0013-7227/91/1284-1960$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 4 Printed in U.S.A.
Effect of Adrenocorticotropin Administration on the Biosynthesis of Corticosteroid-Binding Globulin in Fetal Sheep* ROSS A. JACOBS, VICTOR K. M. HAN, GEOFFREY L. HAMMOND, AND JOHN R. G. CHALLIS Lawson Research Institute, St. Joseph's Health Centre (R.A.J., V.K.M.H., J.R.G.C.), the London Regional Cancer Centre, Victoria Hospital (G.L.H.), the Medical Research Council Group in Fetal and Neonatal Health and Development (V.K.M.H., G.L.H., J.R.G.C.) and the Departments of Obstetrics and Gynecology (R.A.J., G.L.H., J.R.G.C), Physiology (J.R.G.C), Pediatrics (V.K.M.H.), and Biochemistry (V.K.M.H., G.L.H.), University of Western Ontario, London, Ontario, Canada
ABSTRACT. Parturition in sheep is initiated by the fetus through activation of the fetal hypothalamic-pituitary-adrenal axis and is associated with increased concentrations of ACTH, cortisol, and corticosteroid-binding globulin (CBG) in the fetal circulation during the final 10-15 days of pregnancy. Premature parturition and a precocious elevation in fetal plasma CBG are produced by intrafetal ACTH administration, but the possible sources of CBG in the ovine fetus are not known. To determine these sites, CBG mRNA was measured in tissues from fetal sheep in late pregnancy and after intrafetal ACTH treatment, using a sheep CBG cDNA. Fetal ACTH treatment caused a significant increase in the fetal plasma corticosteroid-binding capacity (CBC), although there was no significant difference in CBC between umbilical arterial and umbilical venous plasma. After ACTH treatment, CBC was elevated in fetal liver and
D
URING late pregnancy in the sheep there is maturation of fetal pituitary-adrenal function (1) and a corresponding rise in the concentration of cortisol in the fetal circulation (2). Fetal cortisol provides a signal for the onset of parturition (3) and helps promote maturation of several fetal organ systems (4). The rise in fetal cortisol is associated with an increase in the corticosteroid-binding capacity (CBC) of fetal plasma (5), such that at term the concentration of corticosteroid-binding globulin (CBG) in the fetal circulation exceeds that in the mother (6, 7). The rise in fetal plasma CBC is also stimulated by ACTH administration to fetal sheep, and we have provided evidence that this effect might be mediated by cortisol (8). Others have shown that the normal prepartum increase in CBG was dependent on Received October 22, 1990. Address all correspondence and requests for reprints to: Dr. J. R. G. Challis, Lawson Research Institute, St. Joseph's Health Centre, 268 Grosvenor Street, London, Ontario, Canada N6A 4V2. * This work was supported by the Canadian Medical Research Council (Group Grant in Fetal and Neonatal Health and Development).
kidney. Cortisol binding in these tissues had characteristics similar to those of cortisol binding in fetal sheep plasma. By Northern blot analysis a single mRNA (1.7 kilobases) for CBG was detected in fetal liver, kidney, lung, and adrenal, but not in placenta. The abundance of CBG mRNA in the fetal liver was greater than that in other tissues, but was unchanged by ACTH treatment. The level of CBG mRNA in the fetal kidney, but not in other tissues, increased 3-fold after ACTH. We conclude that the elevation in plasma CBC after intrafetal ACTH, and presumably also at term pregnancy, does not reflect production of CBC by the placenta or transfer from the mother. Rather, it results from production primarily in the fetal liver and kidney, although only in the latter tissue is CBG mRNA accumulation increased by intrafetal ACTH treatment. (Endocrinology 128:1960-1966, 1991)
the presence of the fetal pituitary, but could not be provoked by estrogen treatment (5) and was not related to thyroid status of the fetus (9). In many mammalian species the liver is the major site of CBG synthesis in the adult (10, 11), but the source of plasma CBG in the sheep fetus is not known. Therefore, we have measured the CBC in different tissues of fetal sheep during late pregnancy and after intrafetal administration of ACTH. In addition, we used an ovine CBG cDNA to both identify sites of CBG biosynthesis in the fetal lamb and to examine the effect of ACTH administration on the abundance of CBG mRNA in different fetal tissues.
