Homologous recombinant tilapia growth hormone (rtGH) was tested for its effects on calcium metabolism in freshwater tilapia Oreochromis mossambicus. Fish were fed an optimal ration of 5% of their body mass per day. A positive correlation was found between the amount of food given and the branchial calcium influx. In male tilapia, the mean calcium influxes were 5.80 and 11.71 μmol h1 100 g1 when they were fed 2 % and 5% food, respectively. In female fish fed 5% food, the calcium influx was 6.20 μmol h1 100 g1. Calcium influx via the gills was not affected by rtGH. However, in rtGH-treated fish, the net efflux of calcium was lower than in the controls. Apparently, the calcium taken up from the water was more efficiently stored in the body. GH increased the hepatosomatic index and had mild growth-promoting effects (mass and length increases); it increased the total body calcium pool without affecting bone or scale calcium density. The chloride cell density in the opercular epithelium almost doubled after GH treatment. GH did not influence plasma ion composition. Plasma cortisol levels were lower in rtGH-treated fish. A comparison of the roles of GH and prolactin (the products of the prolactin gene family) in calcium regulation of the tilapia led us to conclude that GH has specific calcitropic effects on freshwater tilapia that differ from those of prolactin.

A characteristic of the members of the prolactin gene family is their pleiotropic nature. In fish, growth hormone (GH) acts as a growth-promoting hormone, but it also appears to be involved in osmoregulation, stress adaptation and reproduction (Le Bail et al. 1991). Growth, osmoregulation, stress adaptation and reproduction are strongly interdependent in fish, and all these processes depend on a well-regulated calcium balance; Ca2+ uptake via the gills is pivotal in calcium handling by fish (Flik et al. 1985). The interdependence of these processes and their dependence on GH may underlie the pleiotropic character of the effects of GH in fish.

Studies on the effects of GH in fish have focused on salmonids, eels, cyprinids and tilapia. The emphasis of the work carried out so far has been on the establishment of sensitive assays for the determination of (plasma) levels of the hormone (e.g. Fryer, 1979; Cook et al. 1983; Bolton et al. 1987; Le Bail et al. 1991; Ayson et al. 1993), on the demonstration of receptors for GH (Hirano, 1991; Gray et al. 1990; Sakamoto and Hirano, 1991) and on the regulation of GH-producing cells (Marchant et al. 1987; Nishioka et al. 1988). A pivotal target for GH is the liver (Hirano, 1991), where it stimulates receptors with nanomolar affinity. Liver cells activated by GH produce a fish insulin-like growth factor (IGF, probably IGF-I; Drakenberg et al. 1989) that may subsequently stimulate growth (Duan and Hirano, 1990; Sakamoto and Hirano, 1991; Gray and Kelley, 1991; McCormick et al. 1992a,b). IGF also has osmoregulatory actions, as has recently been detected in rainbow trout by McCormick et al. (1991). The demonstration of GH receptors in rainbow trout gills (Sakamoto and Hirano, 1991) and of an osmoregulatory role for IGF suggests that an analogous ‘GH–IGF system’ is present in gills and liver. The apparent low density of GH receptors in branchial epithelium (3.1fmolmg1 protein) should be interpreted with care: assuming that the ionocytes in the gills are the target for GH and an average ionocyte density of 3% in the branchial epithelium (G. Flik, personal observation on freshwater rainbow trout and tilapia), the receptor density of ionocytes (3.1X100/3=103fmolmg1 protein) may be even higher than that of hepatocytes (71.0fmolmg1 protein). Clearly, the presence of GH receptors on ionocytes is in good agreement with the consensus that GH exerts osmoregulatory effects in seawater-adapting and seawater-adapted salmonids, and this action could be independent of IGF production by the liver.

To our knowledge, only one study has been published on the effects of GH in tilapia (Clarke et al. 1977): in juvenile tilapia, high doses of homologous GH stimulated growth, as was indicated by increases in length and mass. We have shown before (Flik et al. 1985, 1986a,b) that, in tilapia, calcium uptake from the water via the gills is pivotal for growth and calcium homeostasis. The availability of recombinant tilapia GH (rtGH; Rentier-Delrue et al. 1989) allowed us to test homologous GH for its growth-promoting effect in freshwater tilapia. We focused our study on the effects of rtGH on calcium handling by the tilapia. Freshwater tilapia were weighed and injected with rtGH twice a week for up to 7 weeks. We measured the hepatosomatic index and the calcium balance of the fish and analyzed the plasma ion composition, the opercular ionocyte density and the bone and scale calcium and phosphate contents.

