Plasma immunoreactive-growth hormone in domestic fowl: Measurement by homologous and heterologous radioimmunoassays

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

65,92-98

(1987)

Plasma Immunoreactive-Growth Hormone in Domestic Fowl: Measurement by Homologous and Heterologous Radioimmunoassays S. HARVEY,*,*

S. HOSHINO,?

AND M. SUZUKI~

* Wolfson Institute, University of Hull, Hull HU6 7RX England; tFaculty of Agriculture, Mie University, Tsu, 514, Japan; and *Department of Physiology, Institute of Endocrinology. Gunma University, Maebashi 371, Japan Accepted August

10, 1986

Concentrations of immunoreactive (IR) growth hormone (GH) in the plasma of domestic fowl have been measured by homologous and heterologous radioimmunoassays and the estimates compared. Both assays detected an age-related decline in the circulating IR-GH concentration, an increase in IR-GH secretion following TRH-stimulation or fasting, and a fall in the IR-GH concentration following adrenocorticotropin administration. However, while the overall estimates of IR-GH concentration were significantly correlated, the magnitude of the changes in IR-GH concentration determined by the homologous assay were far greater than those detected by the heterologous system, which failed to show any inhibitory effect of anesthesia or exogenous thyroid hormones on basal or stimulated IR-GH release. These results suggest that the heterologous GH radioimmunoassay lacks the sensitivity of the homologous chicken GH assay and that circulating GH in birds is probably composed of heterogenous moieties with differing immunoreactivities with rat GH antibodies. o 1987 Academic

Press, Inc.

The development of homologous and/or heterologous radioimmunoassays for luteinizing hormone (LH) (Follett et al., 1972; Wentworth et al., 1976), follicle stimulating hormone (FSH) (Croix et al., 1974; Scanes et al., 1977), prolactin (Scanes et al., 1976; McNeilly et al., 1978; Burke and Papkoff, 1980; Proudman and Opel, 1981; Etches and Cheng, 1982; Lea et al., 1981), and growth hormone (GH) (Harvey and Scanes, 1977; Proudman and Wentworth, 1978; Hoshino et al., 1980; Leung et al., 1984a) has promoted considerable research on the physiology of pituitary hormones in birds. Comparisons of the different assay systems for plasma gonadotropins (e.g., Goldsmith and Follett, 1983; Proudman et al., 1984) and prolactin (Etches and Cheng, 1982; Harvey and Bedrak, 1984) have, however, shown discrepancies between the

results obtained. For comparative purposes we have therefore determined plasma immunoreactive (IR) GH concentrations in domestic fowl by homologous and heterologous radioimmunoassay. MATERIALS

Thornber 404 cockerels were reared from hatch under constant light in deep litter, with food and water available ad libitum until used for experimentation (between 3 and 7 weeks of age) or until adult (>24 weeks of age). Experiment 1. Three-week-old birds were bled by brachial vein venipuncture and intravenously injected with 0.9% NaCl (1 ml/kg), with thyroxine (T4, 250 t&kg), or with triiodothyronine (T,, 250 &kg). A second blood sample was collected 60 min after injection, immediately prior to the intravenous administration of thyrotropin releasing hormone (TRH, IO kg/kg) or its 0.9% NaCl vehicle (1 ml/kg). A third blood sample was obtained 10 min later. After separation the plasma was stored at - 20” until required for assay. Experiment 2. To determine the influence of anaesthesia on plasma IR-GH levels another group of 3week-old cockerels (n = 16) was intraveneously injected (3 ml/kg) with sodium pentobarbital and a venous blood sample was taken 30 min later. Plasma

t Present address: Department of Physiology, University of Alberta, Edmonton, Canada T6G 2H7. 92 0016~6480187

