Calcif. Tiss. Res. 20, 173--186 (1976) 9 by Springer-Verlag 1976
Effects of Calcitonin and Uhimobranchialectomy (UBX) on Calcium and Bone Metabolism in the Eel,
Anguilla anguilla L. E. Lopez, J. P e i g n o u x - D e v i l l e , F. Lallier, E. M a r t e l l y a n d C. Milet Laboratoire de Physiologie g6n6rale et eompar@, Mus6um national d'Histoire naturelle and Laboratoire d'Endocrinologie eompar6e associ6 au C.N.R.S., Paris Received December 27, 1974, accepted September 10, 1975 Prolonged administration of synthetic salmon calcitonin (SCT) to immature female silver eels, maintained in sea water, provoked a decrease of the serum calcium concentration and an increase of both the osteoblastic apposition and of the degree of mineralization of the intercellular matrix in the vertebral bone. The osteoclastic resorption and osteocytic osteolysis were not significantly affected, however the osteoclastic index was reduced. The ultimobranchial body, site of CT secretion, was cauterized in immature female silver eels maintained in Ca++ rich tap water. This operation resulted in a rise in serum calcium levels with a maximal response after two weeks. After UBX, the vertebral bone osteoblastic apposition stopped completely but the osteoclastic resorption was not modified. The degree of osteocytic osteolysis did not vary. We also observed a significant decrease in the degree of mineralization of the bone organic matrix. The observations made after UBX confirm those obtained after exogenous CT treatment. SCT administered preventively to immature female eels (maintained in sea water), before experimental maturation, inhibited, at least partially, the acute osteoclastic resorption and completely inhibited the bone demineralization induced by carp pituitalT extract. The increase of osteocytic osteolysis, usually observed, did not appear. Key words: Fish-bone - - Calcitonin - - Ultimobranchialectomy - - Calcium - - Mineralization.
Introduction Teleost fish m a i n t a i n e d u n d e r various conditions are able to regulate t h ei r calcium homeostasis. T h e m e c h a n i s m s of this e n d o c r i n e r e g u l a t i o n are n o t y e t well known. R a s q u i n a n d R o s e n b l o o m (1954) were t h e first to i n d i c a t e t h a t t h e ultimobranchial b o d y (UB) in fish m i g h t be i n v o l v e d in calcium m et ab o l i sm . Later, Copp et al. (1967), e x t r a c t e d calcitonin (CT) f r o m t h e U B of dogfish a n d Squalus suckleyi; h y p o c a l c e m i c responses were s u b s e q u e n t l y o b s e r v e d with U B e x t r a c t s from sharks (Urist, 1967) a n d teleosts ( C o p p e t al., 1968). I t was well established at t h a t t i m e t h a t CT was an i m p o r t a n t h y p o c a l c e m i c f a c t o r in m a m m a l s . Mamm a l i a n CT was r e p o r t e d in a m a j o r i t y of studies to h a v e no h y p o c a l c e m i c effect in teleosts (Pang a n d Pickford, 1967; K e n n y , 1972). W e also failed to elicit a h y p o c a l c e m i c response in t r o u t m a i n t a i n e d in Ca++-rich t a p w a t e r b y i n j e c t i n g porcine CT (Lopez et al., 1971), h o w e v e r we observed a positive effect in t r o u t m a i n t a i n e d in Ca++-frec fl'esh w a t e r ~- t h y r o x i n e . Others au t h o r s showed t h a t m a m m a l i a n CT was effective in Ictalurus melas (Louw et al., 1967) an d in An-
For reprints: Mine Evelyne Lopez, Laboratoire de Physiologic du Mus6um, 7, rue Cuvier F-75005 Paris, France.
174
E. Lopez et al.
guilla japonica (Chan et al., 1968). P a n g a n d Griffith (cf. Pang, 1971, u n p u b l i s h e d data) have observed hypocalcemia a n d h y p o p h o s p h a t e m i a i n fresh w a t e r - a d a p t e d Anguilla rostrata i n j e c t e d with s a l m o n calcitonin (SCT) b u t the same t r e a t m e n t was ineffective i n sea w a t e r - a d a p t e d animals. I n fish a t o t a l lack of hypocalcemic response to fish CT was also reported (Orimo et al., 1971; C o p p c t al., 1968; Pang, 1973) ; however it was p o i n t e d out t h a t hypercalcemia i n d u c e d b y various experim e n t a l m e a n s s t i m u l a t e d U B a c t i v i t y (Lopez et al., 1968; Chan, 1969; Lopez, 1973) a n d t h a t U B a c t i v i t y was related to serum calcium f l u c t u a t i o n s i n Salmo salar (Deville et Lopez, 1970). Dacke et al., (1971) observed t h a t fish plasma CT responded to injected calcium a n d the a d d i t i o n of calcium to the external m e d i u m (Deftos et al., 1972) provoked a n increase in the circulating CT levels. I n previous reports we have described some effects of exogenous CT on the bone catabolism i n the v e r t e b r a l skeleton of some teleosts with cellular bone (Lopez et al., 1971; Lopez, 1973; Lopez a n d Deville, 1973). I t has also been reported t h a t u l t i m o b r a n c h i a l e c t o m y could affect serum calcium c o n c e n t r a t i o n s in teleost fish (Chan, 1969), a n d bone in a m p h i b i a n s (Robertson, 1969 a, b) a n d birds (Copp, 1972). The present s t u d y was carried out to investigate (1) the effect of prolonged a d m i n i s t r a t i o n of SCT on bone apposition a n d a presumed p r e v e n t i v e effect of SCT on bone catabolism provoked b y e x p e r i m e n t a l m a t u r a t i o n i n the female eel, a n d (2) to elucidate the effect of u l t i m o b r a n c h i a l e c t o m y on calcium a n d bone m e t a b o l i s m i n the female silver eel.
Materials and Methods Female silver eels obtained from Peronne (Somme) were kept under laboratory conditions in aquaria at 18-20 ~ for one month. All were immature silver eels ranging in weight from 400 to 600g for the first experiment and 170 to 280g for the second. First Experiment. One week before the beginningof the experiment the eels were progressively adapted to sea water. Eels were divided into four groups a) 6 fish were kept. in sea water during the whole experiment. They received a vehicle gelatine injection 3 times a wk. b) A second group (5 fish) was injected intraperitoneally with SCT (Sandoz labs, S.A. Basel, Switzerland) at dose of 3 MCI~ mU/g bw, dissolved in gelatin solution, 3 times a wk over a 7 wk period. c) a third group (4 fish) was treated similarly; then they were given intraperitoneal injections of carp pituitary extract (CPE, Stoller Fisheries, Spirit Lake, Iowa, USA), lmg/100g bw per injection 3 times a wk, until complete maturation, according to the method described by Fontaine et al. (1964). d) a fourth group (7 fish) received CPE, 3 times a wk (lmg/100g bw per injection) until complete maturation. Immediately after spawning, the eels of the third and fourth groups were killed at the same time as the eels of the first and second groups. Second Experiment. Female silver eels were maintained in Ca++ rich tap water (80mg/1). They were divided into 2 groups and before the operation were anaesthetized with MS 222 (Sandoz labs). The first group (21 fish) was sham operated. The second group (39 fish) was operated on as described in Fig. 1. This operation differed from Chan's method (1969) and was performed without haemorrhage and without damage to the neighbouring tissues such as the heart and liver. The epithelium of the oesophagus healed approximately 2 days after the operation. In the eel, the UB cannot be seen with the naked eye nor with a stereomicroscope. Some fish of both groups were sacrificed one week after the operation. Others were killed after 2, 3 and 5 wks. At the end of the 2 experiments blood was collected from the ventral aorta. Before and during the experiment blood was collected by heart puncture. The blood plasma calcium (total calcium) was determined by the atomic absorption method (Perkin-Elmer). Completeness of the ultimobranchialectomies was estimated by examining serial sections of fixed and
Effect of Calcitonin and UBX on Fish Bone Metabolism
Posterio
175
rior part
Ventral pithelium
Fig. 1. Diagram showing the location of the ultimobranchial body (UB) in eel, and the method of cauterization employed for ultimobranchialeetomy
paraffin-embedded pieces of the transverse septuln and proximal oesophagus. Stannius corpuscles were stained using the Cleveland Wolfe method. The vertebral bone was examined on thin mineralized bone sections (10 to 15 ~z) and on microradiographs. The degree of mineralization of the bone was determined by means of a precise histophotometric method (Baud, 1957) and the morphologic quantification of bone turnover according to the method described by Jowsey et al., (1965). Osteocytic osteolysis was expressed as a percentage of the total number of osteocytic cavities. The osteoclastic index (OI) was estimated, according to the method of Schenk et al., (1969). Details of these techniques are given in a previous paper (Lopez, 1973).
Results
I n t h e first e x p e r i m e n t (Table 1) t h e serum cMeium c o n c e n t r a t i o n of control eels m a i n t a i n e d in sea w a t e r r e m a i n e d c o n s t a n t during t h e whole e x p e r i m e n t . SCT i n j e c t e d into i m m a t u r e f e m a l e eels (group 2), over a period of t h r e e weeks p r o v o k e d a slight b u t significant decrease in s e r u m calcium levels which subseq u e n t l y increased w h e n SCT t r e a t m e n t was stopped. I n t h e t h i r d group C P E i n d u c e d h y p e r c a l c e m i a in spite of t h e p r e v e n t i v e h y p o c a l c e m i c SCT t r e a t m e n t . I n group 4, m a t u r e f e m a l e eels showed a m ~ r k e d h y p e r c a l c e m i a . Table 1. Serum calcium level (rag/l) Groups b No. eels
1 wk before treatment
128.0 ~2 2.79 a
3 weeks after 8 wks after start of start of treatment treatment
1
6
2
5
3
4
111.6~- 1.66 96.6• 2.66 p < 0.01 F test
4
7
117.5 ~: 2.81
93.4~- 1.28 85.6-J_ 3 , 0 1 0 . 0 1 ~ p ~ 0.05 2' test
1 wk after CPE
91.24- 6.05
109.1 ~ 1.64
End of experiment
120.0•
5.36
l14.0-L
4.19
142.7• 5.00
723.5-L 221.26
142.8• 8.24
575.4-t- 155.15
a SEM b Group 1 were controls. Group 2 received SCT (3 MOB, mU/g/bw) over a 7 wk period. Group 3 received similarly SCT then CPE (1 rag/100 g/bw). Group 4 did not received SCT but CPE over all the experiment (1 rag/100 g/bw).