Fish

Tilapia, Oreochromis mossambicus Peters, were obtained from laboratory stock. The fish were held in 120-l all-glass aquaria with a continuous flow of Nijmegen city tapwater (0.7mmol l1 Ca2+, 0.2mmol l1 Mg2+, 0.5mmol l1 Na+, 0.06mmol l1 K+, pH7.8); the water temperature was 26±0.2°C. Lights were on for 12h per day. The loading density of fish did not exceed 1.5kg100 l1. Commercial trout pellets (Trouvit, Putten, The Netherlands) were administered by means of automated food dispensers throughout the light period at 2 or 5% of the recorded total mass of the groups of fish per day. Care was taken that all fish had access to the food and that all food was consumed. Experiments were carried out in May and June.

Hormone treatment and experimental design

Recombinant tilapia growth hormone (rtGH; Rentier-Delrue et al. 1989) was dissolved in 0.02mol l−1 NH4HCO3 (pH9.0) and diluted to the required concentration with phosphate-buffered saline (pH7.8). The dose was 100ng g1 fish. Injections were given intraperitoneally with a Hamilton precision syringe. The carrier volume was 1 μlg1 fish. Control animals received saline injections. Fish were injected twice a week, on Monday morning and Thursday afternoon. The fish were weighed before injection. The use of a Mettler PM34 Delta range balance allowed weighing of free-swimming fish and stress is therefore minimized compared with that seen with netting. Four groups of 15 fish each were included in an experiment: one group was weighed at the start and upon completion of the experiment, and not handled further (group U); one group was handled in the same way as the injected groups, but not injected (group H); one group served as injection controls (group S); and one group received rtGH injections (group GH). The holding conditions for all four groups were identical. The duration of the hormone treatment was 44 days.

Upon completion of the experiments, the fish were quickly anaesthetized in Tris-buffered (pH7.8) 3-aminobenzoic acid ethyl ester (MS-222; 1 gl1) and blood was collected (see below); subsequently, the fish were killed by spinal transection. The liver was removed and weighed to calculate the hepatosomatic index, defined as (Wl/Wf) ×100% (where Wl is the mass of the liver in grams and Wf is the mass of the fish in grams). One operculum was removed for determination of the ionocyte density. Vertebral bone and scale samples were collected to assess calcium and phosphate contents (see below).

Plasma analysis

Mixed arterial and venous blood was collected by puncture of the caudal vessels, using a heparinized tuberculin syringe fitted with a 23-gauge needle. Plasma was separated from cells by centrifugation (1min, 9000 g). Part of the plasma was ultrafiltered (Millipore Ultrafree-MC, molecular cut-off 10000Da) for the estimation of free calcium and magnesium levels. Total calcium and magnesium of plasma and ultrafiltered plasma were determined with commercial colorimetric kits (Sigma, St Louis, MO, USA). Combined calcium/phosphate and magnesium standards (Sigma) were used as reference. Plasma sodium and potassium contents were determined by flame emission spectrophotometry. Plasma osmolality was determined with a Roebling osmometer on fresh 50 μl plasma samples. Cortisol was determined on 5 μl of plasma by radioimmunoassay (Amerlex assay IM-2021, Amersham plc, UK), which is based on a highly specific antibody. The assay is not affected by the anticoagulant heparin. The recovery, defined as the increase in value when a known amount of cortisol is added to the sample and expressed as the percentage recovery (i.e. the measured increase divided by the predicted increase), varied between 98% and 103% in the range of the standard curve. The measurement range of this assay is 0–1700nmol l1. The sensitivity of the standard curve, estimated from the 95% confidence intervals of the within-assay variation of the zero standard counts, was 3±1nmol l1 (N=5). Serial dilutions of plasma samples yielded curves parallel to the standard curve.

Bone calcium and phosphate

Triplicate samples of 10 scales each were taken from both sides at the mid-lateral region, posterior to the operculum. A sample of vertebral bone was taken after removal of adhering muscle tissue by microwave cooking (1min, 700W). All tissues were weighed and dried to constant mass at 90°C, and the dry mass was determined to the nearest 0.01mg. Dried samples were dissolved in 0.5ml of concentrated HNO3 at 60°C for 1h. The sample volume was brought to 5ml with double-distilled water. The total calcium was determined by a Thymol Blue method (for details, see Flik et al. 1986a) and phosphate was determined according to Fiske and Subbarow (1926).