$1.50

Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

AND METHODS

93

IR-GH IN CHICKENS IR-GH concentrations were compared with those observed in conscious birds. Experiment 3. Five-week-old cockerels were intraveneously injected with 0.9% NaCl (1 ml/kg) or with synthetic adrenocorticotophin (ACTH) (Synacthen depot 25 m/kg). Two hours later each bird was bled by brachial vein venipuncture and intraveneously injected with either 0.9% NaCl (1 ml/kg) or TRH (IO kg/kg). A second venous blood sample was collected from each bird 10 min later. A third group of $weekold cockerels was deprived of food for 24 hr prior to the intraveneous administration of TRH (IO wgikg) and was bled before and IO min afterward. Experiment 4. Seven-week-old cockerels were deprived of food for 48 hr and then refed ad libitmz. Venous blood samples were collected before and at intervals after refeeding. Control birds were deprived of food throughout the experiment. Experiment 5. A group of adult (>24-week-old) cockerels was bled by brachial vein venipuncture and its plasma IR-GH levels were compared with those in younger birds used in the earlier studies. Hormone and drugs. Thyroxine and T, were obtained from Sigma Chemical Company (Poole, Dorset), TRH was donated by Reckit and Coleman Ltd. (Hull, North Humberside), and Synacthen was a gift from Ciba (Horsham, Surrey). Radioirnmunoassays. Concentrations of IR-GH in the same plasma samples were determined by homologous (Harvey and Scanes. 1977) and heterologous (Hoshino et al., 1980) radioimmunoassays in England and Japan, respectively. The data were examined for statistical differences by Students c test and analysis of variance, whenever appropriate, and correlated by linear regression.

la

Before After

r* IR:GH P9’l

.¶ E ; 30-

: gzod I5 IOOVehicle

I T4

1

rNSl 251

‘*l

OVehicle

T,

T3

FIG. 1. Concentrations of immunoreactive growth hormone (IR-GH) in the plasma of immature domestic fowl, determined by homologous or heterologous radioimmunoassays (RIAs), before (open bars) and 60 min after (solid bars) the iv administration of thyroxine (T4, 250 ug/kg) or triiodothyronine (T,, 250 pg/kg). Controls were similarly injected with the 0.9% NaCl vehicle (1 ml/kg). Means ? SE (n = 20). Statistical differences (P < 0.05) are indicated by the asterisks; differences not statistically significant (NS) are also indicated.

plasma IR-GH levels, as measured by the homologous assay (by 74%, P < O.OOl), but not when measured by the heterologous system. Overall estimates of plasma IR-GH 60 min after saline, T,, and T, administration were slightly correlated (Y = 0.366, n = 58, P < 0.01). In birds pretreated with saline, T,, and T,, the administration of 0.9% NaCl (1 RESULTS ml/kg) had no significant effect on plasma Experiment 1. Pretreatment concentraIR-GH levels. The administration of TRH, tions of IR-GH measured by the homoloin contrast, markedly elevated IR-GH congous radioimmunoassay (36.6 * 3.0 pg/ centrations, as measured by both assay liter, IZ = 60) were significantly (P < 0.001) systems (Fig. 2). In birds pretreated with higher than those measured by the heterolsaline the IR-GH response (20.07 t 0.25ogous system (19.46 + 0.53 &liter, n = 58) fold, P < 0.001) to TRH challenge was 532 and were unrelated (Y = 0.182, n = 58, P > + 26.0 p.g/liter (n = lo), when measured 0.05). by the homologous assay, but only 15.14 + Whereas IR-GH concentrations (mea- 1.54 kg/liter (n = 10) (2.14 2 0.25-fold), sured by both systems) were unaffected by when measured by the homologous saline administration, they were lowered system. In these birds estimates of the by T, administration (Fig. 1). The decline IR-GH concentrations before and after in the IR-GH level measured by the homolTRH challenge were, nevertheless, posiogous assay (70.0 k 6.8%, P < 0.001) was tively correlated (Y = 0.783, n = 40, P < greater (P < 0.001) than that measured by 0.001). Pretreatment with T, or T, had no the heterologous system (20.9 i 5.9%, P < significant effect on the IR-GH response to 0.05). The injection of T, also suppressed TRH, when measured by the heterologous