176
E. Lopez et al. Table 2 Osteoclastic resorption % surface
OI %
Osteocytic osteolysis %
Osteoblastic apposition % surface
Degree of mineralization of the bone organic matrix g/em3
First group (1) controls n:9
13.78:[: 0.502 a 0.023~ 0.001
4.53 • 0.193
4.20 • 0.141
1.17::t=0.055
Second group (2) SCT n--5
12.02•
0.011:J::0.001
4.30 :J: 0.167
13.14~ 0.223
1.46-4-0.022
Third group (3) SCT then CPE n--4
23.12~ 0.983
0.205-4-0.009
4.95~ 0.171
12.17~ 0.696
1.31~- 0.035
Fourth group (4) CPE n:7
47.40~ 1.170 0.460~ 0.009
40.01:J: 0.325
7.06=[-0.134
0.98• 0.019
Statistical results Osteoclastic resorption
1-4; 1-3; 3-4; 2-3 p ~ 0.001; 1-2 p ~ 0.05.
F test
OI
1-2; 1-3; 1-4; 2 3; 3-4 p~0.001.
Osteoeytic osteolysis
1 4 ; 3-4 p ~ 0.001 ; 1-3 p ~ 0.2 ; 1-2 ; 2-3 p ~ 0.05
Osteoblastic apposition
1-4 ; 1-3 ; 1-2 ; 3-4 p ~ 0.001 ; 2-3 p ~ 0.05
Bone degree of mineralization 1-4; 1-2; 2-3 p~0.01; 3-4 p ~ 0.001; 1-3 p ~ 0.05 aSEM.
The effects of SCT were studied on the various bone parameters. SCT injected into i m m a t u r e silver eels (Table 2, group 2) did n o t significantly affect either osteoclastic resorption or osteoeytic osteolysis b u t it decreased OI a n d conseq u e n t l y the n u m b e r of m u l t i n u c l e a t e d osteoclasts. SCT increased both t h e degree of m i n e r a l i z a t i o n of bone m a t r i x a n d the osteoblastic apposition. I n histological bone sections after SCT t r e a t m e n t of eels (group 2) there was a n increase in the n u m b e r a n d a c t i v i t y of osteoblasts (Figs. 2 a n d 3). B e n e a t h t h e active osteoblasts there was a new bone layer which showed on microradiographs a low m i n e r a l d e n s i t y a n d a smooth surface characteristic of n e w bone formation. E x p e r i m e n t a l m a t u r a t i o n in female silver eels (Table 2, group 4) produced a m a r k e d demineralization of t h e bone m a t r i x as well as osteocytic osteolysis a n d osteoclastic resorption as evidence b y the n u m b e r of m u l t i n u c l e a t e d osteoclasts (OI). The cells which bordered the free bone edges, m a n y of which were osteoblasts, were increased in n u m b e r (Fig. 4). SCT injected as a p r e v e n t i v e t r e a t m e n t before a d m i n i s t r a t i o n of C P E (Table 2, group 3), i n h i b i t e d t h e effects of m a t u r a t i o n on osteocytic osteolysis a n d the degree of m i n e r a l i z a t i o n of the organic m a t r i x (Table 2, groups 3 a n d 4). The t r e a t m e n t partially p r e v e n t e d t h e osteoelastie resorption i n d u c e d b y t h e experim e n t a l m a t u r a t i o n (Table 2, group 4), however t h e osteoclastie resorption a n d OI (group 3) r e m a i n e d higher t h a n i n the controls (group 1). I t was observed t h a t
Fig. 2. I m m a t u r e female silver eel. P a r t of vertebral neural arch. Osteoblasts (--~) showing moderate activity and others which are more active ( 9 ) . (Basis fuehsin • 360)
Fig. 3. I m m a t u r e female silver eel treated with SCT. P a r t of vertebral neural arch. Osteoblasts (--~). New bone layer ( 9 ) . We note here t h a t osteoblasts are active cells (compared to those of Fig. 2) with a clear nucleus, a single nueleolus and a granular cytoplasm. (Basic fuchsin, • 360)
Fig.4. Mature female eel. Part of vertebral hemal arch. Tangential cut showing very activo lining cells. (--~). (Basic fuchsin, • 360)
Fig. 5. Mature female eel. Part of vertebral hemal arch. Large mononuclear cells (preosteoclasts (--~) whose nuclei contain numerous chromatin granules. Their apex (ll~) is always prominent and rounded as compared with that of active osteoblasts (Fig.3). Some preosteoclasts ( 3 ) are fusing to become multinucleated osteoclasts. (Basic fuehsin, X 360)
Fig. 6. Mature female eel. P a r t of hemal arch consisting of cancellous bone; enlarged resorption lacunae containing multinucleated osteoclasts (--)*), preosteoclasts ( ~ ) and osteoprogenitor cells ( ~ ) . (Basic fuchsin, X 360)
Fig. 7. Mature female eel injected with SCT before CPE treatment. P a r t of hemal arch consisting of cancellous bone. Lacunae similar to t h a t of Fig. 6. Note the absence of multinucleated osteoclasts. One preosteoclast is seen (--~). Osteoblasts are evident (11.~). (Basic fuchsin, x 360)
30,
:6
20,
10,
~ . _ n:8
1'
J 2 I n:9
3
4 84
5
~n:6 i
6~:6 weeks i
i
~10.
Fig. 8. Effect of ultimobranchialectomy (UBX) on serum calcium levels of the eel maintained in calcium-rich tap water (80rag/l). Serum calcium is presented as a per cent of the initial values for control eels (-o-) and ultimobranchialectomized eels (--.--). Vertical bars represent the S. E.M. and n equals the number of observations
Fig. 9. U B X female eel. P a r t of neural arch. Osteoblasts (--~) are seen as very flat cells with basophilic nuclei ( 3 ) . There are resting osteoblasts ( ~ ) along highly mineralized bone surfaces. (Basic fuchsin, X 360)
Effect of Calcitonin and UBX on Fish Bone Metabolism
181
Table 3
Sham operated
aSEM UBX
Time after operation (weeks)
Mineral substance concentration g/cm 3 (apatite ~- amorphous phosphate)
Osteoclastic resorption % surface
Osteoplastic apposition % surface
Osteocytic osteolysis %
1 1
1.02 1.01
2 2 3 3 5 5 5
1.10 1.08 1.12 1.07 1.Ol • 0.022 a 1.19 1.08 1.13
11.2 13.8 14.1 14.5 13.2 13.27 13.5 • 0.363 14.3 12.8 12.1
5.2 4.8 4.5 4.9
3.9 3.2 ] 3.8
4.81 5.3 ' -J-0.132 4.8 4.5 5.2 4.1
3.6 I 4.1 4.5
F test
p < 0.001
p > 0.05
p < 0.001
p > 0.05
0.97 0.91
13.41
0.08,
1.00
13.8] 14.2] 14.1 ( 13.88 14.01 • 13.2~ 13.8] 14.5 ] 14.7t
0.13 0.16 0.20 0.13 0.08 -L0.018 0.20 0.18 0.05 0.20
4.2 4.3 4.0 4.1 3.8 4.05 4.0 ~0.058 3.9 3.8 4.1 4.3
0.98 0.92 0.91 0.91 ~0.022 0.83 0.78 0.97 0.87
13.1]
0.091
3.86
m o n o n u c l e a r cells (preosteoclasts) in eel bone fused to become m u l t i n u c l e a t e d osteoclasts (Fig. 5). M i c r o r a d i o g r a p h i c a l l y it a p p e a r e d t h a t these m o n o n u e l e a r cells were a c t i v e in bone resorption. L a r g e r e s o r p t i o n spaces lined b y m u l t i n u e l e a t e d osteoclasts (Fig.6) were seen in t h e v e r t e b r a l archs of t h e m a t u r e female eels (Table 2, group 4). The v e r t e b r a l bone of m a t u r e female eels given SCT before C P E showed smaller r e s o r p t i o n spaces (Fig.7) whose bone surfaces were lined with a few m o n o n u e l e a r preosteoclasts, some of which were active, a n d also b y active osteoblasts. U l t i m o b r a n c h i a l e c t o m y r e s u l t e d in a n increase in s e r u m calcium levels, r e l a t i v e to t h e initial values, reaching a m a x i m u m after two weeks a n d s u b s e q u e n t l y declining to a p p r o x i m a t e l y physiological concentrations. S e r u m calcium concent r a t i o n in t h e controls d i d n o t v a r y b y m o r e t h a n 10 % a n d d i d n o t exceed t h e v a r i a t i o n f o u n d b e t w e e n i n d i v i d u a l s (Fig. 8). I n v e r t e b r a l bone several changes r e l a t e d to t h e osteoblasts a p p e a r e d d u r i n g t h e first week after o p e r a t i o n a n d p e r s i s t e d two, t h r e e a n d five weeks later. W h i l e t h e bone of controls was lined b y active osteoblasts on some surfaces especially along new bone surfaces (Fig.2), in bone of U B X eels, osteoblasts were r e d u c e d in size, n u m b e r a n d in t h e r e g u l a r i t y of t h e i r spacing along t h e bone surfaces (Fig. 9). Osteoblasts in U B X eels were seen as c o m p l e t e l y flat cells lining i n a c t i v e bone surfaces. A b a n d of high m i n e r a l d e n s i t y seen in m i e r o r a d i o g r a p h s i n d i c a t e d t h a t osteoblastic a p p o s i t i o n h a d ceased (Table 3). A f t e r U B X (Table 3) we d i d n o t