Opercular ionocyte density

Opercula were incubated for 1h in 2 μmol l1 2-(dimethylaminostyryl)-1-ethylpyridinium iodine (DASPEI) dissolved in water. DASPEI stains the mitochondrion-rich chloride cells (ionocytes) (Bereiter-Hahn, 1976). After rinsing, the inner opercular epithelium was examined in a Zeiss fluorescence microscope at a magnification of 250×. Cells were counted in 20 different squares of the opercular epithelium with a total surface area of 5mm 2 per fish.

Calcium fluxes

The rtGh-treated fish and their controls were used 44–46 days after the start of hormone treatment. Unidirectional calcium influx was determined on the basis of initial uptake rates of 45Ca2+ from the water and the water 45Ca2+ specific activity, as described in detail elsewhere (Flik et al. 1989). Briefly, fish were exposed to water containing 45Ca2+ of known specific activity. The fish were not fed during the flux determination. After 4h, the fish was anaesthetized, rinsed in 10mmol l1 CaCl2 to remove tracer adsorbed to the body surface and quick-frozen on solid CO2. Next, the fish was partly defrosted and the still frozen intestinal tract was removed. Subsequently, the 45Ca2+ content of the fish and intestinal tract was determined after microwave-cooking and blending the cooked tissues with a known amount of distilled water in a Waring-type blender. Using this method, tracer uptake as a result of drinking is separated from branchial tracer uptake (Pang et al. 1980). The whole-body calcium influx determined in this way was shown to reflect extraintestinal branchial influx. Growing tilapia increase their total body calcium pool, and its size is directly related to the mass of the fish (see below). Therefore, net calcium influx may be determined on the basis of the growth-related increase in the total body calcium pool (the net influx equals the mean accumulation rate of calcium). The treatments reported here did not affect the calcium content of the bone, and therefore the total body calcium pool (Qf) may be calculated as Qf =357.5Wf0.965μmol (W in g; Flik et al. 1985). Net calcium influx was calculated as ΔQft (time in h) over the 44 day period of the experiment. Calcium efflux (Fout) was calculated as the difference between branchial calcium influx (Fin) and net calcium influx (Fnet), Fout=Fin-Fnet.

The fish used in the experiments received food at a dose of 2 or 5% of their mass per day. To evaluate the effects of the amount of food provided on the calcium influx from the water, a comparison was made between the measured calcium influx and the predicted calcium influx at a food dose of 1% per day, given by the equation: Fin=50Wf0.805 nmol h1 (W in g; Flik et al. 1985).

Statistics

Values are presented as means ± S.D. Differences among groups were assessed by means of a one-way analysis of variance. Significance of differences between means was subsequently assessed by the Newman–Keuls test or the Mann–Whitney U-test, where appropriate. Linear regression analysis was based on the least-squares method. Significance was accepted when P<0.05.

rtGH treatment significantly stimulated growth as determined by the increase in body mass and length after 44 days (Table 1). These increases were significant when compared with those of unhandled (P<0.01) as well as with those of handled and saline-injected groups (P<0.05). No differences were observed among the control groups (U, H and S). The percentage increase in body mass over the initial mass after 44 days of treatment was 44.9% for the GH group and 25.6, 28.1 and 28.2% for groups U, H and S, respectively. The percentage increase in length was 15% for GH-treated fish and 8.4%, 9.1% and 9.1% for groups U, H and S, respectively.

Table 1.

Effects of recombinant tilapia growth hormone on body mass (in g) and length (in cm) of freshwater tilapia

Effects of recombinant tilapia growth hormone on body mass (in g) and length (in cm) of freshwater tilapia
Effects of recombinant tilapia growth hormone on body mass (in g) and length (in cm) of freshwater tilapia

Branchial calcium influx increased with the amount of food provided (Table 2). Male fish fed 5% per day had a calcium influx twice as high (P<0.05, Mann–Whitney U-test) as male fish fed 2%, 11.7 and 5.8 μmol h1 100 g1, respectively. Calculated values for branchial calcium influx at a food dose of 1% are around 2 μmolh1 100 g1. For male fish, the uptake of calcium from the water was positively correlated with the amount of food consumed according to: Fin=(2.36±0.18)X(% food)+0.11 μmol h1 100 g1 (r=0.987; P<0.01). Female fish had a calcium influx significantly (P<0.01, Mann–Whitney U-test) lower than that of male fish [both fed 5%, 6.2 and 11.7 μmol h1 100 g1, respectively]. The 5% food regimen represented ad libitum food conditions and was chosen for the GH experiment.

Table 2.

