Calcif. Tissue Int. 32, 83-87 (1980) Calcified Tissue International �9 1980 by Springer-Verlag

Rapid Communication

Paradoxical Effect of 1.25 Dihydroxycholecalciferol on Osteoblastic and Osteoclastic Activity in the Skeleton of the Eel Anguilla anguilla L

E. L o p e z , I. Mac Intyre ~, E. Martel ly , F. Lal l ier , and B. Vidal

Laboratoire de Physiologie g6n6rale et compar6e du Mus6um national d'Histoire naturelle et Laboratoire d'Endocrinologie compar6e associ6 au C.N.R.S., 7, rue Cuvier, 75231 Paris c6dex 05, France

SUMMARY

Female mature eels (300 to 500 g) received

one intraperitoneal injection of 1.25(OH)2D 3

(i0 pg). Their vertebral bone was compared, 8 h

and 24 h after the injection, with vertebral bone

of control mature female eels receiving solvent

alone (ethanol). Sexual maturation in female eels

induces a bone decalcification with hypercalcaemia

and hyperphosphataemia. The control eels showed

marked osteoclastic resorption and osteocytic

osteolysis and the degree of mineralization of

the intercellular substance decreased. Injection

of 1.25(OH)2D 3 into these female mature eels

provoked as early as 8 h : i) an increase in

hypercalcaemia and hyperphosphataemia ; 2) a major

conversion of lining cells to osteoblasts and a

stimulation of osteoblastic activity with new bone

formation ; 3) diminished osteoc]astic resorption

without changing osteocytic osteolysSs or bone

matrix mineralization.

Key words: 1.25 (OH).,D:~ - - Fish - - Bone - -

Osteob las t s

INTRODUCTION

Parathyroids have not been described in fish

(i) but two glands are principally involved in

their phosphocalcic met~olism : tile ultimobran-

chial body secreting calcitonin ; and tile Stannius

corpuscles, hypocalcaemic glands sltuated on the

kidney (2), which act with calcitonin at the sJ te

of the gills (3,4) and bonc (5,6) to ma<ntain

calcium homeostasis. However, we generally forget

that fish liver is extremely rich in vitamin D 3

and very little is known of tile function of the

vitamin in these animals. Recent studies provide

evidence that D 3 is metabolized in fish. Thus,

25(OH)D 3 is present in eel serum (7), bony fish

have a specific plasma transport protein ( @ glo-

bulin) for 25(OH)D 3 and 24R,25(OH)2D 3 (8,9), and

many species contain the l-hydroxylase enzyme in

their kidney (I0). Further, 1.25(OH)2D3-1ike

activity has been detected in eel tissues by

competitive protein binding assay (10a). We have

' Endocrine Unit, Royal Postgradtmte Medical School, Du Cane Road~ London WI2 0HS, England

shown that exogenous 1.25(OH)2D 3 increases plasma

inorganic phosphate (ii) and stimulates bone

resorption in immature yellow and silver eels (12,

13). We now report studies of the effect of

1.25(OH)2D 3 on osteoblastic activity in mature

female silver eels whose skeleton was already

greatly demineralized (14).

MATERIAL AND METHODS

Female silver eels (9 animals) obtained from

Peronne (Somme) were placed in aerated fresh water

and allowed to adapt to laboratory conditions at

18-20~ for one week. All were immature silver

eels ranging in weight from 300 to 500 g. (Note

that eels had never been found naturally mature).

One week before the beginning of the experiment

tile eels were progressively adapted to sea water,

then they received intraperitoneal injections of carp pituitary extract (CPE, Stoller Fisheries,

Sp~r<t Lake, Iowa, USA) 3 times c~ wee]< (i rng/100 g

body weight per injection) until complete matura-

tion (three months) according to a method already

described (15). When CPE treatment was stopped,

just before spawning, 3 mature eels (controls) re-

ceived one intraperitoneal injection of solvent

alone (ethanol), 6 mature eels were given one in-

traperitoneal injection of i0 pg 1.25 (OH)2D3 at

the same time. Three eels injected with 1.25(0H) 2

D 3 were killed 8 h after the injection and three

others 24 h after the same injection. At the end

of the experiment blood was collected from the

ventral aorta and gonadal and body weight was

m e a s u r e d .

For each animal caudal vertebrae were taken

at the level of anus ; the samples were fixed i~l

alcohol 70 ~ and colored with basic fuchsin i %,

embedded in metklyl-methacrylate and cut into sec-

tions which were then ground manually to a

thichness of 10 pm for a morphometrical study.

