0013-7227/97/$03.00/0
Endocrinology
Copyright © 1997 by The Endocrine Society
Vol. 138, No. 12
Printed in U.S.A.
Maternal Growth Hormone Treatment Increases
Placental Diffusion Capacity But Not Fetal or
Placental Growth in Sheep*
J. E. HARDING, P. C. EVANS,
AND
P. D. GLUCKMAN
Research Centre for Developmental Medicine and Biology, Department of Paediatrics, University of
Auckland, Auckland, New Zealand
ABSTRACT
We tested the hypothesis that chronic maternal GH administration
would increase fetal substrate supply, increase maternal and fetal
insulin-like growth factor I (IGF-I) concentrations, and therefore enhance growth in the late gestation fetal sheep. Eleven ewes received
bovine GH 0.1 mg/kg twice daily for 10 days, whereas 10 control ewes
received saline. GH treatment increased placental capacity for simple
diffusion (P , 0.01), with a trend toward an increase in placental
capacity for facilitated diffusion (P 5 0.07). GH treatment also low-
F
ETAL GROWTH in late gestation is limited largely by
maternal substrate supply and mediated by a number
of maternal and fetal hormones. Glucose is the major source
of metabolic substrate for the late gestation fetus (1) and is
supplied from the mother across the placenta. Birth size is
proportional to cord blood insulin-like growth factor 1
(IGF-I) concentrations in a wide range of species (2), and gene
deletion studies have shown clearly that fetal IGF-I is critical
for fetal growth in late gestation (3–5).
Birth size is also related to maternal GH and perhaps IGF-I
concentrations (6 – 8). GH and placental lactogen (PL) both
induce relative insulin resistance in the mother, and thus are
postulated to influence fetal growth by increasing maternal
glucose levels and hence substrate supply available to the
fetus (2). Nevertheless, previous studies of maternal GH
administration have produced conflicting results regarding
effects on fetal growth (9 –15).
We have previously shown in pregnant sheep that shortterm infusion of IGF-I to the mother results in metabolic
changes consistent with an increase in substrate uptake from
mother to fetus (16), whereas IGF-I infusion to the fetus
results in fetal anabolic changes (17). We have therefore
speculated that prolonged elevation of IGF-I concentrations
on both sides of the placenta would be expected to lead to
enhanced fetal growth (16).
We would therefore expect that conditions that both increased fetal glucose supply and increased maternal and fetal
IGF-I concentrations would be most likely to enhance fetal
Received June 24, 1997.
Address all correspondence and requests for reprints to: Prof. J. E.
Harding, Research Centre for Developmental Medicine and Biology,
Department of Paediatrics, University of Auckland, Private Bag 92019,
Auckland, New Zealand. E-mail: [email protected].
* This work was supported by the Auckland Medical Research Foundation and the Health Research Council of New Zealand.
ered maternal and fetal blood urea concentrations, and there was a
trend toward increased fetal protein oxidation (P 5 0.07). Maternal
but not fetal IGF-I and insulin concentrations increased. Fetal and
placental growth were not altered by GH treatment. Maternal and
fetal metabolic status was significantly affected by maternal food
intake. We conclude that maternal GH treatment increases placental
transport capacity, but that anabolic effects in the mother may limit
fetal substrate supply and therefore prevent an increase in fetal
growth. (Endocrinology 138: 5352–5358, 1997)
growth. GH administration to the mother would be expected
to increase maternal circulating blood glucose, fatty acid, and
IGF-I concentrations. In the fetus, circulating IGF-I concentrations are regulated by fetal blood glucose and insulin
concentrations (18 –20). Thus, maternal GH administration,
by increasing maternal and hence fetal blood glucose concentrations, would also be expected to increase both fetal and
maternal IGF-I concentrations. We therefore undertook the
current study to test the hypothesis that chronic maternal GH
administration would chronically increase fetal substrate
supply as well as maternal and fetal IGF-I concentrations and
would therefore enhance the growth of the late gestation fetal
sheep. Among the effects observed was an unanticipated
action of GH to enhance placental diffusion capacity.