Feeding and branchial calcium influx in freshwater tilapia

Feeding and branchial calcium influx in freshwater tilapia
Feeding and branchial calcium influx in freshwater tilapia

rtGH had no effects on calcium influx from the water (Table 3). As a result of the increased growth rate, rtGH-treated fish showed an enhanced net accumulation of calcium (9.45±0.60 μmol h1 100 g1), 36% higher than in the untreated fish (P<0.025) and 25% higher than in the handled fish (P<0.05) and 37% higher than in the saline-injected fish (P<0.01). The calculated net efflux of calcium in rtGH-treated fish was, therefore, lower than in the controls.

Table 3.

Growth hormone and calcium fluxes in freshwater tilapia

Growth hormone and calcium fluxes in freshwater tilapia
Growth hormone and calcium fluxes in freshwater tilapia

Table 4 gives data on plasma ion composition and cortisol levels in fish treated for 44 days with rtGH. The data for the three control groups (U, H and S) have been pooled as no differences were observed among these groups for any of the variables measured. GH did not influence plasma ion composition but had a significant (P<0.025, Mann–Whitney U-test) effect on plasma cortisol levels, which were almost 50% lower than in the controls.

Table 4.

Effects of growth hormone on plasma ion composition and cortisol levels

Effects of growth hormone on plasma ion composition and cortisol levels
Effects of growth hormone on plasma ion composition and cortisol levels

GH treatment increased the hepatosomatic index (HSI) compared with that of saline-injected (P<0.01) and handled and untreated fish (P<0.05) (Fig. 1). No differences were observed among groups U, H and S. In GH-treated fish, the HSI increased by 43 % compared with that of saline-treated fish (from 1.64 to 2.34%).

Fig. 1.

Effects of GH on the hepatosomatic index [(mass of liver/mass of fish) ×100%] after 44 days of treatment with GH. U, unhandled fish; H, handled fish; S, saline-injected fish; GH, growth-hormone-injected fish. *Significantly different from S (P<0.01) and U and H (P<0.05; Newman–Keuls test. Bars indicate standard deviation; N=9.

Fig. 1.

Effects of GH on the hepatosomatic index [(mass of liver/mass of fish) ×100%] after 44 days of treatment with GH. U, unhandled fish; H, handled fish; S, saline-injected fish; GH, growth-hormone-injected fish. *Significantly different from S (P<0.01) and U and H (P<0.05; Newman–Keuls test. Bars indicate standard deviation; N=9.

Analysis of vertebral bone and scales (Table 5) revealed no differences among the four groups of fish (U, H, S and GH) after 44 days of treatment. The values for the calcium content of vertebrae and scales are similar to previously reported values for tilapia (Flik et al. 1986b). The ratios of calcium to phosphate are in all cases close to the value of 1.43, representative for whitlockite, the probable form of apatite in fish bone (Herrmann-Erlee and Flik, 1989).

Table 5.

Recombinant tilapia growth hormone and bone calcium and phosphate content

Recombinant tilapia growth hormone and bone calcium and phosphate content
Recombinant tilapia growth hormone and bone calcium and phosphate content

Opercular ionocyte densities increased in GH-treated fish compared with all other groups (P<0.01) (Fig. 2). The ionocyte density in the saline-injected group was significantly higher than in the unhandled (P<0.01) and handled (P<0.05) groups.

Fig. 2.

Effects of GH on opercular ionocyte density after 44 days of GH treatment. U, unhandled fish; H, handled fish; S, saline-injected fish; GH, growth-hormone-injected fish. *Significantly different from U, H and S (P<0.01); †significantly different from U (P<0.01) and H (P<0.05; Newman–Keuls test). Bars indicate standard deviation; N=9.

Fig. 2.

Effects of GH on opercular ionocyte density after 44 days of GH treatment. U, unhandled fish; H, handled fish; S, saline-injected fish; GH, growth-hormone-injected fish. *Significantly different from U, H and S (P<0.01); †significantly different from U (P<0.01) and H (P<0.05; Newman–Keuls test). Bars indicate standard deviation; N=9.