182
E. Lopez et al.
observe changes in the number of osteoclasts nor were osteoclastic resorption surfaces increased and the degree of the osteocytic osteolysis remained constant. However it was noted that the average rate of mineralization of the bone matrix was noticeably decreased relative to the control values. Stannius corpuscles were found to be very active glands in operated eels. Spheric follicles were generally clumped together into sinuous strands. We have previously described the formation of these strands after stimulation of the Stannius corpuscles (Lopez et Fontaine, 1967). Between the cellular strands extended large blood vessels. Based on these observations and compared with those previously made concerning Stannius corpuscles, there is little doubt that these glands were in their active phase. Discussion
The present results indicate that injections of exogenous CT produce many important effects on the bone of the eel. However, administration of an exogenous hormone cannot exactly reflect the total physiological effect. The existence of the UB as a distinct organ in fish, provided us with an excellent means of evaluating its physiological significance. I n this investigation a decrease of serum calcium concentration in SCT-treated immature silver eels maintained in sea water, was observed. This observation contradicts that reported by Pang (1971) showing that CT treatment was ineffective on serum calcium in sea water-adapted Anguilla rostrata. This variance could be due to the fact that the eels were not collected at the same period of their vital cycle in the two experiments. Furthermore, SCT provokes a marked cell atrophy of the ultimobranchial body in female silver eels (Peignoux-Deville et al., 1975). The experimental maturation of the female eel (Fontaine et al., 1964) provokes an acute hypercalcemia in keeping with the rise of the gonosomatic ratio, a high bone catabolism and a significant stimulation of the UB (Lopez et al., 1968). These results are consistent with those of Watts et al. (1975) on the salmon. The high level of CT in the blood of the mature female salmon and the hyperactivity of UB in the mature female eel and the female conger eel (Lopez, 1973) are probably related to the elevated catabolism observed in these animals. CT in female fish may have a role in the protection of skeletal calcium until spawning, similar to that of mammals during pregnancy and lactation (Lewis et al., 1971). The negative effect on serum calcium observed after both SCT treatment and CPE injections (SCT injected preventively before CPE treatment and SCT injected in addition to CPE, Lopez and Deville, 1973) can be explained, at least partially, by the fact that in spite of the SCT effect in reducing osteoclastic resorption and osteocytic osteolysis, bone catabolism is sufficient to maintain a high serum calcium concentration. Ultimobranchialectomy in the female silver eel, induces a significant rise in serum calcium levels, returning to physiological levels during the third week after operation. Increase in serum calcium concentrations was reported by Chan (1969) in U B X Anguilla japonica, recently by Fenwick in partially U B X Carassius auratus (1975)and by I~obertsonin U B X l~ana pipiens (1969a). The two bone effects obtained in U B X eels: demineralization of the organic matrix and inhibition of the osteoblastic apposition, can partially explain the observed hyper-
Effect of Calcitonin and UBX on Fish Bone Metabolism
183
caleemia during the first week. The prolonged hyperealcemia until the second week can be the result of the failure of CT effect on the kidney (Chan, 1972) and of modifications in calcium exchange across the gills. Preliminary results (Peignoux-Deville et al., unpublished data) lead us ~o believe that CT (SCT) perfnsed in isolated gills from U B X eels, acts on calcium influx and outflux resulting in a lowering of the serum eMeium concentration. The control of hypercalcemia after two weeks in U B X eels cannot be a parathormone response as Robertson has demonstrated in the frog (I~obertson, 1969a) because parathyroid glands appear to be absent in fish. Stannius corpuscles of U B X eels are found to be very active glands in our experiments, we have not been able to show atrophic changes as did Chan (1972). These glands were shown to induce hypocaleemia in teleosts by Fontaine (1964, 1967) principally by acting on the gills (Fontaine etal., 1972; Fenwiek and Peng So, 1974; Milet et al., 1975). We suggest that the hypereMeemia induced by ultimobranehiMectomy in eels is regulated by the Stannius corpuscles. The observations reported here show the effect of exogenous SCT on the osteoblastic bone apposition in female silver eels. This CT effect is confirmed by the drastic decrease of osteoblastic apposition from the first week after ultimobranchialectomy. Robertson (1969a) in U B X Rana pipiens described a similar effect on bone apposition but in 12 week post-operative animals. This variance m a y be due to the fact that in the frog the first target organs for CT are paravertebral lime sacs and not the skeleton. We have previously reported t h a t osteoblastic apposition in fish m a y also be regulated by the Stannius corpuscles (Lopez, 1970a, Lopez, 1973). UB and Stannius corpuscles seem to have an antagonistic effect on eel bone in spite of their similar hypoeMcemic role and we are convinced that serum calcium concentrations do not reflect the complex mechanisms which maintain calcium homeostasis in fish. We have also pointed out that gonadal maturation increases osteoblastie activity and we interpret this as being the effect of the sequential process of the bone remodeling (Frost, 1966). SCT injected in immature female silver eels does not significantly affect the degree of osteoelastic resorption but decreases the pool of multinueleated osteoclasts. We suggest that, in this ease, the measured average degree of osteoelastie resorption results principally from the activity of preosteoelasts. Ultimobranehialeetomy does not modify the number of osteoelasts therefore the osteoclastie resorption is unchanged. These results confirm our previous reports which showed that CT acted effectively on the osteoelastie resorption in fish bone only when bone catabolism was high. Similarly neither SCT nor ultimobranchialectomy modify the physiological degree of osteoeytie osteolysis. In the bone of mature female eels treated preventively with SCT before CPE osteoclastie resorption does not increase as it does in the bone of mature eels injected with CPE. However osteoelastic resorption remains greater than in the controls, probably because the effect of SCT is counteracted by oestrogens as we have shown previously (Lopez and Deville, 1973). We suggest that, when injected after CPE treatment, SCT destroys the mnltinueleated osteoclasts (Lopez, 1973; Lopez and Deville, 1973) but when SCT is given as a preventive treatment it probably prevents preosteoc]asts from fusing into active multinucleated osteoclasts. SCT administered before experimental maturation, completely inhibits the ability of the osteoeyte to resorb its surrounding bone.
184
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I n previous r e p o r t s we h a v e p r o v i d e d evidence of a diffuse decalcification w i t h o u t m o d i f i c a t i o n of t h e organic m a t r i x (Lopez, 1970b; Lopez et M a r t e l l y Bagot, 1971) which is due to t h e loss of some p a r t of t h e a m o r p h o u s calcium phosp h a t e (Lopez et al., 1970) in t h e v e r t e b r a l bone of m a t u r e eels. I n t h e p r e s e n t s t u d y we p o i n t out t h a t SCT increases t h e degree of m i n e r a l i z a t i o n of t h e bone of i m m a t u r e silver eels a n d w h e n i n j e c t e d p r e v e n t i v e l y it p a r t i a l l y i n h i b i t s dem i n e r a l i z a t i o n in t h e bone of m a t u r e eels. Similar o b s e r v a t i o n s on t h e effect of CT on bone m i n e r a l i z a t i o n has been p u b l i s h e d in birds (Copp et al., q u o t e d b y B61anger, 1971) a n d in m a n (Baud et al., 1970). R e c i p r o c a l l y in t h e female silver eel U B X p r o v o k e s a significant d e m i n e r a l i z a t i o n of t h e bone m a t r i x . W e have p r e v i o u s l y suggested (Lopez, 1973) t h a t CT p r o b a b l y acts on t h e calcium exchange at t h e site of t h e " b o n e lining cellular l a y e r " which p r o b a b l y controls calcium ion t r a n s p o r t (Baud, 1968; N e u m a n n , 1969). The role of t h e UB, in t h e silver eel, a p p e a r s to be to effect a release of a h o r m o n e which controls serum calcium, s t i m u l a t e s t h e p r o d u c t i o n a n d a c t i v i t y of osteoblasts a n d regulates t h e degree ef m i n e r a l i z a t i o n of t h e bone a n d in some cases, reduces bone catabolism. W e are convinced, however, considering all of our results concerning fish bone, t h a t Ca ++ ion t r a n s p o r t which r e g u l a t e s the degree of m i n e r a l i z a t i o n of the intercellular s u b s t a n c e is p r o b a b l y one of t h e m a i n m e c h a n i s m s influencing calcium homeostasis in t e l e o s t fish.