Resorption surfaces and bone formation surfaces

were localized to the identification tests propo-

sed by Jowsey (16). Then the measurement of the

bone surfaces crenated and covered by osteoclasts

(mononucleated and plurinucleated) and of the

bone surface extent of the cuboidal form of osteo-

blasts (lining a thin new bone layer~colored by

basic fuschin) was performed, by tile integrating

method. We were using a Zeiss Ii integrating eye-

piece (17, 18) ; according to tile Zeiss counting

0171-967X/80 /0032-0083 $01.00

84 E. Lopez et al.: 1.25 dihydroxycholecalciferol and osteoblastic activity in eel skeleton

table the examination of fields selected at ran-

dom on I0 undecaleified serial sections (the

totality of each vertebral cross section was exa-

mined) and testing 1.600 points of intersection

for each vertebral sample garantes and absolute

error of less than one percent. Osteocytes situa-

ted in enlarged lacunae and presenting on microra-

diographs a demineralized surrounding area (osteo-

lytic osteocytes) were counted and their number

was expressed as a percentage of the total number

of osteocytes. The degree of mineralization of

bone intercellular substance was measured by means

of a microradiographic microdensitometric method

(19,5). All details of these techniques are given

in (20). The plasma calcium (total calcium) was

determined by the atomic absorption method (Perkin

Elmer) and phosphorus according to the method of

Fiske and Subbarow (21).

RESULTS

1.25(OH)2D 3 injected into hypercalcaemic ma-

ture female eels (6) provoked as early as 8 h an increase in calcium and phosphate concentrations

(Table I) which did not rise further subsequently

(24 h).

Table 1 - THE CONCENTRATIONS OF Ca AND INORGANIC

PHOSPHATE IN EELS TREATED WITH

1.25(OH)2D 3.

Body Gonadal Plasma Serum Groups weight g. weight ~ Ca(meq/l) PO4(mg

716 366 16.6 5.3 Controls

554 319 10.5 5.5 ethanol

410 162 ii.0 5.5

580 315 20.5 19.2 1"25 (OH) 2D3 505 215 47.3 35.1

8 h 520 250 19.3 18.3

723 348 18.0 14.0 I'25<OH~2D3'' 540 270 15.8 13.4

24 h 546 256 15.6 7.5

The effect of 1.25(OH)2D 3 was studied on the

various bone parameters. 1.25 (OH)2D 3 did not

change the marked osteocytic osteolysis and the

diminished degree of mineralization observed in

mature female eels but it greatly increased the

surface covered by osteoblasts and consequently

the surfaces affected hy osteoclastic resorption

were reduced (Table 2).

In histological bone sections of mature fema-

le eels (Fig. i) we saw that the bone surfaces were

almost entirely crenated and lined by osteoclasts ;

here, mononucleated osteoclasts very often close

together ~. 8 h after 1.25(OH)2D 3 injection we observed an increase in the number and activity

of osteoblasts (Table 2). Prominent cuboidal os-

teoblasts with rounded apices covered a major part of the bone surface very often previously

The suggested resorptive activity of mononu-

cleated osteoclasts (6) in bone of eel was

identified and confirmed by an electron microsco-

pic study (21a) and also recently shown by Weiss

and Watabe (31) in other fish species.

Table 2 - EFFECT OF 1.25(OH)2D 3 AFTER 8 h AND

24 h.

Osteo- Osteo- Degree Osteo-

clastic cytic of blastic

Groups Resorp- Osteo- minera- apposi-

tion lysis lization tion % S ~ % g/cm 3 % S

45.34 18.6 0.98 8.21 Controls

37.22 37.9 0.95 7.82 ethanol

41.36 38.8 0.96 7.20

30.41 37.1 0.94 40.31 1"25 (OH) 2D3 32.20 36.4 0.98 39.24

8 h 28.14 37.6 0.97 43.12

20.23 36.3 0.98 42.64 I=,,I.2J~OHJ2D 3 19.18 36.8 0.96 43.51

24 h 16.36 37.9 0.98 40.78

S - surface Mineral substance of the

bone organic matrix.

affected by osteoclastic resorption (figs. 2-3).

These osteoblasts lined a thin new bone layer

(fig. 3) also identified on microradiographs.

24 h after 1.25(OH)2D 3 injection, the pro- portion of vertebral bone surfaces lined by active

osteoblasts did not increase further (Table 2),

but beneath the active osteoblasts we observed a

more important new bone layer (Fig. 4) than after

8 h which showed on microradiographs a low mineral

density and a smooth surface characteristic of new

bone formation.

DISCUSSION

It should be emphasised that the cells pre-

sent in the bone of higher vertebrates are also

present in the eel skeleton (20,22) and three

different modes of demineralization can be obser-

ved (23). Sexual maturation in female teleosts,

such as the eel (experimental maturation), and

female conger eel naturally mature, greatly

enhanced the three types of decalcification and

provoked a marked increase in serum calcium and

phosphate levels (22) x. The 1.25(OH)2D 3 injected

into mature female eels induced a further increa-

se in the hypercalcaemia and hyperphosphataemia

Control mature female eel. Vertebral bone

surfaces appeared crenated with typical Howship's

lacunae ~ and osteoclasts (~) x 157.5. Fig. i.