The results presented here demonstrate that tilapia recombinant growth hormone (Rentier-Delrue et al. 1989) is bioactive. The mild growth-promoting activity of rtGH in freshwater tilapia compares well with the mild effects of homologous recombinant GH in carp (Fine et al. 1993). The growth-stimulatory activity is further substantiated by its classical stimulatory effect on the hepatosomatic index. The growth-promoting effect of rtGH, therefore, appears to be mediated through stimulation of the liver, where the IGF is produced that subsequently enhances the growth of cartilage and bone. No change in the plasma ion composition was observed after rtGH treatment. The bone compartments increased in size but no changes in the mineral density occurred. Apparently, sufficient calcium, phosphate and bone matrix were available for calcium deposition to allow enhanced growth without influencing plasma ion composition or bone mineral density. The effect of GH on the calcium balance of tilapia is clearly different from that of prolactin (PRL). PRL does not stimulate growth, but stimulates calcium influx and reduces calcium efflux and, by doing so, induces hypercalcaemia and increases bone calcium density (Flik et al. 1986b; Swennen et al. 1991). We have reported before that low water levels of calcium (i.e. 0.2mmol l1 or less) may influence bone calcium content and plasma ion composition in tilapia (Flik et al. 1986a; Urasa and Wendelaar Bonga, 1987). Furthermore, it has been shown that tilapia mobilize calcium and phosphate from their bone, but only in times of shortage, e.g. during ovarian maturation and when diets are deficient in calcium (Urasa et al. 1985). It appears that fish do not show the strict calcium homeostasis observed in terrestrial vertebrates and rely primarily on external calcium (Perry and Flik, 1988). The observation that the unidirectional influx of calcium from the water is always larger than the calculated net influx (i.e. the growth-related calcium accumulation rate) into the fish suggests that fish rely on branchial calcium influx for growth and homeostasis: the amount of calcium offered in the food would have been sufficient to allow the growth observed (see below), yet the fish increased their branchial calcium influx. We realize that an estimation of the efficiency of intestinal calcium absorption may ultimately shed light on the role of intestinal calcium handling for growth.

A remarkable observation is that branchial calcium influx increased linearly with the amount of food offered to the fish. Given a food calcium content of 850 μmol g1 at an amount of 5% of its body mass per day, a 100g fish obtains 5×850=4250 μmol calcium. The Ca2+ influx via the gills per day is Fin×24=11.7×24=280.8 μmol for a 100g fish. Thus, although the food-associated calcium would suffice to cover the fish’s extra need for (enhanced) growth, a larger dietary calcium input does not lead to a decreased branchial calcium influx. Ample evidence has been given that calcium transport occurs in the proximal intestine of the tilapia (Flik et al. 1990). However, we have no information on the net movements of calcium in more distal segments of the intestine, where calcium secretion must occur. The role of dietary calcium in the calcium metabolism of the fish remains enigmatic: a calcium-deficient diet does not hamper calcium homeostasis or growth (Berg, 1970; Urasa and Wendelaar Bonga, 1987). Another line of evidence suggesting that branchial calcium uptake is adjusted to the growth rate of the fish comes from our data on female fish: under identical conditions, female fish have a lower calcium influx from the water, grow more slowly and do not reach the size of males (in our laboratory).

Recombinant tilapia GH did not enhance calcium influx (Fin) from the water. We realize that this statement is based on data that reflect a single time point, i.e. 44 days after the start of the treatment. It could well be that the unidirectional influx was stimulated earlier in the 44-day period. Such an increase could have accounted for an increased integrated net flux over the 44-day period, whereas the increased unidirectional influx had attenuated by day 44. The conclusion would be that rtGH transiently stimulated Ca2+ influx from the water to enhance calcium accumulation for growth. However, on the basis of the consistent stimulation of growth observed in this experiment, we consider this possibility to be unlikely. Moreover the Ca2+ influxes determined 16 days after the start of treatment on six saline-injected and five rtGH-injected fish were not significantly different (Fin=8.9±3.4 and 10.3±4.1 μmol h1 100 g1 fish, for saline-and GH-treated fish, respectively; P>0.10), nor were these values different from those determined after 44 days of treatment. The Ca2+ influx in a fish provided with 5% food per day appears to be high enough to cover the growth-related accumulation of calcium. The rtGH-treated fish grew faster and enlarged their bone compartment (the bone calcium content did not change); these fish apparently accumulated more calcium per unit time (Fnet increased). As a result, the calculated total efflux of calcium is lower in rtGH-treated fish. Direct measurements of branchial and extrabranchial calcium efflux are required to discriminate between branchial, renal and intestinal efflux routes as targets for rtGH.

The stimulation by rtGH of the opercular, and presumably the branchial, ionocyte density suggests an osmoregulatory action of the hormone. The total, DASPEI-stainable ionocyte population is not necessarily a good indicator of ion-transport activity of the gills. It may well be that the ionocyte turnover and number were stimulated by rtGH, but that at the same time the number of functional ionocytes, i.e. fully developed cells in contact with the water (Wendelaar Bonga et al. 1990), remained unchanged. However, we favour the idea that this rtGH effect in freshwater fish was an IGF-dependent mitogenic effect of exogenous GH. Clearly, these data require future evaluation of IGF levels. A stimulation of branchial ionocytes has been reported for seawater trout (Madsen, 1990); injection of GH into seawater fish improves hypo-osmoregulatory mechanisms and therefore the stimulation by GH of branchial ionocytes may be of functional significance.

