 ZnSO,--7H,O 0.357 0.357 MnSO,4 7 6Hz0 0.08 0.08 CUCI, 0.011 0.011 KI 0.017 0.017 CoCI,--6H,O 0.105 0.105 KCI 5.0 5.0 Cellulose 77.07

Total (g) 100 100

Rib

0 3 14 28

Days on diet

Fig. 1. Effects of the calcium- and phosphorus-deficient (CaDPD) diet on the growth and mineralization of scales and ribs. Bars represent standard errors of the means. Figures in parentheses refer to the number of fish

examined.

Ca regulatory systems in Ca- and P-deficient goldfish 711

Fig. 2. Scales (upper) and ribs (lower) 4 wk after the rearing experiment. Growth zones, indicated by arrows, were nar- row and not calcified in calcium- and phosphorus-deficient diet groups (right) compared with those in control diet

groups (left). von Kossa stain. Scales, x 90; ribs, x 150.

of the fish and after 2 and 4 wk in all fish (Fig. 4); the cell nuclei were larger in diameter and the cytoplasm stained deeper with hematoxylin compared to those of the control diet group. The active cells at day 3 showed frequent mitotic figures.

The ultimobranchial gland showed no histological difference between the control and CaD-PD diet groups through the experimental period. The prolac- tin cells of the pituitary examined histologically at day 3 were found to be unaffected by the CaD-PD diet.

Experiment 2

There were only 8 and 6% weight increases in the control and CaD-PD diet groups, respectively, 14 days after the surgical operation because of a tran- sient weight loss caused by the stress of the operation and the subsequent fasting for 5 days. The growth of

0 3 14 28 Days on CaD-PD diet

Fig. 3. Effects of the calcium- and phosphorus-deficient (CaBPD) diet on serum calcium and inorganic phos- phorus levels represented by percentage values to controls.

the scales and ribs was not of sufficient magnitude to distinguish differences between the two groups. All fish looked healthy and were behaving normally at the end of the experiment. The CaD-PD diet resulted in hypophosphatemia but not hypercalcemia in the sham-operated fish (Fig. 5). This is similar to the results with the CaD-PD fish of 2 wk feeding in Ex- periment 1. The CaD-PD diet brought about hyper- calcemia in stanniectomized fish, especially in those whose corpuscles were totaily removed. In the control diet group, however, the stanniectomy had no signifi- cant effect on serum calcium. Stanniectomy appeared not to affect serum inorganic phosphate in either diet group. The prolactin cells showed no histological dif- ference between the four groups (i.e. the stanniecto- mized and the sham on each diet).

DISCUSSION

Watanabe et al. (1980) reported that a deficiency in dietary calcium did not affect the body growth of chum salmon when they were maintained in fresh- water containing calcium at around 20ppm. Dietary calcium is not necessary for the survival of fish because they can more actively absorb calcium across the gills and the integument from ambient water than from diets (cited from Simkiss, 1974). Dietary phos- phorus, however, is important because only a small amount of phosphorus is contained in freshwater or seawater. PD diets have been reported to retard weight gain in channel catfish (Andrews et al., 1973; Lovell, 1975), carp (Ogino & Takeda, 1976), rainbow trout (Ogino & Takeda, 1978) and chum salmon (Watanabe et nl., 1980). Dietary phosphorus also

Fig. 4. Stannius corpuscles 2 wk after starting the rearing experiment (l), From control diet group. (2), From calcium- and phosphorus-deficient diet group. The nuclei became larger and paler, and the

cytoplasm became more basophilic in (2). H-E stain. x 600.

c .“.I’.,*, 72 4 +i

712 SHIGETAKA YAMANE et ul.

‘St- Ca - ”

.

Sham STX Sham STX

Control CaD-PD diet diet

Fig. 5. Serum calcium and inorganic phosphorus levels (mg/lOO ml) in fish 2 wk after stanniectomy (STX). The STX and sham operated fish were fed the control or cal- cium- and phosphorus-deficient (CaD-PD) diet for 9 days.

greatly affects the ash, calcium and phosphorus con- tent of bones and the whole body (Ogino & Takeda, 1978; Watanabe et al., 1980).