Materials and Methods Animals Acute studies. Tissues were collected from fetuses of four sheep during late pregnancy (>140 days; term = 145 days). The animals were anesthetized with sodium pentobarbitone (Abbott Laboratories, Montreal, Quebec, Canada). General anesthesia
1960
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CBG IN FETAL SHEEP was maintained by using a 50:50 nitrous oxide-oxygen mixture and 2-3% halothane with a flow rate of 2-3 liters/min. The fetuses were exposed, and blood samples (3-10 ml) were obtained by needle puncture into chilled heparinized tubes from an umbilical artery and an umbilical vein. Amniotic and allantoic fluid samples were also collected by needle puncture. The mother and fetuses were then killed with an overdose of pentobarbitone. Placentomes, fetal kidney, liver, and adrenals were dissected out rapidly, frozen immediately, and stored at —70 C. Chronic studies. Eleven pregnant sheep of mixed breed with known insemination dates and carrying twins were used. Between days 115-118 gestation and under general anaesthesia, both fetuses in each sheep were implanted surgically with polyvinyl catheters, inserted 8-10 cm into a femoral artery and femoral vein. The maternal femoral vessels were catheterized in a similar manner. Details of the surgical procedure have been described previously (12). After a recovery period of at least 7 days, ACTH-(l-24) (Cortrosyn, Organon Pharmaceuticals, Toronto, Ontario, Canada) was infused at 1 ng for 15 min every 2 h (pulsatile ACTH; P-ACTH) into the femoral vein of one of the fetuses (13). In the first series of experiments, P-ACTH infusion into one of the twins was continued until the animals were in active labor, as judged by the uterine electromyographic record, measured continuously from stainless steel electrodes sutured into the myometrium of each uterine horn (13). In these animals, the mean time (±SEM) from the start of P-ACTH to the onset of labor was 204.0 ± 29.5 h (n = 6). With the animal in active labor, the ewes were anaesthetized with sodium pentobarbitone, as described above. Samples of umbilical artery and umbilical venous blood were collected by venipuncture. The mother and fetuses were then killed with an overdose of pentobarbitone, and fetal liver and kidney were collected for CBC determination. In the second series of experiments (n = 5 twin pregnancies), P-ACTH administration continued as in the first experimental series for 96 h into one of each twin pair, while the remaining twin received similar pulses of saline for 96 h. Beginning 24 h before the start of P-ACTH or saline treatment, blood samples were collected from the fetal (2.5 ml) and maternal (5.0 ml) femoral arteries at 0800 and 2000 h. Sampling was continued at these times just before the next ACTH or saline pulse for the remaining 96 h of the study. Blood samples were collected into chilled plastic tubes containing heparin and immediately centrifuged at 1500 X g for 10 min at 4 C, and plasma was stored at -20 C until analysis. At 96 h the animals were killed with an overdose of pentobarbitone. Fetal liver, kidney, lung, adrenal, and whole placentomes were collected from three saline-treated and three P-ACTHtreated fetuses and rapidly frozen for later determination of CBC or CBG mRNA levels. Analysis Steroids. Nonradioactive steroids were obtained from Sigma Chemical Co. (St. Louis, MO). [6,7-3-H]Cortisol (SA, 80-100 Ci/mmol) was purchased from New England Nuclear (Boston, MA) and purified by TLC in the solvent system chloroform-ethanol (94:6) before use.
1961
Cortisol. The concentration of cortisol was measured by RIA after extraction of the samples with diethyl ether. The antibody characteristics and assay validation for fetal sheep plasma have been described previously (12). The intra- and interassay coefficients of variation were 9-13%. CBC. The CBCs of fetal and maternal plasma were determined by the procedure of Ballard et al. (5) with modifications (8). Briefly, duplicate aliquots (50 /x\) of plasma were added to glass testtubes containing 10,000 cpm [3H]cortisol and 32 ng nonradioactive cortisol, previously added in ethanol and dried under nitrogen. The tubes were agitated briefly on a vortex mixer, incubated at 39 C for 30 min, then at 4 C for 12 h. Bound and free cortisol were separated using dextran-coated charcoal (5, 8). For each sample nonspecific binding of [3H]cortisol was determined in the presence of 1 fig nonradioactive cortisol. Values for nonspecific binding were 1.0-1.5% of the total radioactivity added. The CBC was calculated from the fraction of bound cortisol, minus nonspecific binding, multiplied by the total concentration of cortisol (endogenous plus unlabeled plus [3H]cortisol) in 50-/Ltl plasma samples. The assay does not measure cortisol binding to albumin, since this complex dissociates rapidly after the addition of charcoal, and the unbound cortisol is absorbed and precipitated with the charcoal upon centrifugation. The CBC was measured in liver and kidney extracts from PACTH- and saline-infused fetuses. Tissue was homogenized in Tris-HCl (0.1 M; 5 g tissue/10 ml buffer) using three 10-sec bursts with a Polytron homogenizer (Brinkmann Instruments, Rexdale, Canada). The homogenates were centrifuged at 1000 x g for 15 min at 4 C, lyophilized, and reconstituted in distilled water, and CBC was determined in 50-^1 aliquots of reconstituted material, as described above. The specificity of cortisol binding in tissues and plasma was determined by the ability of graded amounts of different nonradioactive steroids to compete with [3H]cortisol. The results are expressed as the percent cross-reactivity, calculated from the mass of cortisol required to produce 50% binding of [3H] cortisol divided by the mass of potential competing steroid that displaced to 50% binding of [3H]cortisol. Sephadex gel filtration. Aliquots (50 /zl) of plasma or reconstituted homogenate were added to tubes containing [3H] cortisol (~30,000cpm), [3H]cortisol plus 32 ng cortisol, [3H]cortisol plus 32 ng dexamethasone, [3H]dexamethasone (SA, 49 Ci/mmol; New England Nuclear, Boston, MA), [3H]dexamethasone plus 32 ng dexamethasone. [3H]Cortisol was also incubated with 50 Ml 1% BSA (Sigma Chemical Co., St. Louis, MO). The contents were mixed and incubated for 30 min at 39 C, then for 12 h at 4 C. Samples were applied to Sephadex G-100 columns, swollen in Tris-HCl (0.1 M; pH 7.4) contained in 10-ml pipettes. The bed volume of the columns was 5 ml. Gel filtration was performed at 4 C, and successive 1-ml fractions were collected during elution with 0.1 M Tris-HCl, pH 7.4. Aliquots of each fraction were taken for the measurement of radioactivity on a /^-scintillation counter (Tricarb, TM Analytica, Elk Grove Village, IL) or for protein determination either from the spectrophotometric optical density reading at 280 nm or quantitatively, using BSA as standard (14).