The opercular ionocyte density in the control groups was somewhat higher than the densities reported before, 120–188mm2versus less than 100mm2 in control freshwater tilapia (Wendelaar Bonga et al. 1990). We tentatively relate this increased ionocyte density to the relatively high cortisol levels (discussed below) seen in these fish (around 1000nmol l1). However, an increased cortisol level does not explain the increased ionocyte density in the GH-treated fish, as cortisol levels were about 50 % lower in these fish than in the controls. It would be interesting to know the dynamics of cortisol under these conditions. The lower level of cortisol could reflect an increased utilization of cortisol in rtGH-treated fish.

In general, the plasma cortisol levels in all four groups were high compared with those of completely unstressed fish (typical cortisol levels around 100nmol l1; G. Flik, unpublished observation). The present experimental set-up with frequent handling and an intensive feeding regimen, in all likelihood, leads to increased cortisol levels. Indeed the levels in groups U, H and S are elevated to a level normally seen in fish 10min after the start of netting. An alternative explanation for the lower cortisol levels in the GH group may be that the stress-related cortisol response in these fish has become suppressed. We did not observe differences between unhandled and handled control fish with respect to cortisol levels and, therefore, tentatively conclude that the elevated plasma cortisol levels somehow relate to the high dose of food provided for our fish. Again, this topic needs further research.

Lower cortisol levels in GH-treated fish may further indicate that GH exerts a negative control over the interrenals, a conclusion in line with the data of Carsia et al. (1985) on domestic fowl. It contrasts, however, with data on coho salmon by Young (1988), who reported a stimulatory action of (ovine) GH on the interrenals, both in vivo and in vitro. A negative feedback of GH on the interrenals, as suggested by our data, completes the functional interrelationship between cortisol and GH-producing cells proposed by Nishioka et al. (1985), who reported stimulatory effects of cortisol on GH release by tilapia pituitary gland in vitro.

The authors thank Tom Spanings for the excellent organization of the fish husbandry.