References Baud, C.A. : Radiographies et microradiographies osseuses quantitatives. Praxis 46, 329-331 (1957) Baud, C.A.: Submicroscopic structure and functional aspects of the osteocyte. Clin. Orthop. ~6, 227-336 (1968) Baud, C.A., Siebenthal J.de, Langer, B., Tupling, M.R., Mach, R. S. : The effects of prolonged administration of thyrocalcitonin in human senile osteoporosis. In: Caleitonin 1969. Proceedings of the second symposium (S. Taylor, ed.), p. 540-546. London: Heineman 1970 Belanger, L.F. : The ultimobranehial gland of birds and the effects of nutritional variations. The J. exp. Zool. 178, 125-137 (1971) Chan, D.K.O.: Endocrine regulation of calcium and inorganic phosphate balance in freshwater adapted teleost fish, Anguilla anguilla and Anguilla japonica. In: Progress in Endocrinology. Proc. 3rd Intern. Congress Endocrinol. (C. Gual ed.), p. 709-716. Amsterdam: Excerpta Med. 1969 Chan, D.K.O.: Hormonal regulation of calcium balance in teleost fish. VI gme Symposium international d'Endocrinologie Comparge. Gen. comp. Endocr. Suppl. 3, 411-420 (1972) Chan, D.K.O., Chester-Jones, I., Smith, R.N.: The effect of mammalian calcitonin on the plasma levels of calcium and inorganic phosphate in the European eel (Anguilla anguilla L.). Gen. comp. Endocr. l l , 243-245 (1968) Copp, D.H.: Endocrine regulation of calcium metabolism. Ann. Rev. Physiol. 32, 61-86 (1970) Copp, D.H. : Calcium regulation in birds. Gen. comp. Endocr. Suppl. 3, 441-447 (1972) Copp, D.H., Cockroft, D.W., Kueh, Y.: Calcitonin from ultimobranchial glands of dogfish and chicken. Science 1~8, 924-925 (1967) Copp, D.H., Cockroft, D.W., Kueh, Y., Melville, M.: Calcitonin-ultimobranchial hormone In: Calcitonin 1967 (S. Taylor, ed.), p. 306-321. Berlin-Heidelberg-NewYork: Springer 1968 Dacke, C.G., Fleming, W.R., Kenny, A.D.: Plasma calcitonin levels in fish. Physiologist 14, 127 (Abstract) (1971) Deftos, L.J., Murray, T.M., Powell, D.A., Habener, J.F., Singer, F.R., Mayer, G.P., Potts, J.T. Jr.: Calcium, parathyroid hormone and the calcitonins. (R.V. Talmage, Munson P.L. eds.), p. 140. Amsterdam: Excerpta Med. Found. 1972
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Deville, J., Lopez, E.: Le corps ultimobranchial du Saumon Salmo salar L. Etude histophysiologique s diverses 6tapes de son cycle vital en eau douce. C.R. Acad. Sci. (Paris) 270, 2347-2350 (1970) Fenwick, J.C. : Effect of partial ultimobranchialectomy on plasma calcium concentration and on some related parameters in Goldfish (Carassius auratus L.) during acute transfer from fresh water to 30 % sea water. Gen. comp. Endocr. 25, 60-64 (1975) Fenwick, J.C., Peng So, u A perfusion study of the effect of stanniectomy on the net influx of calcium 45 across an isolated eel gill. J. exp. Zool. 188, 125-131 (1974) Fontaine, M. : Corpuscules de Stannius et r~gulation ionique (Ca, K, Na) du milieu int6rieur de l'Anguille (Anguilla anguilla L.). C.R. Acad. Sci. (Paris) 259, 875-878 (1964) Fontaine, M. : Intervention des corpuscules de Stannius dans F6quilibre phosphocalcique du milieu int4rieur d'un poisson t~l~ost~en, l'Anguille. C.R. Acad. Sci. (Paris) 264, 736-737 (1967) FontMne, M., Bertrand, E., Lopez, E., Callamand, O.: Sur la maturation des organes g~nitaux de l'Anguille femelle Anguilla anguilla L. et l'6mission spontan~e des oeufs en aquarium. C.R. Acad. Sci. (Paris) 259, 2907-2910 (1964) Fontaine, M., Delerue, N., Martelly, E., Marchelidon, J., Miler, C.: R6le des corpuscules de Stannius dans les @hanges de calcium d'un poisson t616ost@n, l'Anguille (Anguilla anguilla L.) avec le milieu ambiant. C.R. Acad. Sei. (Paris) 275, 1523-1528 (1972) Frost, H.M.: Bone dynamics in os~oporosis and osteomalacia, p. 1-176. Springfield: C. Thomas 1966 Jowsey, J., Kelly, P . J . , Riggs, B.L., Bianco, A.J., Scholl~z, D.A., Gershon-Cohen, J.: Quantitative microradiographic studies of normal and osteoporotic bone. J. Bone J t Surg. 47, 785-806 (1965) Kenny, A.D.: Calcium, parathyroid hormone and the calcitonins. Proc. 4th parathyroid conference. Chapel Hill, W.C., USA, March 15-19 (R.V. Talmage, P.L. Munson eds.), p. 9-11. Amsterdam: Excerpta Med. Found. 1972 Lewis, P., Rafferty, B., Shelley, M., Robinson, C.J.: Suggested physiological role of calcitonin. The protection of the skeleton during pregnancy and lactation. J. Endocr. 49, 9-10 (1971) Lopez, E. : L'os cellulaire d'un poisson t416ost6en, Anguilla anguilla L. II-Action de l'ablation des corpuscules de Stannius. Z. Zellforsch. 109, 566--572 (1970a) Lopez, E. : Demonstration of several forms of decalcification in bone of the teleost fish, Anguilla anguilla L. Calcif. Tiss. Res. 4 (Suppl.), 83 (1970b) Lopez, E. : Etude morphologique et physiologique de l'os cellulaire des Poissons t61~ost6ens (th~se de Doctorat d'Etat, 1972). M4moires du Mus6um National d'Histoire Naturelle (nouvelle s6rie), s6rie A, Zoologic, tome L X X X (1973) Lopez, E., Chartier-Baraduc, M.M., Deville, J. : Mise en 4vidence de Faction de la calcitonine porcine sur l'os de la truite Salmo gairdnerii soumise s un traitement d4min~ralisant. C.R. Acad. Sci. (Paris) 272, 2600~603 (1971) Lopez, E., Deville, J. : Effect of prolonged administration of synthetic sahnon caleitonin (SCT) on vertebral bone morphology and on the ultimobranchial body (UB) activity of the mature female eel (Anguilla anguilla L.). Proc. 9th european symposium on calcified tissues, Baden (A. Czitober, J. Eschberger eds.), p. 169-174. Vienna: H. Egermann, Facta-Publication 1973 Lopez, E., Deville, 3 , Bagot, E. : Etude histophysiologique du corps ultimobranchial d'un T~l~ost~en (Anguilla anguilla L.) au cours d'hypercalc@fies expSrimentales. C.R. Acad. Sei. (Paris) 267, 1531--1534 (1968) Lopez, E., Fontaine, M. : RSponse des corpuscules de Sl~annius de l'Anguille (Anguilla anguilla L.) s des blessures exp~rimentales. C.R. Soc. Biol. (Paris) 161, 36-39 (1967) Lopez, E., Lee, I-I.S., Baud, C.A.: Etude histophysique de l'os d'un T~lSostSen, Anguilla anguilla L., au cours d'une hypercalc6mie provoqu6e par la maturation exp~rimentale. C.R. Acad. Sci. (Paris) 270, 2015-2017 (1970) Lopez, E., Martelly-Bagot, E. : L'os cellulaire d'un poisson T61~ost6en, Anguilla anguilla L. III-Etude histologique et histophysique au cours de la maturation provoqu6e par injections d'extrait hypophysaire de Carpe. Z. Zellforsch. 117, 176-190 (1971) 6 Calcif. Tiss. Res.
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Louw, G.N., Sutton, W. S., Kenny, A.D. : Action of thyrocalcitonin in the teleost fish Ictalurus melas. Nature (Lond.) 215, 888 889 (1967) Milet, C., Martelly, E., Fontaine, M.: Corpuscules de Stannius (CS) et flux de calcium au niveau des branehies perfus6es d'Anguilla vulgaris. J. Physiol. (Paris) 71, 141A (1975) Neuma~, W. F. : The milieu interieur of bone : Claude Bernard revisited. Fed. Proe. 28, 18461850 (1969) Orimo, H., Ohata, M., Yoshikawa, M., Abe, J., Watanabe, S., Kotani, M., Higashi, T.: Ultimobranehial caleitonin. II-Physiologieal role of ealeitonin. Igaky No Ayumi 79, 480 (1971) Pang, P.K,T.: Caleitonin and ultimnbranchial glands in fishes. J. exp. Zool 178, 89 100 (1971) Pang, P . K . T . : Endocrine control of calcium metabolism in teleosts. Amer. Zool. 13, 775-792 (1973) Pang, P.K.T., Pickford, G.E.: Failure of hog thyroealcitonin to elicit hypocalcemia in the teleost fish, Fundulus heteroclitus. Comp. Biochem. Physiol. 21, 573 578 (1967) Peignoux-Deville, J., Lopez, E., Lallier, F., Martelly-Bagot, E., Milet, C.: Responses of the ultimobranchial body in eels (Anguilla anguilla L.) maintained in sea water and experimentally matured, to injections of synthetic salmon ealeitonin. Cell Tiss. Res. 164, 73-83 (1975) Rasquin, P., Rosenbloom, L. : Endocrine imbalance and tissue hyperplasia in teleost maintained in darkness. Bull. Amer. Mus. Nat. Hist. 104, 363 425 (1954) Robertson, D.R.: The ultimobranchial body of Rana pipiens. VIII-Effects of extirpation upon calcimn distribution and bone cell types. Gen. comp. Endoer. 12, 479-490 (1969a) Robertson, D.R.: The ultimobranehial body of Rana pipiens. X-Effect of glandular extirpation on fracture healing. J. exp. Zoo]. 172, 425-441 (1969b) Schenk, R., Merz, W.A., Muller, J.: A quantitative histological study on bone resorption in human cancellous bone. Aeta anat. (Basel) 74, 44 53 (1969) Urist, M.R.: Avian parathyroid physiology including a special comment on caleitonin. Amer. Zool. 7, 883 895 (1967) Watts, E.G., Copp, D.H., Deftos, L . J . : Changes in plasma ealcitonin and calcium during the migration of sahnon. Endocrinology 96, 214-218 (1975)
Effects of Calcitonin and Uhimobranchialectomy (UBX) on Calcium and Bone Metabolism in the Eel,
Anguilla anguilla L. E. Lopez, J. P e i g n o u x - D e v i l l e , F. Lallier, E. M a r t e l l y a n d C. Milet Laboratoire de Physiologie g6n6rale et eompar@, Mus6um national d'Histoire naturelle and Laboratoire d'Endocrinologie eompar6e associ6 au C.N.R.S., Paris Received December 27, 1974, accepted September 10, 1975 Prolonged administration of synthetic salmon calcitonin (SCT) to immature female silver eels, maintained in sea water, provoked a decrease of the serum calcium concentration and an increase of both the osteoblastic apposition and of the degree of mineralization of the intercellular matrix in the vertebral bone. The osteoclastic resorption and osteocytic osteolysis were not significantly affected, however the osteoclastic index was reduced. The ultimobranchial body, site of CT secretion, was cauterized in immature female silver eels maintained in Ca++ rich tap water. This operation resulted in a rise in serum calcium levels with a maximal response after two weeks. After UBX, the vertebral bone osteoblastic apposition stopped completely but the osteoclastic resorption was not modified. The degree of osteocytic osteolysis did not vary. We also observed a significant decrease in the degree of mineralization of the bone organic matrix. The observations made after UBX confirm those obtained after exogenous CT treatment. SCT administered preventively to immature female eels (maintained in sea water), before experimental maturation, inhibited, at least partially, the acute osteoclastic resorption and completely inhibited the bone demineralization induced by carp pituitalT extract. The increase of osteocytic osteolysis, usually observed, did not appear. Key words: Fish-bone - - Calcitonin - - Ultimobranchialectomy - - Calcium - - Mineralization.