Materials and Methods
Animals
Twenty-one Coopworth-Border cross-bred ewes with singleton pregnancies of known gestational age were brought into the laboratory at
approximately 105 days of gestation. They were housed in individual
metabolic cages with free access to water and fed ad libitum once daily
with weighed amounts of concentrates (NRM Multifeed sheep nuts,
NRM Feeds, Ltd., Auckland, New Zealand) and chopped barley straw.
The residual feed was weighed each day and the net feed intake
recorded.
At 115 6 4 days gestation (mean 6 sem) ewes underwent surgery as
described previously (17). Briefly, hysterotomy was performed under
general anaesthetic. Catheters were placed in the fetal femoral artery and
vein via tarsal vessels and the common umbilical vein via a paraumbilical incision. Two growth measuring devices (growth catheters)
were placed, one around each half of the fetal chest from sternum to
spine (21). Catheters were also placed in the uterine vein via the uteroovarian vein draining the pregnant horn, the maternal carotid artery and
jugular vein and the maternal femoral artery and vein via the tarsal
vessels.
Antibiotics [250 mg streptomycin and 250 mg penicillin (Streptopen,
Pitman Moore, Upper Hutt, New Zealand)] im to the ewe and 80 mg
gentamycin iv to the fetus) were administered at the time of surgery and
5352
MATERNAL GH TREATMENT IN SHEEP
daily for the next 3 days. Catheters were flushed on alternate days with
saline containing 10 U/ml of heparin. Growth catheters were measured
twice daily after the ewe had been standing quietly for several minutes,
and the mean of the two measurements recorded. Every 5 days ewes
were weighed and body condition scores recorded (22).
At the end of the experiment (134 6 0.3 days) ewes were killed with
an overdose of pentobarbitone. The uterus and its contents were dissected, weighed and measured. All procedures were approved by the
institutional Animal Ethics Committee.
Experimental procedures
Beginning at 125 6 0.3 days gestation, ewes were divided into GH
treated and control groups. GH treated animals received 0.1 mg/kg
recombinant bovine GH (courtesy of Dr. D. Chaleff, American Cyanamid, Princeton, NJ) sc twice daily for 10 days. Control animals received
an equivalent volume of normal saline. Blood samples were taken from
the fetus (2.5 ml) and the ewe (3 ml) in the morning before feeding on
days 0, 3, 5, 7, and 10 for metabolic and hormone measurements.
On days 0, 5, and 10 feto-placental substrate uptakes were measured.
A solution containing antipyrine 6.5 mg/ml, 3–O-[methyl-3H]glucose 40
mCi/ml and [14C]urea 7.3 mCi/ml in normal saline was infused through
a 0.45 mm filter to the fetus via the fetal venous catheter at 3 ml/h for
3.5 h after a 3-ml bolus. After 150 min of infusion to establish steady state,
five sets of blood samples were taken at 15-min intervals. Each set
consisted of samples taken from the maternal artery (2.5 ml), uterine vein
(2.5 ml), umbilical vein (1 ml), and fetal femoral artery (1.3 ml).
All blood samples for metabolic measurements were aliquoted into
Eppendorf tubes on ice and immediately frozen at 270 C until assay.
Blood for hormone measurements was centrifuged at 4 C and the plasma
separated and frozen at 270 C until assay. Samples for blood gas measurement were kept in capped syringes on ice until the measurements
were made within 30 min using a Radiometer ABL330 blood gas analyzer and Radiometer OSM2 hemoximeter (Radiometer, Copenhagen,
Denmark).
5353
All baseline measurements and postmortem measurements were compared using t tests. Not all data were available for every animal at each timepoint, largely because
catheters failed to sample or there were technical problems
with the assays. Furthermore, although all animals were fed
ad libitum, there was a wide variation in food intake both
between animals and from day to day in the same animal.