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CBG IN FETAL SHEEP
1962
Ovine CBG cDNA cloning. A sheep cDNA for CBG was obtained by screening an ovine liver cDNA library (Clontech Laboratories, Inc., Palo Alto, CA) with a rat CBG cDNA (11) at reduced stringency (40% formamide; 37 C). Positive plaques were cloned, recombinant phage were purified (15), and their cDNA inserts were subcloned in pBluescript vectors (Stratagene Cloning Systems, La Jolla, CA). Single stranded templates were obtained and partially sequenced using the dideoxy chain termination method (16). The nucleotide sequence obtained from the 5' end of the ovine cDNA displayed sequence identity with the rat CBG cDNA (11). RNA extraction and Northern blot analysis. RNA was extracted using guanidine thiocyanate and cesium chloride (17). Poly(A)+ RNA was purified using oligo(dT)-cellulose (Collaborative Research, Bedford, MA) chromatography, followed by precipitation with ethanol, and an aliquot (20 ng) was denatured by heating to 55 C for 15 min in the presence of 1.3 x 3-[iVmorpholinolpropranesulfonic acid, 8.9% formaldehyde, and 69% formamide. Samples were then electrophoresed through a 1% agarose gel in the presence of formaldehyde (18) and transferred to a nylon membrane (Zetaprobe, Bio-Rad, Mississauga, Ontario, Canada) by capillary blotting (19). The membrane was prehybridized for 24 h at 42 C in 4 x SSPE (20 x SSPE = 174 g NaCl, 27.6 g NaH 2 PO 4 H 2 O, and 7.4 g EDTA in 1 liter water, pH 7.4)-7% sodium doedecyl sulfate (SDS), 50% formamide, and 100 ng/m\ sheared salmon sperm DNA. Hybridization of the membrane was performed for 24 h at 42 C in the same buffer with the addition of 32P-labeled ovine CBG cDNA (106 cpm/ml buffer). Radiolabeling of ovine CBG cDNA was performed by random priming using [32P]dCTP (New England Nuclear, DuPont Canada, Mississauga, Ontario, Canada). After hybridization, the membrane was washed in 2 x SSC (20 x SSC = 175.3 g NaCl and 88.2 g sodium citrate in 1 liter water, pH 7.0)-0.1% SDS for 15 min (three times), then in 1 x SSC-0.1% SDS for 30 min at 42 C, then in 0.1 X SSC0.1% SDS for 30 min at 42 C. Autoradiography was carried out using Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY) at -70 C. Subsequently, the DNA was removed from the filter by washing in 0.1 X SSC-0.1% SDS at 95 C. As an internal control for the amount of RNA loaded in each well and of transfer efficiency, the filter was reprobed with radiolabeled cDNA encoding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; American type culture collection, Rockville, MD) (20) to determine the relative amount of RNA bound to the filter. Hybridization and washing conditions were as described above. A densitometer (Ultrascan XL, Pharmacia LKB Canada, Inc., Montreal, Canada) was used to estimate the relative intensity of autoradiographic signals. Data analysis In the second series of intrafetal ACTH infusions, average values for plasma CBC were calculated for each animal from the two samples collected on any 1 day. The means of these values from all five animals are presented. When appropriate, results were examined by analysis of variance. Differences in CBC in paired samples from P-ACTH- and saline-treated twin fetuses were examined using a paired t test. Values represent the mean ± SEM unless otherwise stated. Statistical significance was set at P < 0.05. The relative abundance of CBG mRNA
Endo«1991 Voll28«No4
was obtained by dividing the densitometric readings for the autoradiographic signals obtained for CBG by that for GAPDH.