Introduction Teleost fish m a i n t a i n e d u n d e r various conditions are able to regulate t h ei r calcium homeostasis. T h e m e c h a n i s m s of this e n d o c r i n e r e g u l a t i o n are n o t y e t well known. R a s q u i n a n d R o s e n b l o o m (1954) were t h e first to i n d i c a t e t h a t t h e ultimobranchial b o d y (UB) in fish m i g h t be i n v o l v e d in calcium m et ab o l i sm . Later, Copp et al. (1967), e x t r a c t e d calcitonin (CT) f r o m t h e U B of dogfish a n d Squalus suckleyi; h y p o c a l c e m i c responses were s u b s e q u e n t l y o b s e r v e d with U B e x t r a c t s from sharks (Urist, 1967) a n d teleosts ( C o p p e t al., 1968). I t was well established at t h a t t i m e t h a t CT was an i m p o r t a n t h y p o c a l c e m i c f a c t o r in m a m m a l s . Mamm a l i a n CT was r e p o r t e d in a m a j o r i t y of studies to h a v e no h y p o c a l c e m i c effect in teleosts (Pang a n d Pickford, 1967; K e n n y , 1972). W e also failed to elicit a h y p o c a l c e m i c response in t r o u t m a i n t a i n e d in Ca++-rich t a p w a t e r b y i n j e c t i n g porcine CT (Lopez et al., 1971), h o w e v e r we observed a positive effect in t r o u t m a i n t a i n e d in Ca++-frec fl'esh w a t e r ~- t h y r o x i n e . Others au t h o r s showed t h a t m a m m a l i a n CT was effective in Ictalurus melas (Louw et al., 1967) an d in An-
For reprints: Mine Evelyne Lopez, Laboratoire de Physiologic du Mus6um, 7, rue Cuvier F-75005 Paris, France.
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guilla japonica (Chan et al., 1968). P a n g a n d Griffith (cf. Pang, 1971, u n p u b l i s h e d data) have observed hypocalcemia a n d h y p o p h o s p h a t e m i a i n fresh w a t e r - a d a p t e d Anguilla rostrata i n j e c t e d with s a l m o n calcitonin (SCT) b u t the same t r e a t m e n t was ineffective i n sea w a t e r - a d a p t e d animals. I n fish a t o t a l lack of hypocalcemic response to fish CT was also reported (Orimo et al., 1971; C o p p c t al., 1968; Pang, 1973) ; however it was p o i n t e d out t h a t hypercalcemia i n d u c e d b y various experim e n t a l m e a n s s t i m u l a t e d U B a c t i v i t y (Lopez et al., 1968; Chan, 1969; Lopez, 1973) a n d t h a t U B a c t i v i t y was related to serum calcium f l u c t u a t i o n s i n Salmo salar (Deville et Lopez, 1970). Dacke et al., (1971) observed t h a t fish plasma CT responded to injected calcium a n d the a d d i t i o n of calcium to the external m e d i u m (Deftos et al., 1972) provoked a n increase in the circulating CT levels. I n previous reports we have described some effects of exogenous CT on the bone catabolism i n the v e r t e b r a l skeleton of some teleosts with cellular bone (Lopez et al., 1971; Lopez, 1973; Lopez a n d Deville, 1973). I t has also been reported t h a t u l t i m o b r a n c h i a l e c t o m y could affect serum calcium c o n c e n t r a t i o n s in teleost fish (Chan, 1969), a n d bone in a m p h i b i a n s (Robertson, 1969 a, b) a n d birds (Copp, 1972). The present s t u d y was carried out to investigate (1) the effect of prolonged a d m i n i s t r a t i o n of SCT on bone apposition a n d a presumed p r e v e n t i v e effect of SCT on bone catabolism provoked b y e x p e r i m e n t a l m a t u r a t i o n i n the female eel, a n d (2) to elucidate the effect of u l t i m o b r a n c h i a l e c t o m y on calcium a n d bone m e t a b o l i s m i n the female silver eel.
Materials and Methods Female silver eels obtained from Peronne (Somme) were kept under laboratory conditions in aquaria at 18-20 ~ for one month. All were immature silver eels ranging in weight from 400 to 600g for the first experiment and 170 to 280g for the second. First Experiment. One week before the beginningof the experiment the eels were progressively adapted to sea water. Eels were divided into four groups a) 6 fish were kept. in sea water during the whole experiment. They received a vehicle gelatine injection 3 times a wk. b) A second group (5 fish) was injected intraperitoneally with SCT (Sandoz labs, S.A. Basel, Switzerland) at dose of 3 MCI~ mU/g bw, dissolved in gelatin solution, 3 times a wk over a 7 wk period. c) a third group (4 fish) was treated similarly; then they were given intraperitoneal injections of carp pituitary extract (CPE, Stoller Fisheries, Spirit Lake, Iowa, USA), lmg/100g bw per injection 3 times a wk, until complete maturation, according to the method described by Fontaine et al. (1964). d) a fourth group (7 fish) received CPE, 3 times a wk (lmg/100g bw per injection) until complete maturation. Immediately after spawning, the eels of the third and fourth groups were killed at the same time as the eels of the first and second groups. Second Experiment. Female silver eels were maintained in Ca++ rich tap water (80mg/1). They were divided into 2 groups and before the operation were anaesthetized with MS 222 (Sandoz labs). The first group (21 fish) was sham operated. The second group (39 fish) was operated on as described in Fig. 1. This operation differed from Chan's method (1969) and was performed without haemorrhage and without damage to the neighbouring tissues such as the heart and liver. The epithelium of the oesophagus healed approximately 2 days after the operation. In the eel, the UB cannot be seen with the naked eye nor with a stereomicroscope. Some fish of both groups were sacrificed one week after the operation. Others were killed after 2, 3 and 5 wks. At the end of the 2 experiments blood was collected from the ventral aorta. Before and during the experiment blood was collected by heart puncture. The blood plasma calcium (total calcium) was determined by the atomic absorption method (Perkin-Elmer). Completeness of the ultimobranchialectomies was estimated by examining serial sections of fixed and
Effect of Calcitonin and UBX on Fish Bone Metabolism
Posterio
175
rior part
Ventral pithelium
Fig. 1. Diagram showing the location of the ultimobranchial body (UB) in eel, and the method of cauterization employed for ultimobranchialeetomy
paraffin-embedded pieces of the transverse septuln and proximal oesophagus. Stannius corpuscles were stained using the Cleveland Wolfe method. The vertebral bone was examined on thin mineralized bone sections (10 to 15 ~z) and on microradiographs. The degree of mineralization of the bone was determined by means of a precise histophotometric method (Baud, 1957) and the morphologic quantification of bone turnover according to the method described by Jowsey et al., (1965). Osteocytic osteolysis was expressed as a percentage of the total number of osteocytic cavities. The osteoclastic index (OI) was estimated, according to the method of Schenk et al., (1969). Details of these techniques are given in a previous paper (Lopez, 1973).
Results
I n t h e first e x p e r i m e n t (Table 1) t h e serum cMeium c o n c e n t r a t i o n of control eels m a i n t a i n e d in sea w a t e r r e m a i n e d c o n s t a n t during t h e whole e x p e r i m e n t . SCT i n j e c t e d into i m m a t u r e f e m a l e eels (group 2), over a period of t h r e e weeks p r o v o k e d a slight b u t significant decrease in s e r u m calcium levels which subseq u e n t l y increased w h e n SCT t r e a t m e n t was stopped. I n t h e t h i r d group C P E i n d u c e d h y p e r c a l c e m i a in spite of t h e p r e v e n t i v e h y p o c a l c e m i c SCT t r e a t m e n t . I n group 4, m a t u r e f e m a l e eels showed a m ~ r k e d h y p e r c a l c e m i a . Table 1. Serum calcium level (rag/l) Groups b No. eels
1 wk before treatment
128.0 ~2 2.79 a
3 weeks after 8 wks after start of start of treatment treatment
1
6
2
5
3
4
111.6~- 1.66 96.6• 2.66 p < 0.01 F test
4
7
117.5 ~: 2.81
93.4~- 1.28 85.6-J_ 3 , 0 1 0 . 0 1 ~ p ~ 0.05 2' test
1 wk after CPE
91.24- 6.05
109.1 ~ 1.64
End of experiment
120.0•
5.36
l14.0-L
4.19
142.7• 5.00
723.5-L 221.26
142.8• 8.24
575.4-t- 155.15
a SEM b Group 1 were controls. Group 2 received SCT (3 MOB, mU/g/bw) over a 7 wk period. Group 3 received similarly SCT then CPE (1 rag/100 g/bw). Group 4 did not received SCT but CPE over all the experiment (1 rag/100 g/bw).