94

HARVEY,

L*i* J Pretreatment ‘vehlcieT4 T3

HOSHINO,

LNSdNS J Pretreotmenf "ehcle

T4

T3

FIG. 2. Concentrations of immunoreactive growth hormone (IR-GH) in the plasma of immature domestic fowl, determined by homologous or heterologous radioimmunoassays (RIAs), IO min after the iv administration of thyrotropin releasing hormone (TRH IO p.g/kg solid bars) or its 0.9% NaCl vehicle (I.0 ml/kg open bars). Prior to saline or TRH administration the birds were pretreated. 60 min earlier. with an iv injection of thyroxine (T,. 250 k/kg). triiodothyronine IT,. 250 kg/kg). or 0.9% NaCl (vehicle. I .O ml/kg). Statistically significant (P < 0.05) effects of T, or T, are indicated by the asterisks. while significant (P < 0.01) effects of TRH are indicated by the arrows. Means ? SE (n = IO).

AND SUZUKI

Experiment 3. Sixty minutes after ACTH administration a reduction (P < 0.01) in the plasma IR-GH concentration, relative to that in vehicle-injected controls, was detected by both assay systems (Fig. 3a). The magnitude of the suppression determined by the homologous assay (84.1%), was greater than that determined by the heterologous system (54.9%), although a significant correlation between the plasma IR-GH estimates was observed (v = 0.634, n = 26, P < 0.001). In birds pretreated with the 0.9% vehicle or with the ACTH, the administration of TRH again increased plasma IR-GH concentrations (Fig. 3b). The increase in the vehicle-pretreated controls, determined by the homologous assay (6.84 t 1.26-fold, II = 7, 148.2 ? 14.9 pg/liter) was greater (P < 0.001) than that determined by heterologous assay (1.66 t 0.3-fold, II = 7; 4.38 ? 1.68 kg/liter). Treatment with ACTH impaired (P < 0.001) the GH response (a 66.8 kg/liter increase) to TRH determined by homologous assay (Fig. 3b) but did not reduce the response measured by heterologous assay. Despite these differences, a significant correlation between the assay systems used was observed (1. = 0.597, II = 52. P < O.OOl), when all the estimates of IR-GH before and after TRH challenge were compared. In birds which had been deprived of food for 24 hr prior to the administration of TRH, the plasma IR-GH concentration, measured by both homologous and heterologous assays, was elevated (P < 0.05) in comparison with the fed controls and by similar magnitudes (by 89.5 and 60.6%, respectively) (Table 1). The administration of TRH to the fasted birds was followed by an augmentation of the GH response, as determined by both assay systems (Table 1). Homologous and heterologous IR-GH levels throughout the experiment were therefore correlated (Y = 0.597, n = 29, P

system, but markedly reduced (P < 0.001) the IR-GH response measured by the homologous assay. Estimates of IR-GH 10 min after TRH challenge were, however, correlated (Y = 0.725, II = 60, P < O.OOl), as were the overall IR-GH levels measured throughout the experiment (Y = 0.677, iz = 174, P < 0.001). Experiment 2. Thirty minutes after sodium pentobarbital anesthesia the IR-GH concentration in a group of 3-week-old cockerels (measured by homologous assay), 5.8 + 0.6 kg/liter (n = 16) was less (P < 0.001) than that in conscious controls (34.68 + 3.89 pg/liter, n = 20). Concentrations of IR-GH determined by heterologous assay (17.64 +- 0.98 kg/liter, n = 16) did not differ significantly from those observed in the conscious birds (18.24 + 0.6 pgiliter. n = 20). There was, therefore, no overall correlation between estimates of plasma < 0.001). Experiment IR-GH (v = 0.353, n = 20, P > 0.05).