E. Lopez et al.: 1.25 dihydroxycholecalciferol and osteoblastic activity in eel skeleton 85

Vertebral bone of mature female eel, 8 h after

the injection of 1.25(OH)2D 3. Osseous borders

were lined by bery active osteoblasts (~. K

Some osteoclasts were still present (~) x 157.5. Fig. 2.

already present in these mature fish. However,

surfaces affected by osteoclastic resorption were

reduced and we observed a very rapid and dramatic

stimulation of the osteoblastic activity, with

new bone formation, although the osteocytic osteo-

lysis and the degree of mineralization of the

intercellular substance appeared unchanged. Pre-

sumably the effect of 2.25(OH)2D 3 on plasma cal-

cium concentration was due, in part, to the action

of this metabolite on the intestine absorption of

calcium as we showed recently in the eel (24).

In manm~als, 1.25(OH)2D 3 is generally thought

to stimulate resorption (24,25). However, it has

also been suggested that some other D metabolite

may exert a direct effect in controlling and

enhancing mineralization (27). With regard to

osteoblastic activity itself, pharmacologic doses

of vitamin D3, in rats, induced an increase in

osteoblastic bone formation at vascular canals,

but despite dlis effect an increased porosity

occured (28).

In the eel when bone turnover was rather low,

1.25(OH)2D 3 stimulated the various forms of os-

seous resorption (12,13) ; in contrast when bone

Vertebral bone of mature female eel, 8 h after the

injection of 1.25(OH)2D 3. High magnification

x 283.5. Very active osteoblasts ~). New bone

layer ( .... )). Fig. 3.

N o t e t h a t s e r u m c a l c i u m c o n c e n t r a t i o n i n iKmla-

t u r e s i l v e r e e l i s on a n a v e r a g e 7 . 5 m e q / 1 .

Mature eel 24 h after the injection of 1.25(OH)2D 3.

Active osteoblasts ('~;. New bone layer with

osteoblasts entrapped in it (---) x630. Fig. 4.

turnover was activated, as in the case of sexual

maturation of the female eel (6), calcium and phos-

phate serum concentrations being already markedly

elevated, 1.25(OH)2D~ results in a drastic effect

on conversion of linlng cells to osteoblasts and

on osteoblastic activity. Thus in the eel, the res-

ponse to 1.25(OH)2D 3 appears dependent on the se- xual phase. This induces a stimulation of all the

osteoprogenitor cells surrounding vertebral bone

surfaces (14) and may modify the response of the

target cells. Just now (21a) an electron microsco-

pic study on eel vertebral bone show that lining

cells may have a very active resorptive function ;

they can be mononucleated osteoclasts, functionna-

ly similar to multinucleated osteoclasts of mamm~als.

We can presume that, in eel bone, lining cells may

be alternatively implicated in bone resorption or

formation. However, we must consider the possibili-

ty that the action on osteoblasts observed was

secondary to the further increase in calcium and

phosphate produced, rather than to a direct effect.

Further studies are necessary to distinguish bet-

ween these possibilities. Finally, one further qua-

lification in interpreting our results may be pru-

dent. This is that the amounts employed of 1.25

(OH)2D 3 were much greater than is usual in mamma-

lian experiments. For this reason further study

will be required to exclude the possibility that

our findings reflect pharmacological rather than

physiological events.

It is well established now that bone minerali-

zation is under cellular control (29). In fish,

however, in contradistinction to other vertebrates,

matrix vesicles (the nucleation site for mineral)

appear very numerous in secondary osteoblastic

accretion (30). In studies in progress we are exa-

mining the possibility that 1.25(OH)2D 3 affects the

formation of matrix vesicles by osteoblasts and the

sequence of mineralization.

In higher vertebrates the mode of action of

the vitamin D 3 (or metabolites) on cell metabolism

and its possible effect on bone formation in gene-

ral and calcification in particular is still far

from clear. The degree to which 1.25(OH)2D 3 and pa-

rathormone are dependent on each other to affect

bone mobilization is still a problem. A study of

the effect of vitamin D 3 and of its metabolites in

fish, devoid of parathyroid glands, may provide a

86 E. Lopez et al.: 1.25 dihydroxycholecalciferol and osteoblastic activity in eel skeleton

useful phylogenetie interpretation of its mode of action.

ACKNOWLEDGEMENTS

We thank C.N.R.S. for financial assistance (ATP Physiologie et Pathologie des Tissus calci- fi@s). We thank also Hoffman La Roche for the gift of synthetic 1.25 dihydroxycholecalciferol.

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