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E. Lopez et al. Table 2 Osteoclastic resorption % surface
OI %
Osteocytic osteolysis %
Osteoblastic apposition % surface
Degree of mineralization of the bone organic matrix g/em3
First group (1) controls n:9
13.78:[: 0.502 a 0.023~ 0.001
4.53 • 0.193
4.20 • 0.141
1.17::t=0.055
Second group (2) SCT n--5
12.02•
0.011:J::0.001
4.30 :J: 0.167
13.14~ 0.223
1.46-4-0.022
Third group (3) SCT then CPE n--4
23.12~ 0.983
0.205-4-0.009
4.95~ 0.171
12.17~ 0.696
1.31~- 0.035
Fourth group (4) CPE n:7
47.40~ 1.170 0.460~ 0.009
40.01:J: 0.325
7.06=[-0.134
0.98• 0.019
Statistical results Osteoclastic resorption
1-4; 1-3; 3-4; 2-3 p ~ 0.001; 1-2 p ~ 0.05.
F test
OI
1-2; 1-3; 1-4; 2 3; 3-4 p~0.001.
Osteoeytic osteolysis
1 4 ; 3-4 p ~ 0.001 ; 1-3 p ~ 0.2 ; 1-2 ; 2-3 p ~ 0.05
Osteoblastic apposition
1-4 ; 1-3 ; 1-2 ; 3-4 p ~ 0.001 ; 2-3 p ~ 0.05
Bone degree of mineralization 1-4; 1-2; 2-3 p~0.01; 3-4 p ~ 0.001; 1-3 p ~ 0.05 aSEM.
The effects of SCT were studied on the various bone parameters. SCT injected into i m m a t u r e silver eels (Table 2, group 2) did n o t significantly affect either osteoclastic resorption or osteoeytic osteolysis b u t it decreased OI a n d conseq u e n t l y the n u m b e r of m u l t i n u c l e a t e d osteoclasts. SCT increased both t h e degree of m i n e r a l i z a t i o n of bone m a t r i x a n d the osteoblastic apposition. I n histological bone sections after SCT t r e a t m e n t of eels (group 2) there was a n increase in the n u m b e r a n d a c t i v i t y of osteoblasts (Figs. 2 a n d 3). B e n e a t h t h e active osteoblasts there was a new bone layer which showed on microradiographs a low m i n e r a l d e n s i t y a n d a smooth surface characteristic of n e w bone formation. E x p e r i m e n t a l m a t u r a t i o n in female silver eels (Table 2, group 4) produced a m a r k e d demineralization of t h e bone m a t r i x as well as osteocytic osteolysis a n d osteoclastic resorption as evidence b y the n u m b e r of m u l t i n u c l e a t e d osteoclasts (OI). The cells which bordered the free bone edges, m a n y of which were osteoblasts, were increased in n u m b e r (Fig. 4). SCT injected as a p r e v e n t i v e t r e a t m e n t before a d m i n i s t r a t i o n of C P E (Table 2, group 3), i n h i b i t e d t h e effects of m a t u r a t i o n on osteocytic osteolysis a n d the degree of m i n e r a l i z a t i o n of the organic m a t r i x (Table 2, groups 3 a n d 4). The t r e a t m e n t partially p r e v e n t e d t h e osteoelastie resorption i n d u c e d b y t h e experim e n t a l m a t u r a t i o n (Table 2, group 4), however t h e osteoclastie resorption a n d OI (group 3) r e m a i n e d higher t h a n i n the controls (group 1). I t was observed t h a t
Fig. 2. I m m a t u r e female silver eel. P a r t of vertebral neural arch. Osteoblasts (--~) showing moderate activity and others which are more active ( 9 ) . (Basis fuehsin • 360)
Fig. 3. I m m a t u r e female silver eel treated with SCT. P a r t of vertebral neural arch. Osteoblasts (--~). New bone layer ( 9 ) . We note here t h a t osteoblasts are active cells (compared to those of Fig. 2) with a clear nucleus, a single nueleolus and a granular cytoplasm. (Basic fuchsin, • 360)
Fig.4. Mature female eel. Part of vertebral hemal arch. Tangential cut showing very activo lining cells. (--~). (Basic fuchsin, • 360)
Fig. 5. Mature female eel. Part of vertebral hemal arch. Large mononuclear cells (preosteoclasts (--~) whose nuclei contain numerous chromatin granules. Their apex (ll~) is always prominent and rounded as compared with that of active osteoblasts (Fig.3). Some preosteoclasts ( 3 ) are fusing to become multinucleated osteoclasts. (Basic fuehsin, X 360)
Fig. 6. Mature female eel. P a r t of hemal arch consisting of cancellous bone; enlarged resorption lacunae containing multinucleated osteoclasts (--)*), preosteoclasts ( ~ ) and osteoprogenitor cells ( ~ ) . (Basic fuchsin, X 360)
Fig. 7. Mature female eel injected with SCT before CPE treatment. P a r t of hemal arch consisting of cancellous bone. Lacunae similar to t h a t of Fig. 6. Note the absence of multinucleated osteoclasts. One preosteoclast is seen (--~). Osteoblasts are evident (11.~). (Basic fuchsin, x 360)
30,
:6
20,
10,
~ . _ n:8
1'
J 2 I n:9
3
4 84
5
~n:6 i
6~:6 weeks i
i
~10.
Fig. 8. Effect of ultimobranchialectomy (UBX) on serum calcium levels of the eel maintained in calcium-rich tap water (80rag/l). Serum calcium is presented as a per cent of the initial values for control eels (-o-) and ultimobranchialectomized eels (--.--). Vertical bars represent the S. E.M. and n equals the number of observations
Fig. 9. U B X female eel. P a r t of neural arch. Osteoblasts (--~) are seen as very flat cells with basophilic nuclei ( 3 ) . There are resting osteoblasts ( ~ ) along highly mineralized bone surfaces. (Basic fuchsin, X 360)
Effect of Calcitonin and UBX on Fish Bone Metabolism
181
Table 3
Sham operated
aSEM UBX
Time after operation (weeks)
Mineral substance concentration g/cm 3 (apatite ~- amorphous phosphate)
Osteoclastic resorption % surface
Osteoplastic apposition % surface
Osteocytic osteolysis %
1 1
1.02 1.01
2 2 3 3 5 5 5
1.10 1.08 1.12 1.07 1.Ol • 0.022 a 1.19 1.08 1.13
11.2 13.8 14.1 14.5 13.2 13.27 13.5 • 0.363 14.3 12.8 12.1
5.2 4.8 4.5 4.9
3.9 3.2 ] 3.8
4.81 5.3 ' -J-0.132 4.8 4.5 5.2 4.1
3.6 I 4.1 4.5
F test
p < 0.001
p > 0.05
p < 0.001
p > 0.05
0.97 0.91
13.41
0.08,
1.00
13.8] 14.2] 14.1 ( 13.88 14.01 • 13.2~ 13.8] 14.5 ] 14.7t
0.13 0.16 0.20 0.13 0.08 -L0.018 0.20 0.18 0.05 0.20
4.2 4.3 4.0 4.1 3.8 4.05 4.0 ~0.058 3.9 3.8 4.1 4.3
0.98 0.92 0.91 0.91 ~0.022 0.83 0.78 0.97 0.87
13.1]
0.091
3.86
m o n o n u c l e a r cells (preosteoclasts) in eel bone fused to become m u l t i n u c l e a t e d osteoclasts (Fig. 5). M i c r o r a d i o g r a p h i c a l l y it a p p e a r e d t h a t these m o n o n u e l e a r cells were a c t i v e in bone resorption. L a r g e r e s o r p t i o n spaces lined b y m u l t i n u e l e a t e d osteoclasts (Fig.6) were seen in t h e v e r t e b r a l archs of t h e m a t u r e female eels (Table 2, group 4). The v e r t e b r a l bone of m a t u r e female eels given SCT before C P E showed smaller r e s o r p t i o n spaces (Fig.7) whose bone surfaces were lined with a few m o n o n u e l e a r preosteoclasts, some of which were active, a n d also b y active osteoblasts. U l t i m o b r a n c h i a l e c t o m y r e s u l t e d in a n increase in s e r u m calcium levels, r e l a t i v e to t h e initial values, reaching a m a x i m u m after two weeks a n d s u b s e q u e n t l y declining to a p p r o x i m a t e l y physiological concentrations. S e r u m calcium concent r a t i o n in t h e controls d i d n o t v a r y b y m o r e t h a n 10 % a n d d i d n o t exceed t h e v a r i a t i o n f o u n d b e t w e e n i n d i v i d u a l s (Fig. 8). I n v e r t e b r a l bone several changes r e l a t e d to t h e osteoblasts a p p e a r e d d u r i n g t h e first week after o p e r a t i o n a n d p e r s i s t e d two, t h r e e a n d five weeks later. W h i l e t h e bone of controls was lined b y active osteoblasts on some surfaces especially along new bone surfaces (Fig.2), in bone of U B X eels, osteoblasts were r e d u c e d in size, n u m b e r a n d in t h e r e g u l a r i t y of t h e i r spacing along t h e bone surfaces (Fig. 9). Osteoblasts in U B X eels were seen as c o m p l e t e l y flat cells lining i n a c t i v e bone surfaces. A b a n d of high m i n e r a l d e n s i t y seen in m i e r o r a d i o g r a p h s i n d i c a t e d t h a t osteoblastic a p p o s i t i o n h a d ceased (Table 3). A f t e r U B X (Table 3) we d i d n o t
182
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observe changes in the number of osteoclasts nor were osteoclastic resorption surfaces increased and the degree of the osteocytic osteolysis remained constant. However it was noted that the average rate of mineralization of the bone matrix was noticeably decreased relative to the control values. Stannius corpuscles were found to be very active glands in operated eels. Spheric follicles were generally clumped together into sinuous strands. We have previously described the formation of these strands after stimulation of the Stannius corpuscles (Lopez et Fontaine, 1967). Between the cellular strands extended large blood vessels. Based on these observations and compared with those previously made concerning Stannius corpuscles, there is little doubt that these glands were in their active phase. Discussion