Ayson
,
F. G.
,
Kaneko
,
T.
,
Tagawa
,
M.
,
Hasegawa
,
S.
,
Grau
,
E. G.
,
Nishioka
,
R. S.
,
King
,
D. S.
,
Bern
,
H. A.
and
Hirano
,
T.
(
1993
).
Effects of acclimation to hypertonic environment on plasma and pituitary levels of two prolactins and growth hormone in two species of tilapia, Oreochromis mossambicus and Oreochromis niloticus
.
Gen. comp. Endocr.
89
,
138
148
.
Bereiter-Hahn
,
J.
(
1976
).
Dimethylaminostyrylmethylpyridinium-iodine as a fluorescent probe for mitochondria in situ
.
Biochim. biophys. Acta
423
,
1
14
.
Berg
,
A.
(
1970
).
Studies on the metabolism of calcium and strontium in freshwater fish. II. Relative contribution of direct and intestinal absorption in growth conditions
.
Me. Inst. Ital. Idrobiol.
26
,
241
255
.
Bolton
,
J. P.
,
Collie
,
N. L.
,
Kawauchi
,
T.
and
Hirano
,
T.
(
1987
).
Osmoregulatory actions of growth hormone in rainbow trout (Salmo gairdneri)
.
J. Endocr.
112
,
63
68
.
Carsia
,
R. V.
,
Weber
,
H.
,
King
,
D. B.
and
Scanes
,
C. G.
(
1985
).
Adrenocortical cell function in hypophysectomized domestic fowl: effects of growth hormone and 3,5,3-triiodothyronine replacement
.
Endocrinol.
117
,
928
933
.
Clarke
,
W. C.
,
Walker Farmer
,
S.
and
Hartwell
,
K. M.
(
1977
).
Effect of teleost pituitary growth hormone on growth of Tilapia mossambica and on growth and seawater adaptation of sockeye salmon (Oncorhynchus nerka)
.
Gen. comp. Endocr
.
33
,
174
178
.
Cook
,
A. F.
,
Wilson
,
S. W.
and
Peter
,
R. E.
(
1983
).
Development and validation of a carp growth hormone radioimmunoassay
.
Gen. comp. Endocr.
50
,
335
339
.
Drakenberg
,
K.
,
Sara
,
V. R.
,
Lindahl
,
K. I.
and
Kewish
,
B.
(
1989
).
The study of insulin-like growth factors in tilapia Oreochromis mossambicus
.
Gen. comp. Endocr.
74
,
173
180
.
Duan
,
C.
and
Hirano
,
T.
(
1990
).
Stimulation of 35S-sulphate uptake by mammalian insulin-like growth factors I and II in cultured cartilages of the Japanese eel, Anguilla japonica
.
J. exp. Zool.
256
,
347
350
.
Fine
,
M.
,
Sakal
,
E.
,
Vashdi
,
D.
,
Daniel
,
V.
,
Levanon
,
A.
,
Lipshitz
,
O.
and
Gertler
,
A.
(
1993
).
Recombinant carp (Cyprinus carpio) growth hormone: expression, purification and determination of biological activity in vitro and in vivo
.
Gen. comp. Endocr.
89
,
51
61
.
Fiske
,
C. H.
and
Subbarow
,
Y.
(
1926
).
The colorimetric determination of phosphorus
.
J. biol. Chem.
66
,
375
400
.
Flik
,
G.
,
Fenwick
,
J. C.
,
Kolar
,
Z.
,
Mayer-Gostan
,
N.
and
Wendelaar Bonga
,
S.E.
(
1985
).
Whole body calcium flux rates in the cichlid teleost fish Oreochromis mossambicus, adapted to fresh water
.
Am. J. Physiol.
249
,
R432
R437
.
Flik
,
G.
,
Fenwick
,
J. C.
,
Kolar
,
Z.
,
Mayer-Gostan
,
N.
and
Wendelaar Bonga
,
S.E.
(
1986a
).
Effects of low ambient calcium levels on whole-body Ca2+-flux rates and internal calcium pools in the freshwater cichlid teleost, Oreochromis mossambicus
.
J. exp. Biol.
120
,
249
266
.
Flik
,
G.
,
Fenwick
,
J. C.
,
Kolar
,
Z.
,
Mayer-Gostan
,
N.
and
Wendelaar Bonga
,
S.E.
(
1986b
).
Effects of ovine prolactin on calcium uptake and distribution in Oreochromis mossambicus
.
Am. J. Physiol.
250
,
R161
R166
.
Flik
,
G.
,
Fenwick
,
J. C.
and
Wendelaar Bonga
,
S.E.
(
1989
).
Calcitropic actions of prolactin in freshwater North American eel (Anguilla rostrata LeSueur)
.
Am. J. Physiol.
257
,
R74
R79
.
Flik
,
G.
,
Schoenmakers
,
TH. J. M.
,
Groot
,
J. A.
,
Van Os
,
C. H.
and
Wendelaar Bonga
,
S.E.
(
1990
).
Calcium absorption by fish intestine: the involvement of ATP- and sodium-dependent calcium extrusion mechanisms
.
J. Membr. Biol.
113
,
13
22
.
Fryer
,
J. N.
(
1979
).
A radioreceptor assay for purified teleost growth hormone
.
Gen. comp. Endocr.
39
,
123
130
.
Gray
,
E. S.
and
Kelley
,
K. M.
(
1991
).
Growth regulation in the gobiid teleost, Gillichthys mirabilis: roles of growth hormone, hepatic growth hormone receptors and insulin-like growth factor-I
.
J. Endocr.
131
,
57
66
.
Gray
,
E. S.
,
Young
,
G.
and
Bern
,
H. A.
(
1990
).
Radioreceptor assay for growth hormone in coho salmon (Oncorhynchus kisutch) and its application to the study of stunting
.
J. exp. Zool.
256
,
290
296
.
Herrmann-Erlee