Results CBC in tissues of late pregnant sheep The Sephadex G-100 elution profiles of [3H]cortisol or [ H]dexamethasone binding to homogenates of liver and kidney and to umbilical arterial plasma from a fetal sheep in late pregnancy are shown in Fig. 1. In fetal plasma incubated with [3H]cortisol, a radioactivity peak eluted with the plasma protein peak, and it was displaced after the addition of nonradioactive cortisol, but not dexamethasone. Radioactivity eluted in the position of free steroid when [3H]dexamethasone was incubated with plasma, and there was no [3H]dexamethasone associated with the protein elution peak. Similarly, when fetal liver and kidney preparations were incubated with [3H]cortisol, radioactivity coeluted from the Sephadex G-100 column with the main protein peak. This peak of radioactivity was reduced in the presence of nonradioactive cortisol, but not by dexamethasone. There was no radioactivity eluted with the protein peak after incubation with [3H]dexamethasone or when [3H]cortisol was incubated with albumin (data not shown). Similar elution profiles were obtained with tissues and fetal plasma from three other animals. The specificity characteristics of [3H]cortisol binding to fetal kidney and liver preparations and to samples of umbilical arterial and venous plasma are shown in Table 1. In general, the specificity characteristics were similar. [3H]Cortisol binding was displaced by corticosterone and by 11-desoxycortisol in fetal plasma and in liver and kidney. There was no detectable reduction of [3H]cortisol binding in the liver or kidney preparations by dexamethasone, estrone, or estradiol. We were unable to detect specific [3H]cortisol binding in samples of amniotic or allantoic fluid after overnight incubation at 4 C, either directly after charcoal separation or after gel filtration on Sephadex G-100. 3
Effects of P-ACTH treatment on plasma and tissue CBC P-ACTH was infused into one fetus of twins in order to provoke premature labor (13). In animals in active labor there was a significant increase in the CBC of umbilical arterial plasma in the P-ACTH-treated twins compared to that in the saline-infused twins {P < 0.02; Table 2), but there was no significant umbilical arteriovenous difference. There was also a significant increase in the CBC in the liver and kidney of the P-ACTHtreated fetuses (Table 2). To examine the time course of this response and to identify sites of CBG gene expression, P-ACTH was infused for 96 h into one fetus of twin pregnancies in a second series of experiments. The mean fetal CBC in
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CBG IN FETAL SHEEP
1963
Fetal Plasma
Liver
Kidney
2.0
O
FIG. 1. Sephadex G-100 elution profiles after incubation of [3H]cortisol or [3H] dexamethasone with fetal sheep plasma and homogenates of fetal liver and kidney {top panel). Cold cortisol displaces the elution of radioactivity from a protein-bound to a free steroid position (second panel), but there is no displacement after incubation with dexamethasone (third panel), nor binding of [3H] dexamethasone alone (bottom panel).
e
6
a 10
8
10
6
B
10
0.0
Fraction No. TABLE 1. Specificity of CBC in fetal tissues and plasma
Cortisol 11-Desoxycortisol Corticosterone Progesterone Testosterone Dexamethasone Estrone Estradiol
Umbilical
Umbilical artery
Kidney
Liver
100 33 ±16 29 ± 4 2.9 ± 0.4 1.9 ± 0.6 NS NS NS
100 56 ± 31 26 ± 9 2.6 ± 1.0 1.5 ± 0.3 NS NS NS
100 32 ± 23 24 ±17 0.6 ± 0.2 0.6 ± 0.2 NS NS NS
100 34 ± 23 6±3 1.9 ± 1.4 4.3 ± 3.6 NS NS NS
Values represent the mean ± SEM; n = 4 replicates. NS, No significant cross-reactivity, TABLE 2. CBC in fetal plasma, liver, and kidney after P-ACTH administration to one of twin fetal sheep CBC (ng/mg protein) P-ACTH-treated twin Saline-treated twin
Umbilical artery
Umbilical vein
Fetal liver
Fetal kidney
0.837 ± 0.175 (4)° 0.349 ± 0.096 (6)
0.738 ± 0.219 (5) 0.453 ± 0.122 (6)
0.182 ± 0.036 (5)* 0.023 ± 0.007 (6)
0.159 ± 0.038 (6)c 0.079 ± 0.028 (6)
Values are the mean ± SEM; the number of fetuses is in parentheses. " P < 0.02 us. saline-treated twin (by t test). b P < 0.001 us. saline-treated twin (by t test). c P < 0.05 us. saline-treated twin (by t test).
twins was not significantly different before treatment, and the fetal CBC was similar to that in the maternal plasma (Table 3). Within 24 h of P-ACTH administration, plasma CBC in the treated fetus rose significantly above that in the saline-treated fetus; after 96 h of PACTH treatment, the fetal CBC was 53.9 ± 4.9 ng/ml (n = 5), compared to 18.8 ± 2.4 ng/ml in the salinetreated fetuses (P < 0.001). The CBC of maternal plasma
was similar to that in the saline-treated fetuses and at 96 h was significantly lower than that in fetuses treated with P-ACTH (Table 3). Measurement of CBG mRNA Northern blot analysis of tissues from three sets of twin fetuses indicated the presence of a single mRNA [1.7 kilobases (kb)] for CBG in the liver, kidney, lung,
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CBG IN FETAL SHEEP
1964
TABLE 3. CBC (nanograms per ml) in plasma from ewes and twin fetuses infused with either ACTH-(l-24) or saline for 96 h Time (h)
P-ACTH-treated twin
Saline-treated twin
Pre 24 48 72 96
20.3 ± 2.7 41.0 ± 10.7° 42.4 ± 3.4° 39.9 ± 8.4* 53.9 ± 4.9C
17.5 ± 2.5 13.5 ± 2.7 18.8 ± 3.5 21.1 ±4.0 18.8 ± 2.4
Endo • 1991 Voll28«No4
istration increased the amount of CBG mRNA by 3-fold in kidneys from paired fetuses (Figs. 2-4).
Maternal plasma 18.9 ± 25.8 ± 37.9 ± 23.5 ± 24.5 ±
Discussion
5.1 5.8 14.2 7.8 5.1
We have shown previously that ACTH administration to singleton sheep fetuses in late pregnancy causes an increase in the CBC of fetal sheep plasma (8). This observation has been extended in the present study to
Values are the mean ± SEM; n = 5 for each group. P < 0.01 us. saline-treated fetuses. * P < 0.05 vs. saline-treated fetuses. c P < 0.001 us. saline-treated fetuses. 0
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O
Vol. 128, No. 4 Printed in U.S.A.