In mammals, PD diets cause hypercalcemia and hypophosphatemia (Freeman & McLean, 1941; Lotz et al., 1968; Baylink et al., 1971; Bruin et al., 1975; Massry, 1978; Brautbar et al., 1979; Rader et al., 1979), whereas CaD diets cause hypocalcemia and hyperphosphatemia (Stauffer et al., 1973; Rader et al., 1979). In rats reared on a PD or CaD diet, there was a marked stimulation of bone resorption and a marked inhibition of bone matrix formation and mineralization accompanied by an increase in width of the periosteal osteoid and decreases in bone cal- cium and phosphorus content (Baylink et al., 1971; Stauffer et al., 1973; Bruin et al., 1975; Ivey et al., 1978). These findings in rats are consistent with our results in goldfish where an inhibition of mineraliza- tion a reduced rate of matrix formation, and an in- .7- crease in width of the osteoids in ribs and scales were observed accompanied by normo- or hypercalcemia and hypophosphatemia. Baylink et al. (1971) indi- cated that hypophosphatemia causes an immediate increase in transfer of calcium from bones to blood through an acceleration of bone resorption. They also observed that the increase of total serum calcium caused by a PD diet was due in part to an increase in ionized calcium.

Rearing of goldfish in diluted seawater stimulates their Stannius corpuscles and the stanniectomy results in an increase in the plasma calcium level (Ogawa, 1963, 1968). The hypercalcemia following stanniec- tomy is dependent on the availability of exogenous calcium (Pang, 1973; Fenwick, 1974). We previously observed, in goldfish reared in distilled water, that the cells of the Stannius corpuscles contained ultrastruc- turally vacuolated secretory granules which indicate a low synthetizing activity of the cells (unpublished). The activity of the Stannius corpuscles is also de- pressed in guppy reared in deionized water (Tomasulo et al., 1970). The present study showed that the stan- niectomy did not induced hypercalcemia in goldfish

maintained in distilled water when they were fed on the normal diet. On the other hand, the goldfish fed on the CaD-PD diet showed highly active Stannius corpuscles in spite of having been reared in distilled water. We also observed previously that the cells had well-developed rough endoplasmic reticulum in the cytoplasm under the same condition. The stanniec- tomy of these fish caused an increase in serum cal- cium after 2 wk. The normocalcemia observed for the first 2 wk in the intact CaD-PD diet group may be explained as follows: serum calcium levels were main- tained by the activated Stannius corpuscles. The hypercalcemia after 4 wk was a result of Ca and P deficiency beyond the controlling capacity of the hypocalcemic effect of the corpuscles of Stannius. This indicates that the Stannius corpuscles are stimulated not only by exogenous calcium but also by endogen- ous calcium. In another experiment, we found that the corpuscles of Stannius of goldfish were stimulated by an increase in ionized calcium in the serum but not in bound-calcium; cytoplasmic granules in the corpuscle cells were released by an intraperitoneal injection of calcium chloride but not of estradiol-l7/$ though in both cases the total serum calcium was raised. The source of endogenous ionized calcium which stimu- lated the Stannius corpuscles may, at least in part, be hard tissues where mobilization of calcium was induced by hypophosphatemia, as in the case of the PD rats (Baylink et al.. 1971). The Stannius corpuscles which were stimulated by calcium from hard tissues possibly prevented an excessive rise in serum calcium over the normal range for at least 2 wk. Removal of the Stannius corpuscles in eels did not stimulate bone resorption in spite of an increase in serum calcium (Lopez, 1970; Lwowski, 1978). Stanniectomy or rear- ing in acalcemic water elevated the rate of renal cal- cium excretion in eels (Butler, 1969; Fenwick, 1974, 1978). On the other hand, stanniectomy increased gill calcium influx and decreased calcium outflux (So & Fenwick, 1977; Milet et al., 1978). Therefore, the hypercalcemia observed in the stanniectomized gold- fish fed on the CaD-PD diet in acalcemic water may be due to an increased plasma calcium retention caused by a decrease in gill calcium outflux.

The ultimibranchial gland of fish produces and se- cretes calcitonin which exerts a powerful and rapid hypocalcemic response in mammals. On the other hand, prolactin in the pituitary has a hypercalcemic effect in fish (Pang, 1973). Greven et al. (1978) reported that removal of the Stannius corpuscles from freshwater sticklebacks led to a significant increase in plasma calcium and a marked reduction in prolactin cell activity. In our experiments, however, histological changes were detected neither in prolactin cells nor in the ultimobranchial gland despite remarkable alter- ations in serum calcium and inorganic phosphate concentrations. The ultimobranchial gland or calcito- nin in fishes appears to be involved in the process of maturation of the ovary or in some related processes (Yamane, 1980). Even if the ultimobranchial gland plays a role in calcium metabolism in goldfish, its function must be much weaker than that of the cor- puscles of Stannius.

Acknowledgements-We wish to express our deepest appreciation to Professor Juri, Yamada of the Faculty of

Ca regulatory systems in Ca - and P-deficient goldfish 713

Fisheries, Hokkaido University, for his kindness in reading phosphorus-depletion syndrome in man. New Engl. J. the manuscript. Med. 278,409-415.

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