,
M. P. M.
and
Flik
,
G.
(
1989
).
Bone: comparative studies on endocrine involvement in bone metabolism
. In
Vertebrate Endocrinology: Fundamentals and Biomedical Implications
, vol.
3
(ed.
P. K. T.
Pang
and
M.
Schreibman
), pp.
211
242
. New York,
London
:
Academic Press
.
Hirano
,
T.
(
1991
).
Hepatic receptors for homologous growth hormone in the eel
.
Gen. comp. Endocr.
81
,
383
390
.
Le Bail
,
P. Y.
,
Sumpter
,
J. P.
,
Carragher
,
J. F.
,
Mourot
,
B.
,
Niu
,
P. D.
and
Weil
,
C.
(
1991
).
Development and validation of a highly sensitive radioimmunoassay for chinook salmon (Oncorhynchus tshawytscha) growth hormone
.
Gen. comp. Endocr.
83
,
75
85
.
Madsen
,
S. S.
(
1990
).
The role of cortisol and growth hormone in seawater adaptation and development of hypoosmoregulatory mechanisms in sea trout parr (Salmo trutta trutta)
.
Gen. comp. Endocr.
79
,
1
11
.
Marchant
,
T. A.
,
Fraser
,
R. A.
,
Andrews
,
P. C.
and
Peter
,
R. E.
(
1987
).
The influence of mammalian and teleost somatostatins on the secretion of growth hormone from goldfish (Carassius auratus L.) pituitary fragments in vitro
.
Regul. Peptides
17
,
41
47
.
Mccormick
,
S. D.
,
Kelley
,
K. M.
,
Young
,
G.
,
Nishioka
,
R. S.
and
Bern
,
H. A.
(
1992a
).
Stimulation of coho salmon growth by insulin-like growth factor I
.
Gen. comp. Endocr.
86
,
398
406
.
Mccormick
,
S. D.
,
Sakamoto
,
T.
,
Hasegawa
,
S.
and
Hirano
,
T.
(
1991
).
Osmoregulatory actions of insulin-like growth factor-I in rainbow trout (Oncorhynchus mykiss)
.
J. Endocr.
130
,
87
92
.
Mccormick
,
S. D.
,
Tsai
,
P. I.
,
Kelley
,
K. M.
,
Nishioka
,
R. S.
and
Bern
,
H. A.
(
1992b
).
Hormonal control of sulfate uptake by branchial cartilage of coho salmon: role of IGF-I
.
J. exp. Zool.
262
,
166
171
.
Nishioka
,
R. S.
,
Grau
,
E. G.
and
Bern
,
H. A.
(
1985
).
In vitro release of growth hormone from the pituitary gland of tilapia, Oreochromis mossambicus
.
Gen. comp. Endocr
.
60
,
90
94
.
Nishioka
,
R. S.
,
Kelly
,
K. M.
and
Bern
,
H. A.
(
1988
).
Control of prolactin and growth hormone secretion in teleost fishes
.
Zool. Sci.
5
,
267
272
.
Pang
,
P. K. T.
,
Griffith
,
R. W.
,
Maetz
,
J.
and
Pic
,
P.
(
1980
).
Calcium uptake in fishes
. In
Epithelial Transport in the Lower Vertebrates
(ed.
B.
Lahlou
), pp.
121
131
. Malta: Cambridge University Press.
Perry
,
S. F.
and
Flik
,
G.
(
1988
).
Characterization of branchial transepithelial calcium fluxes in freshwater trout, Salmo gairdneri
.
Am. J. Physiol
.
254
,
R491
R498
.
Rentier-Delrue
,
F.
,
Swennen
,
D.
,
Philippart
,
J. C.
,
L’hoir
,
C.
,
Lion
,
M.
,
Benrubi
,
O.
and
Martial
,
J. A.
(
1989
).
Tilapia growth hormone: molecular cloning of cDNA and expression in Escherichia coli
.
DNA
8
,
271
278
.
Sakamoto
,
T.
and
Hirano
,
T.
(
1991
).
Growth hormone receptors in the liver and osmoregulatory organs of rainbow trout: characterization and dynamics during adaptation to seawater
.
J. Endocr.
130
,
425
433
.
Swennen
,
D.
,
Rentier-Delrue
,
F.
,
Auperin
,
B.
,
Prunet
,
P.
,
Flik
,
G.
,
Wendelaar Bonga
,
S. E.
,
Lion
,
M.
and
Martial
,
J. A.
(
1991
).
Production and purification of biologically active recombinant tilapia (Oreochromis niloticus) prolactins
.
J. Endocr
.
131
,
219
227
.
Urasa
,
F. M.
,
Flik
,
G.
and
Wendelaar Bonga
,
S.E.
(
1985
).
Selective mobilization of phosphate from bone during ovarian development in the teleost Oreochromis mossambicus
. In
Fish Culture. Proceedings of the 7th conference of the ESCPB
, pp.
1
9
. Barcelona: Promociones Publicaciones Universitarias.
Urasa
,
F. M.
and
Wendelaar Bonga
,
S.E.
(
1987
).
Effects of calcium and phosphate on the corpuscles of Stannius of the teleost fish, Oreochromis mossambicus
.
Cell Tissue Res.
249
,
681
690
.
Wendelaar Bonga
,
S. E.
,
Flik
,
G.
,
Balm
,
P. H. M.
and
Van Der Meij
,
J. C. A.
(
1990
).
The ultrastructure of chloride cells in the gills of the teleost Oreochromis mossambicus during exposure to acidified water
.
Cell Tissue Res.
259
,
575
585
.
Young
,
G.
(
1988
).
Enhanced response of the interrenal of coho salmon (Oncorhynchus kisutch) to ACTH after growth hormone treatment in vivo and in vitro
.
Gen. comp. Endocr
.
71
,
85
92
.