Effect of Adrenocorticotropin Administration on the Biosynthesis of Corticosteroid-Binding Globulin in Fetal Sheep* ROSS A. JACOBS, VICTOR K. M. HAN, GEOFFREY L. HAMMOND, AND JOHN R. G. CHALLIS Lawson Research Institute, St. Joseph's Health Centre (R.A.J., V.K.M.H., J.R.G.C.), the London Regional Cancer Centre, Victoria Hospital (G.L.H.), the Medical Research Council Group in Fetal and Neonatal Health and Development (V.K.M.H., G.L.H., J.R.G.C.) and the Departments of Obstetrics and Gynecology (R.A.J., G.L.H., J.R.G.C), Physiology (J.R.G.C), Pediatrics (V.K.M.H.), and Biochemistry (V.K.M.H., G.L.H.), University of Western Ontario, London, Ontario, Canada
ABSTRACT. Parturition in sheep is initiated by the fetus through activation of the fetal hypothalamic-pituitary-adrenal axis and is associated with increased concentrations of ACTH, cortisol, and corticosteroid-binding globulin (CBG) in the fetal circulation during the final 10-15 days of pregnancy. Premature parturition and a precocious elevation in fetal plasma CBG are produced by intrafetal ACTH administration, but the possible sources of CBG in the ovine fetus are not known. To determine these sites, CBG mRNA was measured in tissues from fetal sheep in late pregnancy and after intrafetal ACTH treatment, using a sheep CBG cDNA. Fetal ACTH treatment caused a significant increase in the fetal plasma corticosteroid-binding capacity (CBC), although there was no significant difference in CBC between umbilical arterial and umbilical venous plasma. After ACTH treatment, CBC was elevated in fetal liver and
D
URING late pregnancy in the sheep there is maturation of fetal pituitary-adrenal function (1) and a corresponding rise in the concentration of cortisol in the fetal circulation (2). Fetal cortisol provides a signal for the onset of parturition (3) and helps promote maturation of several fetal organ systems (4). The rise in fetal cortisol is associated with an increase in the corticosteroid-binding capacity (CBC) of fetal plasma (5), such that at term the concentration of corticosteroid-binding globulin (CBG) in the fetal circulation exceeds that in the mother (6, 7). The rise in fetal plasma CBC is also stimulated by ACTH administration to fetal sheep, and we have provided evidence that this effect might be mediated by cortisol (8). Others have shown that the normal prepartum increase in CBG was dependent on Received October 22, 1990. Address all correspondence and requests for reprints to: Dr. J. R. G. Challis, Lawson Research Institute, St. Joseph's Health Centre, 268 Grosvenor Street, London, Ontario, Canada N6A 4V2. * This work was supported by the Canadian Medical Research Council (Group Grant in Fetal and Neonatal Health and Development).
kidney. Cortisol binding in these tissues had characteristics similar to those of cortisol binding in fetal sheep plasma. By Northern blot analysis a single mRNA (1.7 kilobases) for CBG was detected in fetal liver, kidney, lung, and adrenal, but not in placenta. The abundance of CBG mRNA in the fetal liver was greater than that in other tissues, but was unchanged by ACTH treatment. The level of CBG mRNA in the fetal kidney, but not in other tissues, increased 3-fold after ACTH. We conclude that the elevation in plasma CBC after intrafetal ACTH, and presumably also at term pregnancy, does not reflect production of CBC by the placenta or transfer from the mother. Rather, it results from production primarily in the fetal liver and kidney, although only in the latter tissue is CBG mRNA accumulation increased by intrafetal ACTH treatment. (Endocrinology 128:1960-1966, 1991)
the presence of the fetal pituitary, but could not be provoked by estrogen treatment (5) and was not related to thyroid status of the fetus (9). In many mammalian species the liver is the major site of CBG synthesis in the adult (10, 11), but the source of plasma CBG in the sheep fetus is not known. Therefore, we have measured the CBC in different tissues of fetal sheep during late pregnancy and after intrafetal administration of ACTH. In addition, we used an ovine CBG cDNA to both identify sites of CBG biosynthesis in the fetal lamb and to examine the effect of ACTH administration on the abundance of CBG mRNA in different fetal tissues.