The present results indicate that injections of exogenous CT produce many important effects on the bone of the eel. However, administration of an exogenous hormone cannot exactly reflect the total physiological effect. The existence of the UB as a distinct organ in fish, provided us with an excellent means of evaluating its physiological significance. I n this investigation a decrease of serum calcium concentration in SCT-treated immature silver eels maintained in sea water, was observed. This observation contradicts that reported by Pang (1971) showing that CT treatment was ineffective on serum calcium in sea water-adapted Anguilla rostrata. This variance could be due to the fact that the eels were not collected at the same period of their vital cycle in the two experiments. Furthermore, SCT provokes a marked cell atrophy of the ultimobranchial body in female silver eels (Peignoux-Deville et al., 1975). The experimental maturation of the female eel (Fontaine et al., 1964) provokes an acute hypercalcemia in keeping with the rise of the gonosomatic ratio, a high bone catabolism and a significant stimulation of the UB (Lopez et al., 1968). These results are consistent with those of Watts et al. (1975) on the salmon. The high level of CT in the blood of the mature female salmon and the hyperactivity of UB in the mature female eel and the female conger eel (Lopez, 1973) are probably related to the elevated catabolism observed in these animals. CT in female fish may have a role in the protection of skeletal calcium until spawning, similar to that of mammals during pregnancy and lactation (Lewis et al., 1971). The negative effect on serum calcium observed after both SCT treatment and CPE injections (SCT injected preventively before CPE treatment and SCT injected in addition to CPE, Lopez and Deville, 1973) can be explained, at least partially, by the fact that in spite of the SCT effect in reducing osteoclastic resorption and osteocytic osteolysis, bone catabolism is sufficient to maintain a high serum calcium concentration. Ultimobranchialectomy in the female silver eel, induces a significant rise in serum calcium levels, returning to physiological levels during the third week after operation. Increase in serum calcium concentrations was reported by Chan (1969) in U B X Anguilla japonica, recently by Fenwick in partially U B X Carassius auratus (1975)and by I~obertsonin U B X l~ana pipiens (1969a). The two bone effects obtained in U B X eels: demineralization of the organic matrix and inhibition of the osteoblastic apposition, can partially explain the observed hyper-
Effect of Calcitonin and UBX on Fish Bone Metabolism
183
caleemia during the first week. The prolonged hyperealcemia until the second week can be the result of the failure of CT effect on the kidney (Chan, 1972) and of modifications in calcium exchange across the gills. Preliminary results (Peignoux-Deville et al., unpublished data) lead us ~o believe that CT (SCT) perfnsed in isolated gills from U B X eels, acts on calcium influx and outflux resulting in a lowering of the serum eMeium concentration. The control of hypercalcemia after two weeks in U B X eels cannot be a parathormone response as Robertson has demonstrated in the frog (I~obertson, 1969a) because parathyroid glands appear to be absent in fish. Stannius corpuscles of U B X eels are found to be very active glands in our experiments, we have not been able to show atrophic changes as did Chan (1972). These glands were shown to induce hypocaleemia in teleosts by Fontaine (1964, 1967) principally by acting on the gills (Fontaine etal., 1972; Fenwiek and Peng So, 1974; Milet et al., 1975). We suggest that the hypereMeemia induced by ultimobranehiMectomy in eels is regulated by the Stannius corpuscles. The observations reported here show the effect of exogenous SCT on the osteoblastic bone apposition in female silver eels. This CT effect is confirmed by the drastic decrease of osteoblastic apposition from the first week after ultimobranchialectomy. Robertson (1969a) in U B X Rana pipiens described a similar effect on bone apposition but in 12 week post-operative animals. This variance m a y be due to the fact that in the frog the first target organs for CT are paravertebral lime sacs and not the skeleton. We have previously reported t h a t osteoblastic apposition in fish m a y also be regulated by the Stannius corpuscles (Lopez, 1970a, Lopez, 1973). UB and Stannius corpuscles seem to have an antagonistic effect on eel bone in spite of their similar hypoeMcemic role and we are convinced that serum calcium concentrations do not reflect the complex mechanisms which maintain calcium homeostasis in fish. We have also pointed out that gonadal maturation increases osteoblastie activity and we interpret this as being the effect of the sequential process of the bone remodeling (Frost, 1966). SCT injected in immature female silver eels does not significantly affect the degree of osteoelastic resorption but decreases the pool of multinueleated osteoclasts. We suggest that, in this ease, the measured average degree of osteoelastie resorption results principally from the activity of preosteoelasts. Ultimobranehialeetomy does not modify the number of osteoelasts therefore the osteoclastie resorption is unchanged. These results confirm our previous reports which showed that CT acted effectively on the osteoelastie resorption in fish bone only when bone catabolism was high. Similarly neither SCT nor ultimobranchialectomy modify the physiological degree of osteoeytie osteolysis. In the bone of mature female eels treated preventively with SCT before CPE osteoclastie resorption does not increase as it does in the bone of mature eels injected with CPE. However osteoelastic resorption remains greater than in the controls, probably because the effect of SCT is counteracted by oestrogens as we have shown previously (Lopez and Deville, 1973). We suggest that, when injected after CPE treatment, SCT destroys the mnltinueleated osteoclasts (Lopez, 1973; Lopez and Deville, 1973) but when SCT is given as a preventive treatment it probably prevents preosteoc]asts from fusing into active multinucleated osteoclasts. SCT administered before experimental maturation, completely inhibits the ability of the osteoeyte to resorb its surrounding bone.
184
E. Lopez et al.
I n previous r e p o r t s we h a v e p r o v i d e d evidence of a diffuse decalcification w i t h o u t m o d i f i c a t i o n of t h e organic m a t r i x (Lopez, 1970b; Lopez et M a r t e l l y Bagot, 1971) which is due to t h e loss of some p a r t of t h e a m o r p h o u s calcium phosp h a t e (Lopez et al., 1970) in t h e v e r t e b r a l bone of m a t u r e eels. I n t h e p r e s e n t s t u d y we p o i n t out t h a t SCT increases t h e degree of m i n e r a l i z a t i o n of t h e bone of i m m a t u r e silver eels a n d w h e n i n j e c t e d p r e v e n t i v e l y it p a r t i a l l y i n h i b i t s dem i n e r a l i z a t i o n in t h e bone of m a t u r e eels. Similar o b s e r v a t i o n s on t h e effect of CT on bone m i n e r a l i z a t i o n has been p u b l i s h e d in birds (Copp et al., q u o t e d b y B61anger, 1971) a n d in m a n (Baud et al., 1970). R e c i p r o c a l l y in t h e female silver eel U B X p r o v o k e s a significant d e m i n e r a l i z a t i o n of t h e bone m a t r i x . W e have p r e v i o u s l y suggested (Lopez, 1973) t h a t CT p r o b a b l y acts on t h e calcium exchange at t h e site of t h e " b o n e lining cellular l a y e r " which p r o b a b l y controls calcium ion t r a n s p o r t (Baud, 1968; N e u m a n n , 1969). The role of t h e UB, in t h e silver eel, a p p e a r s to be to effect a release of a h o r m o n e which controls serum calcium, s t i m u l a t e s t h e p r o d u c t i o n a n d a c t i v i t y of osteoblasts a n d regulates t h e degree ef m i n e r a l i z a t i o n of t h e bone a n d in some cases, reduces bone catabolism. W e are convinced, however, considering all of our results concerning fish bone, t h a t Ca ++ ion t r a n s p o r t which r e g u l a t e s the degree of m i n e r a l i z a t i o n of the intercellular s u b s t a n c e is p r o b a b l y one of t h e m a i n m e c h a n i s m s influencing calcium homeostasis in t e l e o s t fish.