4. When

48 hr fasted

7-

95

IR-GH IN CHICKENS Vehicle TRH

-*

b

Pretreatment Vehicle ACTti

Pretreatment Vehicle ACTH

FIG. 3. (a) Concentrations of immunoreactive growth hormone (IR-GH) in the plasma of immature domestic fowl. determined by homologous or heterologous radioimmunoassays (RlAs) I20 min after iv administration of synthetic adrenocorticotropin (ACTH) 25 iuikg. or the 0.9’7c NaCl vehicle. Means 2 SE (n = 14). Statistically significant (P < 0.05) differences are indicated by the asterisks. (b) Concentrations of immunoreactive growth hormone (IR-GH) in the plasma of immature domestic fowl, determined by homologus and heterologous radioimmunoassays (RIAsl IO min after the iv administration of thyrotropin releasing hormone (TRH. IO (*g/kg; solid bars) or its vehicle (0.9% NaCI, 1.O ml/kg; open bars). Prior to TRH or saline administration the birds were pretreated. I20 min earlier. with synthetic adrenocorticotropin (ACTH, 25 iuikgl or with its 0.9% NaCl vehicle. Statistically significant (P < 0.05) differences between vehicle and ACTH groups are indicated by the asterisks, while significant (P < 0.05) IR-GH repsonse to TRH are indicated by the arrows. Means ? SE (11 = 7).

week-old cockerels were allowed to refeed ad libitum, a significant (P < 0.05) fall in the IR-GH concentration, measured by both homologous and heterologous assays, was observed within 2 hr (Fig. 4). The IR-GH concentration, determined by homologous assay, progressively fell thereafter. Heterologous radioimmunoassay of the same plasma samples also showed that IR-GH levels in the birds refed for 4 and 8 hr were lower than those in the food-deprived controls, although no significant difference

was observed after 6 hr. Because of this discrepancy the overall correlation between homologous and heterologous estimates of IR-GH concentration during the experiment (Y = 0.212, tz = 66) was not significant . Experiment 5. In adult birds estimates of plasma IR-GH concentrations by both homologous and heterologous systems, were lower than those in immature birds (Table 2). Although homologous radioimmunoassay measurements of plasma IR-GH in

TABLE

1

PLASMAIMMUNOREACTIVEGROWTHHORMONE(IR-GH)CONCENTRATIONSIN ~-WEEK-OLD CHICKS

Plasma IR-GH (pgiliter) Basal Treatment Fed ad libirum Fasted 24 hr

Homologous

Stimulatedb

RIA

Heterologous RIA

Homologous RIA

Heterologous

30.6 t 5.4 (7)” 58.0 t 4.1 (8)c

10.04 2 1.88 (7) 16.12 2 1.38 (8p

178.8 t 14.4 (7) 370.4 2 36.0 (8)c

15.52 ? 1.08 (7) 23.52 t 2.68 (8)’

a Means 2 SEM (n). b Plasma IR-GH concentration IO min after iv administration c Significantly different from corresponding fed controls.

of thyrotropin releasing hormone (IO &kg).

RIA

HARVEY,

96

HOSHINO,

AND SUZUKI

mates IR-GH pared, = 444,

of homologous and heterologous from all the experiments were coma positive correlation (u = 0.513, n P < 0.001) was also evident. DISCUSSION

More than a decade ago it was demonstrated that purified chicken GH competitively inhibited the binding of labelled rat 0 2 4 6 8 hours 0 2 4 6 Bhaurs GH to antiserum raised against rat GH FIG. 4. Concentrations of immunoreactive growth (Farmer ef al., 1974). This observation prohormone (IR-GH) in the plasma of immature domestic moted the use of a rat GH radioimmufowl. determined by homologous and heterologous ra- noassay for the measurement of plasma dioimmunoassays (RIAs), at intervals after a 4%hr peIR-GH in chickens (Hoshino et al., 1980). riod of starvation in controls (0) and birds allowed to The results of the present studies indicate refeed ad lihifunl (0). Means 2 SE (n = 7). The asterisks indicated significant (P < 0.05) reductions in that under a variety of physiological condithe plasma IR-GH level. tions the circulating IR-GH measured by the heterologous radioimmunoassay reby homologous immature birds were consistently higher (P sembles that determined assay. < 0.01) than those made by heterologous It is well established that TRH is a potent radioimmunoassay, estimates of IR-GH GH secretagogue in chickens (e.g., Harvey concentrations in the plasma of adult birds et al., 1978a; Burke, 1983; Leung et al., did not differ significantly. Variability in the 1984b) and the heterologous GH radioimbasal IR-GH concentrations determined by munoassay clearly detects an increase in the homologous assay was greater than that the circulating IR-GH level following TRH determined by heterologous assay. While no correlation between homologous and challenge, as briefly reported (Hoshino et heterologous estimates of IR-GH was ob- al., 1984). The deprivation of food also eleserved in the plasma of 3-week and 7- vated the circulating IR-GH concentration week-old cockerels (Table 2). when the measured by both assay systems (Table I, basal IR-GH levels in all ages were com- Fig. 4), in agreement with earlier reports (Harvey et al., 1978b; Hoshino et al., 1980) pared, a significant (P < 0.001) correlation was observed. Moreover. when all the esti- and other studies using a different homoloTABLE 2 PLASMAIMMUNOREACTIVEGROWTHHORMONE(IR-GH) CONCENTRATIONSIN~MMATUREAND MATURE COCKERELS Plasma IR-GH &liter Age (weeks) 3