Materials and Methods Animals Acute studies. Tissues were collected from fetuses of four sheep during late pregnancy (>140 days; term = 145 days). The animals were anesthetized with sodium pentobarbitone (Abbott Laboratories, Montreal, Quebec, Canada). General anesthesia
1960
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CBG IN FETAL SHEEP was maintained by using a 50:50 nitrous oxide-oxygen mixture and 2-3% halothane with a flow rate of 2-3 liters/min. The fetuses were exposed, and blood samples (3-10 ml) were obtained by needle puncture into chilled heparinized tubes from an umbilical artery and an umbilical vein. Amniotic and allantoic fluid samples were also collected by needle puncture. The mother and fetuses were then killed with an overdose of pentobarbitone. Placentomes, fetal kidney, liver, and adrenals were dissected out rapidly, frozen immediately, and stored at —70 C. Chronic studies. Eleven pregnant sheep of mixed breed with known insemination dates and carrying twins were used. Between days 115-118 gestation and under general anaesthesia, both fetuses in each sheep were implanted surgically with polyvinyl catheters, inserted 8-10 cm into a femoral artery and femoral vein. The maternal femoral vessels were catheterized in a similar manner. Details of the surgical procedure have been described previously (12). After a recovery period of at least 7 days, ACTH-(l-24) (Cortrosyn, Organon Pharmaceuticals, Toronto, Ontario, Canada) was infused at 1 ng for 15 min every 2 h (pulsatile ACTH; P-ACTH) into the femoral vein of one of the fetuses (13). In the first series of experiments, P-ACTH infusion into one of the twins was continued until the animals were in active labor, as judged by the uterine electromyographic record, measured continuously from stainless steel electrodes sutured into the myometrium of each uterine horn (13). In these animals, the mean time (±SEM) from the start of P-ACTH to the onset of labor was 204.0 ± 29.5 h (n = 6). With the animal in active labor, the ewes were anaesthetized with sodium pentobarbitone, as described above. Samples of umbilical artery and umbilical venous blood were collected by venipuncture. The mother and fetuses were then killed with an overdose of pentobarbitone, and fetal liver and kidney were collected for CBC determination. In the second series of experiments (n = 5 twin pregnancies), P-ACTH administration continued as in the first experimental series for 96 h into one of each twin pair, while the remaining twin received similar pulses of saline for 96 h. Beginning 24 h before the start of P-ACTH or saline treatment, blood samples were collected from the fetal (2.5 ml) and maternal (5.0 ml) femoral arteries at 0800 and 2000 h. Sampling was continued at these times just before the next ACTH or saline pulse for the remaining 96 h of the study. Blood samples were collected into chilled plastic tubes containing heparin and immediately centrifuged at 1500 X g for 10 min at 4 C, and plasma was stored at -20 C until analysis. At 96 h the animals were killed with an overdose of pentobarbitone. Fetal liver, kidney, lung, adrenal, and whole placentomes were collected from three saline-treated and three P-ACTHtreated fetuses and rapidly frozen for later determination of CBC or CBG mRNA levels. Analysis Steroids. Nonradioactive steroids were obtained from Sigma Chemical Co. (St. Louis, MO). [6,7-3-H]Cortisol (SA, 80-100 Ci/mmol) was purchased from New England Nuclear (Boston, MA) and purified by TLC in the solvent system chloroform-ethanol (94:6) before use.
1961
Cortisol. The concentration of cortisol was measured by RIA after extraction of the samples with diethyl ether. The antibody characteristics and assay validation for fetal sheep plasma have been described previously (12). The intra- and interassay coefficients of variation were 9-13%. CBC. The CBCs of fetal and maternal plasma were determined by the procedure of Ballard et al. (5) with modifications (8). Briefly, duplicate aliquots (50 /x\) of plasma were added to glass testtubes containing 10,000 cpm [3H]cortisol and 32 ng nonradioactive cortisol, previously added in ethanol and dried under nitrogen. The tubes were agitated briefly on a vortex mixer, incubated at 39 C for 30 min, then at 4 C for 12 h. Bound and free cortisol were separated using dextran-coated charcoal (5, 8). For each sample nonspecific binding of [3H]cortisol was determined in the presence of 1 fig nonradioactive cortisol. Values for nonspecific binding were 1.0-1.5% of the total radioactivity added. The CBC was calculated from the fraction of bound cortisol, minus nonspecific binding, multiplied by the total concentration of cortisol (endogenous plus unlabeled plus [3H]cortisol) in 50-/Ltl plasma samples. The assay does not measure cortisol binding to albumin, since this complex dissociates rapidly after the addition of charcoal, and the unbound cortisol is absorbed and precipitated with the charcoal upon centrifugation. The CBC was measured in liver and kidney extracts from PACTH- and saline-infused fetuses. Tissue was homogenized in Tris-HCl (0.1 M; 5 g tissue/10 ml buffer) using three 10-sec bursts with a Polytron homogenizer (Brinkmann Instruments, Rexdale, Canada). The homogenates were centrifuged at 1000 x g for 15 min at 4 C, lyophilized, and reconstituted in distilled water, and CBC was determined in 50-^1 aliquots of reconstituted material, as described above. The specificity of cortisol binding in tissues and plasma was determined by the ability of graded amounts of different nonradioactive steroids to compete with [3H]cortisol. The results are expressed as the percent cross-reactivity, calculated from the mass of cortisol required to produce 50% binding of [3H] cortisol divided by the mass of potential competing steroid that displaced to 50% binding of [3H]cortisol. Sephadex gel filtration. Aliquots (50 /zl) of plasma or reconstituted homogenate were added to tubes containing [3H] cortisol (~30,000cpm), [3H]cortisol plus 32 ng cortisol, [3H]cortisol plus 32 ng dexamethasone, [3H]dexamethasone (SA, 49 Ci/mmol; New England Nuclear, Boston, MA), [3H]dexamethasone plus 32 ng dexamethasone. [3H]Cortisol was also incubated with 50 Ml 1% BSA (Sigma Chemical Co., St. Louis, MO). The contents were mixed and incubated for 30 min at 39 C, then for 12 h at 4 C. Samples were applied to Sephadex G-100 columns, swollen in Tris-HCl (0.1 M; pH 7.4) contained in 10-ml pipettes. The bed volume of the columns was 5 ml. Gel filtration was performed at 4 C, and successive 1-ml fractions were collected during elution with 0.1 M Tris-HCl, pH 7.4. Aliquots of each fraction were taken for the measurement of radioactivity on a /^-scintillation counter (Tricarb, TM Analytica, Elk Grove Village, IL) or for protein determination either from the spectrophotometric optical density reading at 280 nm or quantitatively, using BSA as standard (14).