References Baud, C.A. : Radiographies et microradiographies osseuses quantitatives. Praxis 46, 329-331 (1957) Baud, C.A.: Submicroscopic structure and functional aspects of the osteocyte. Clin. Orthop. ~6, 227-336 (1968) Baud, C.A., Siebenthal J.de, Langer, B., Tupling, M.R., Mach, R. S. : The effects of prolonged administration of thyrocalcitonin in human senile osteoporosis. In: Caleitonin 1969. Proceedings of the second symposium (S. Taylor, ed.), p. 540-546. London: Heineman 1970 Belanger, L.F. : The ultimobranehial gland of birds and the effects of nutritional variations. The J. exp. Zool. 178, 125-137 (1971) Chan, D.K.O.: Endocrine regulation of calcium and inorganic phosphate balance in freshwater adapted teleost fish, Anguilla anguilla and Anguilla japonica. In: Progress in Endocrinology. Proc. 3rd Intern. Congress Endocrinol. (C. Gual ed.), p. 709-716. Amsterdam: Excerpta Med. 1969 Chan, D.K.O.: Hormonal regulation of calcium balance in teleost fish. VI gme Symposium international d'Endocrinologie Comparge. Gen. comp. Endocr. Suppl. 3, 411-420 (1972) Chan, D.K.O., Chester-Jones, I., Smith, R.N.: The effect of mammalian calcitonin on the plasma levels of calcium and inorganic phosphate in the European eel (Anguilla anguilla L.). Gen. comp. Endocr. l l , 243-245 (1968) Copp, D.H.: Endocrine regulation of calcium metabolism. Ann. Rev. Physiol. 32, 61-86 (1970) Copp, D.H. : Calcium regulation in birds. Gen. comp. Endocr. Suppl. 3, 441-447 (1972) Copp, D.H., Cockroft, D.W., Kueh, Y.: Calcitonin from ultimobranchial glands of dogfish and chicken. Science 1~8, 924-925 (1967) Copp, D.H., Cockroft, D.W., Kueh, Y., Melville, M.: Calcitonin-ultimobranchial hormone In: Calcitonin 1967 (S. Taylor, ed.), p. 306-321. Berlin-Heidelberg-NewYork: Springer 1968 Dacke, C.G., Fleming, W.R., Kenny, A.D.: Plasma calcitonin levels in fish. Physiologist 14, 127 (Abstract) (1971) Deftos, L.J., Murray, T.M., Powell, D.A., Habener, J.F., Singer, F.R., Mayer, G.P., Potts, J.T. Jr.: Calcium, parathyroid hormone and the calcitonins. (R.V. Talmage, Munson P.L. eds.), p. 140. Amsterdam: Excerpta Med. Found. 1972
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Deville, J., Lopez, E.: Le corps ultimobranchial du Saumon Salmo salar L. Etude histophysiologique s diverses 6tapes de son cycle vital en eau douce. C.R. Acad. Sci. (Paris) 270, 2347-2350 (1970) Fenwick, J.C. : Effect of partial ultimobranchialectomy on plasma calcium concentration and on some related parameters in Goldfish (Carassius auratus L.) during acute transfer from fresh water to 30 % sea water. Gen. comp. Endocr. 25, 60-64 (1975) Fenwick, J.C., Peng So, u A perfusion study of the effect of stanniectomy on the net influx of calcium 45 across an isolated eel gill. J. exp. Zool. 188, 125-131 (1974) Fontaine, M. : Corpuscules de Stannius et r~gulation ionique (Ca, K, Na) du milieu int6rieur de l'Anguille (Anguilla anguilla L.). C.R. Acad. Sci. (Paris) 259, 875-878 (1964) Fontaine, M. : Intervention des corpuscules de Stannius dans F6quilibre phosphocalcique du milieu int4rieur d'un poisson t~l~ost~en, l'Anguille. C.R. Acad. Sci. (Paris) 264, 736-737 (1967) FontMne, M., Bertrand, E., Lopez, E., Callamand, O.: Sur la maturation des organes g~nitaux de l'Anguille femelle Anguilla anguilla L. et l'6mission spontan~e des oeufs en aquarium. C.R. Acad. Sci. (Paris) 259, 2907-2910 (1964) Fontaine, M., Delerue, N., Martelly, E., Marchelidon, J., Miler, C.: R6le des corpuscules de Stannius dans les @hanges de calcium d'un poisson t616ost@n, l'Anguille (Anguilla anguilla L.) avec le milieu ambiant. C.R. Acad. Sei. (Paris) 275, 1523-1528 (1972) Frost, H.M.: Bone dynamics in os~oporosis and osteomalacia, p. 1-176. Springfield: C. Thomas 1966 Jowsey, J., Kelly, P . J . , Riggs, B.L., Bianco, A.J., Scholl~z, D.A., Gershon-Cohen, J.: Quantitative microradiographic studies of normal and osteoporotic bone. J. Bone J t Surg. 47, 785-806 (1965) Kenny, A.D.: Calcium, parathyroid hormone and the calcitonins. Proc. 4th parathyroid conference. Chapel Hill, W.C., USA, March 15-19 (R.V. Talmage, P.L. Munson eds.), p. 9-11. Amsterdam: Excerpta Med. Found. 1972 Lewis, P., Rafferty, B., Shelley, M., Robinson, C.J.: Suggested physiological role of calcitonin. The protection of the skeleton during pregnancy and lactation. J. Endocr. 49, 9-10 (1971) Lopez, E. : L'os cellulaire d'un poisson t416ost6en, Anguilla anguilla L. II-Action de l'ablation des corpuscules de Stannius. Z. Zellforsch. 109, 566--572 (1970a) Lopez, E. : Demonstration of several forms of decalcification in bone of the teleost fish, Anguilla anguilla L. Calcif. Tiss. Res. 4 (Suppl.), 83 (1970b) Lopez, E. : Etude morphologique et physiologique de l'os cellulaire des Poissons t61~ost6ens (th~se de Doctorat d'Etat, 1972). M4moires du Mus6um National d'Histoire Naturelle (nouvelle s6rie), s6rie A, Zoologic, tome L X X X (1973) Lopez, E., Chartier-Baraduc, M.M., Deville, J. : Mise en 4vidence de Faction de la calcitonine porcine sur l'os de la truite Salmo gairdnerii soumise s un traitement d4min~ralisant. C.R. Acad. Sci. (Paris) 272, 2600~603 (1971) Lopez, E., Deville, J. : Effect of prolonged administration of synthetic sahnon caleitonin (SCT) on vertebral bone morphology and on the ultimobranchial body (UB) activity of the mature female eel (Anguilla anguilla L.). Proc. 9th european symposium on calcified tissues, Baden (A. Czitober, J. Eschberger eds.), p. 169-174. Vienna: H. Egermann, Facta-Publication 1973 Lopez, E., Deville, 3 , Bagot, E. : Etude histophysiologique du corps ultimobranchial d'un T~l~ost~en (Anguilla anguilla L.) au cours d'hypercalc@fies expSrimentales. C.R. Acad. Sei. (Paris) 267, 1531--1534 (1968) Lopez, E., Fontaine, M. : RSponse des corpuscules de Sl~annius de l'Anguille (Anguilla anguilla L.) s des blessures exp~rimentales. C.R. Soc. Biol. (Paris) 161, 36-39 (1967) Lopez, E., Lee, I-I.S., Baud, C.A.: Etude histophysique de l'os d'un T~lSostSen, Anguilla anguilla L., au cours d'une hypercalc6mie provoqu6e par la maturation exp~rimentale. C.R. Acad. Sci. (Paris) 270, 2015-2017 (1970) Lopez, E., Martelly-Bagot, E. : L'os cellulaire d'un poisson T61~ost6en, Anguilla anguilla L. III-Etude histologique et histophysique au cours de la maturation provoqu6e par injections d'extrait hypophysaire de Carpe. Z. Zellforsch. 117, 176-190 (1971) 6 Calcif. Tiss. Res.
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Louw, G.N., Sutton, W. S., Kenny, A.D. : Action of thyrocalcitonin in the teleost fish Ictalurus melas. Nature (Lond.) 215, 888 889 (1967) Milet, C., Martelly, E., Fontaine, M.: Corpuscules de Stannius (CS) et flux de calcium au niveau des branehies perfus6es d'Anguilla vulgaris. J. Physiol. (Paris) 71, 141A (1975) Neuma~, W. F. : The milieu interieur of bone : Claude Bernard revisited. Fed. Proe. 28, 18461850 (1969) Orimo, H., Ohata, M., Yoshikawa, M., Abe, J., Watanabe, S., Kotani, M., Higashi, T.: Ultimobranehial caleitonin. II-Physiologieal role of ealeitonin. Igaky No Ayumi 79, 480 (1971) Pang, P.K,T.: Caleitonin and ultimnbranchial glands in fishes. J. exp. Zool 178, 89 100 (1971) Pang, P . K . T . : Endocrine control of calcium metabolism in teleosts. Amer. Zool. 13, 775-792 (1973) Pang, P.K.T., Pickford, G.E.: Failure of hog thyroealcitonin to elicit hypocalcemia in the teleost fish, Fundulus heteroclitus. Comp. Biochem. Physiol. 21, 573 578 (1967) Peignoux-Deville, J., Lopez, E., Lallier, F., Martelly-Bagot, E., Milet, C.: Responses of the ultimobranchial body in eels (Anguilla anguilla L.) maintained in sea water and experimentally matured, to injections of synthetic salmon ealeitonin. Cell Tiss. Res. 164, 73-83 (1975) Rasquin, P., Rosenbloom, L. : Endocrine imbalance and tissue hyperplasia in teleost maintained in darkness. Bull. Amer. Mus. Nat. Hist. 104, 363 425 (1954) Robertson, D.R.: The ultimobranchial body of Rana pipiens. VIII-Effects of extirpation upon calcimn distribution and bone cell types. Gen. comp. Endoer. 12, 479-490 (1969a) Robertson, D.R.: The ultimobranehial body of Rana pipiens. X-Effect of glandular extirpation on fracture healing. J. exp. Zoo]. 172, 425-441 (1969b) Schenk, R., Merz, W.A., Muller, J.: A quantitative histological study on bone resorption in human cancellous bone. Aeta anat. (Basel) 74, 44 53 (1969) Urist, M.R.: Avian parathyroid physiology including a special comment on caleitonin. Amer. Zool. 7, 883 895 (1967) Watts, E.G., Copp, D.H., Deftos, L . J . : Changes in plasma ealcitonin and calcium during the migration of sahnon. Endocrinology 96, 214-218 (1975)