5 76 >24 3->24

Homologous RIA 36.6 32.0 30.6 5.3 32.32

k + + k k

Heterologous RIA

3.0 (60)" 3.3 (14) 3.0 (7)

19.46 II.18 17.82

0.9 (8) 2.26 (89)

7.70 16.64

Correlation (v)

2 0.53 (58) t 1.11(14) 2 0.94(7) 2 lSO(8) " 0.60 (87)

D Means 5 SEM (n). b Plasma IR-GH level in 48 hr fasted birds following 8 hr crd libiturn c NS, Not statistically significant.

0.182

0.500 0.382 0.715 0.375

refeeding.

Significance

NS' P < 0.05 NS P < 0.05 P < 0.001

97

IR-GH IN CHICKENS

gous assay (Leung and Taylor, 1983). The results of the present study additionally indicate that fasting induced GH secretion in chickens is suppressed following refeeding. The elevated plasma concentrations in fasted birds may partly result from an increase in sensitivity to provocative stimuli, since both assays indicated that the IR-GH response to exogenous TRH was augmented during fasting (Table 1). Proudman and Opel (1981) also found that the GH response of immature turkeys was enhanced during dietary deprivation. In addition to pharmacological (TRH) and stressful (fasting) elevations in GH secretion, high basal levels of plasma IR-GH were found in young birds (Table 2). This finding confirms our previous, independent observations (Harvey et al., 1979; Hoshino et al., 1982) and those of subsequent investigators (e.g., Burke and Marks, 1982; Leung et al., 1984b) and suggests that the heterologous and homologous assays are measuring similar (if not identical) circulating GH moieties in young birds or that an age-related change in the concentrations of all plasma GH moieties occurs during growth. The observed decline in the IR-GH concentration following ACTH challenge (Fig. 3) measured by both assays, similarly suggests that both assays can measure the same GH moieties or that ACTH has similar effects or a number of GH moieties. The inhibitory effect of ACTH on GH secretion has previously only been observed using the homologous system (Davidson et al., 1979). In certain physiological and pharmacological conditions the homologous and heterologous GH radioimmunoassays do, therefore, give qualitatively similar results. It is, however, clear that changes in the plasma IR-GH concentration detected by the homologous radioimmunoassay are not necessarily measured by the heterologous assay. The magnitude of the changes in IR-GH level determined by the homologous system (both increases and decreases) is far greater than that observed using

heterologous assay, probably because the cross-reaction of chicken GH with rat GH antiserum is not completely parallel (Farmer et al., 1974). However, as the heterologous radioimmunoassay failed to detect the inhibitory effect of anesthesia on basal GH concentrations (Harvey and Scanes, 1977b), as it failed to show significant thyroidal inhibition of basal and stimulated GH secretion (Harvey. 1983; Leung et al., 1985) and did not detect an inhibitory effect on ACTH on TRH-induced GH release (Fig. 3). it is possible that some of the GH moieties in chicken plasma have poor cross-reactivity with rat GH antiserum. Growth hormone heterogeniety in mammalian species is well documented (e.g., Bauman, 1984; Lewis et al., 1984) and the different GH moieties have been shown to differ in their immunoreactivity. It is therefore possible that the differences between the IR-GH measured by the homologous and heterologous radioimmunoassays are due to the molecular heterogeniety of plasma GH and to differences in antisera specificity. REFERENCES

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