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CBG IN FETAL SHEEP
1962
Ovine CBG cDNA cloning. A sheep cDNA for CBG was obtained by screening an ovine liver cDNA library (Clontech Laboratories, Inc., Palo Alto, CA) with a rat CBG cDNA (11) at reduced stringency (40% formamide; 37 C). Positive plaques were cloned, recombinant phage were purified (15), and their cDNA inserts were subcloned in pBluescript vectors (Stratagene Cloning Systems, La Jolla, CA). Single stranded templates were obtained and partially sequenced using the dideoxy chain termination method (16). The nucleotide sequence obtained from the 5' end of the ovine cDNA displayed sequence identity with the rat CBG cDNA (11). RNA extraction and Northern blot analysis. RNA was extracted using guanidine thiocyanate and cesium chloride (17). Poly(A)+ RNA was purified using oligo(dT)-cellulose (Collaborative Research, Bedford, MA) chromatography, followed by precipitation with ethanol, and an aliquot (20 ng) was denatured by heating to 55 C for 15 min in the presence of 1.3 x 3-[iVmorpholinolpropranesulfonic acid, 8.9% formaldehyde, and 69% formamide. Samples were then electrophoresed through a 1% agarose gel in the presence of formaldehyde (18) and transferred to a nylon membrane (Zetaprobe, Bio-Rad, Mississauga, Ontario, Canada) by capillary blotting (19). The membrane was prehybridized for 24 h at 42 C in 4 x SSPE (20 x SSPE = 174 g NaCl, 27.6 g NaH 2 PO 4 H 2 O, and 7.4 g EDTA in 1 liter water, pH 7.4)-7% sodium doedecyl sulfate (SDS), 50% formamide, and 100 ng/m\ sheared salmon sperm DNA. Hybridization of the membrane was performed for 24 h at 42 C in the same buffer with the addition of 32P-labeled ovine CBG cDNA (106 cpm/ml buffer). Radiolabeling of ovine CBG cDNA was performed by random priming using [32P]dCTP (New England Nuclear, DuPont Canada, Mississauga, Ontario, Canada). After hybridization, the membrane was washed in 2 x SSC (20 x SSC = 175.3 g NaCl and 88.2 g sodium citrate in 1 liter water, pH 7.0)-0.1% SDS for 15 min (three times), then in 1 x SSC-0.1% SDS for 30 min at 42 C, then in 0.1 X SSC0.1% SDS for 30 min at 42 C. Autoradiography was carried out using Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY) at -70 C. Subsequently, the DNA was removed from the filter by washing in 0.1 X SSC-0.1% SDS at 95 C. As an internal control for the amount of RNA loaded in each well and of transfer efficiency, the filter was reprobed with radiolabeled cDNA encoding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH; American type culture collection, Rockville, MD) (20) to determine the relative amount of RNA bound to the filter. Hybridization and washing conditions were as described above. A densitometer (Ultrascan XL, Pharmacia LKB Canada, Inc., Montreal, Canada) was used to estimate the relative intensity of autoradiographic signals. Data analysis In the second series of intrafetal ACTH infusions, average values for plasma CBC were calculated for each animal from the two samples collected on any 1 day. The means of these values from all five animals are presented. When appropriate, results were examined by analysis of variance. Differences in CBC in paired samples from P-ACTH- and saline-treated twin fetuses were examined using a paired t test. Values represent the mean ± SEM unless otherwise stated. Statistical significance was set at P < 0.05. The relative abundance of CBG mRNA
Endo«1991 Voll28«No4
was obtained by dividing the densitometric readings for the autoradiographic signals obtained for CBG by that for GAPDH.
Results CBC in tissues of late pregnant sheep The Sephadex G-100 elution profiles of [3H]cortisol or [ H]dexamethasone binding to homogenates of liver and kidney and to umbilical arterial plasma from a fetal sheep in late pregnancy are shown in Fig. 1. In fetal plasma incubated with [3H]cortisol, a radioactivity peak eluted with the plasma protein peak, and it was displaced after the addition of nonradioactive cortisol, but not dexamethasone. Radioactivity eluted in the position of free steroid when [3H]dexamethasone was incubated with plasma, and there was no [3H]dexamethasone associated with the protein elution peak. Similarly, when fetal liver and kidney preparations were incubated with [3H]cortisol, radioactivity coeluted from the Sephadex G-100 column with the main protein peak. This peak of radioactivity was reduced in the presence of nonradioactive cortisol, but not by dexamethasone. There was no radioactivity eluted with the protein peak after incubation with [3H]dexamethasone or when [3H]cortisol was incubated with albumin (data not shown). Similar elution profiles were obtained with tissues and fetal plasma from three other animals. The specificity characteristics of [3H]cortisol binding to fetal kidney and liver preparations and to samples of umbilical arterial and venous plasma are shown in Table 1. In general, the specificity characteristics were similar. [3H]Cortisol binding was displaced by corticosterone and by 11-desoxycortisol in fetal plasma and in liver and kidney. There was no detectable reduction of [3H]cortisol binding in the liver or kidney preparations by dexamethasone, estrone, or estradiol. We were unable to detect specific [3H]cortisol binding in samples of amniotic or allantoic fluid after overnight incubation at 4 C, either directly after charcoal separation or after gel filtration on Sephadex G-100. 3
Effects of P-ACTH treatment on plasma and tissue CBC P-ACTH was infused into one fetus of twins in order to provoke premature labor (13). In animals in active labor there was a significant increase in the CBC of umbilical arterial plasma in the P-ACTH-treated twins compared to that in the saline-infused twins {P < 0.02; Table 2), but there was no significant umbilical arteriovenous difference. There was also a significant increase in the CBC in the liver and kidney of the P-ACTHtreated fetuses (Table 2). To examine the time course of this response and to identify sites of CBG gene expression, P-ACTH was infused for 96 h into one fetus of twin pregnancies in a second series of experiments. The mean fetal CBC in
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CBG IN FETAL SHEEP
1963
Fetal Plasma
Liver
Kidney
2.0
O
FIG. 1. Sephadex G-100 elution profiles after incubation of [3H]cortisol or [3H] dexamethasone with fetal sheep plasma and homogenates of fetal liver and kidney {top panel). Cold cortisol displaces the elution of radioactivity from a protein-bound to a free steroid position (second panel), but there is no displacement after incubation with dexamethasone (third panel), nor binding of [3H] dexamethasone alone (bottom panel).
e
6
a 10
8
10
6
B
10
0.0
Fraction No. TABLE 1. Specificity of CBC in fetal tissues and plasma
Cortisol 11-Desoxycortisol Corticosterone Progesterone Testosterone Dexamethasone Estrone Estradiol
Umbilical
Umbilical artery
Kidney
Liver
100 33 ±16 29 ± 4 2.9 ± 0.4 1.9 ± 0.6 NS NS NS
100 56 ± 31 26 ± 9 2.6 ± 1.0 1.5 ± 0.3 NS NS NS
100 32 ± 23 24 ±17 0.6 ± 0.2 0.6 ± 0.2 NS NS NS
100 34 ± 23 6±3 1.9 ± 1.4 4.3 ± 3.6 NS NS NS
Values represent the mean ± SEM; n = 4 replicates. NS, No significant cross-reactivity, TABLE 2. CBC in fetal plasma, liver, and kidney after P-ACTH administration to one of twin fetal sheep CBC (ng/mg protein) P-ACTH-treated twin Saline-treated twin
Umbilical artery
Umbilical vein
Fetal liver
Fetal kidney
0.837 ± 0.175 (4)° 0.349 ± 0.096 (6)
0.738 ± 0.219 (5) 0.453 ± 0.122 (6)
0.182 ± 0.036 (5)* 0.023 ± 0.007 (6)
0.159 ± 0.038 (6)c 0.079 ± 0.028 (6)
Values are the mean ± SEM; the number of fetuses is in parentheses. " P < 0.02 us. saline-treated twin (by t test). b P < 0.001 us. saline-treated twin (by t test). c P < 0.05 us. saline-treated twin (by t test).
twins was not significantly different before treatment, and the fetal CBC was similar to that in the maternal plasma (Table 3). Within 24 h of P-ACTH administration, plasma CBC in the treated fetus rose significantly above that in the saline-treated fetus; after 96 h of PACTH treatment, the fetal CBC was 53.9 ± 4.9 ng/ml (n = 5), compared to 18.8 ± 2.4 ng/ml in the salinetreated fetuses (P < 0.001). The CBC of maternal plasma
was similar to that in the saline-treated fetuses and at 96 h was significantly lower than that in fetuses treated with P-ACTH (Table 3). Measurement of CBG mRNA Northern blot analysis of tissues from three sets of twin fetuses indicated the presence of a single mRNA [1.7 kilobases (kb)] for CBG in the liver, kidney, lung,
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CBG IN FETAL SHEEP
1964
TABLE 3. CBC (nanograms per ml) in plasma from ewes and twin fetuses infused with either ACTH-(l-24) or saline for 96 h Time (h)
P-ACTH-treated twin
Saline-treated twin
Pre 24 48 72 96
20.3 ± 2.7 41.0 ± 10.7° 42.4 ± 3.4° 39.9 ± 8.4* 53.9 ± 4.9C
17.5 ± 2.5 13.5 ± 2.7 18.8 ± 3.5 21.1 ±4.0 18.8 ± 2.4
Endo • 1991 Voll28«No4
istration increased the amount of CBG mRNA by 3-fold in kidneys from paired fetuses (Figs. 2-4).
Maternal plasma 18.9 ± 25.8 ± 37.9 ± 23.5 ± 24.5 ±
Discussion
5.1 5.8 14.2 7.8 5.1
We have shown previously that ACTH administration to singleton sheep fetuses in late pregnancy causes an increase in the CBC of fetal sheep plasma (8). This observation has been extended in the present study to
Values are the mean ± SEM; n = 5 for each group. P < 0.01 us. saline-treated fetuses. * P < 0.05 vs. saline-treated fetuses. c P < 0.001 us. saline-treated fetuses. 0
UJ
O