Food intake in sheep is an important regulator both of fetal
growth and of circulating metabolites such as glucose, lactate, and fatty acids. We therefore undertook all analyses
using multiple linear regression, including daily metabolizable energy intake as well as treatment group and study day
as independent variables. The effect of repeated samples was
taken into account by including sheep number nested within
treatment group in the regression model, and the effect of
treatment was assessed by the treatment by study day interaction term.
Differences in growth rates were tested using multiple
linear regression using a similar approach. To test for a
significant change in growth rate, two time variables were
used (before and during treatment) each with their own
constant. The second time variable took values only during
GH treatment. Animal number was nested within group as
the independent variable and the treatment by study day
interaction was the statistic of interest.
All values are given as the mean 6 se of the mean. All
statistical analyses were carried out using commercial software packages, Statview (Abacus Concepts, Berkley, CA)
and JMP (SAS Institute, Cary, NC).
Assays
Glucose (23) and urea (24) were measured by standard enzymatic
colorimetric methods modified for assay using a 96-well plate reader
(25). Lactate was measured in microplates using an enzymatic procedure
based on the reduction of NAD. Amino nitrogen was assayed by colorimetric reaction with b-napthoquinone sulfonate (26). Free fatty acids
were measured by a modification of a commercial kit assay (27). Antipyrine was measured in duplicate by HPLC (28). [14C]urea and 3–O[methyl-3H]glucose activities were determined in duplicate aliquots of
blood deproteinized with sulfuric acid and sodium tungstate and
counted in a dual channel liquid scintillation counter (Rak-Beta model
1219, LKB Wallac, Turku, Finland) with external standard quench correction for 10 min or to a dpm error of less than 3%.
IGF-I was measured by double antibody RIA after acid ethanol cryoprecipitation extraction validated for fetal sheep plasma (18, 29). Insulin
(18) and ovine placental lactogen (30) were measured by RIA.
Data analysis
Blood oxygen content was calculated from measured hemoglobin, oxygen saturation, and pO2. Blood flows were
calculated from measured antipyrine concentrations according to the Fick principle (31). Uptakes of oxygen, glucose, and
lactate were calculated for the uterus and its contents (referred to as uterine uptake 5 maternal artery-uterine vein
concentration difference 3 uterine blood flow), fetus (umbilical vein-femoral artery concentration difference 3 umbilical blood flow), and placenta (uterine uptake-fetal uptake). Clearances of [14C]urea and 3-O-[methyl-3H]glucose
were calculated by steady state diffusion techniques. Fetal
urea production rate was calculated as the product of [14C]urea clearance and the (fetal artery-maternal artery) urea concentration difference (32, 33).
Results
There was no difference between GH treated and control
animals for any metabolic or endocrine measurements at the
beginning of the treatment period (Tables 1–3).
GH treated and control ewes did not differ in body weight,
condition score, or food intake at the time of surgery, nor at
the beginning of the treatment period (Table 4). There was
also no difference between groups in any of these measurements during the study period.
Maternal GH treatment approximately doubled maternal
plasma concentrations of IGF-I and insulin but did not
change maternal plasma placental lactogen concentrations
(Table 1). Fetal concentrations of IGF-I, insulin, and placental
lactogen were unchanged by GH treatment. Maternal and
fetal plasma IGF-I concentrations also rose in both groups
over the study period independent of GH treatment
(Table 1).
Maternal GH treatment decreased maternal and fetal
blood urea concentrations by approximately 45% (Table 2)
but did not alter circulating concentrations of oxygen, glucose, lactate, amino nitrogen, or fatty acids in the maternal or
fetal blood. Maternal and fetal blood urea concentrations and
fetal blood oxygen content fell with increasing gestational
age in both groups. Maternal and fetal fatty acid concentrations and fetal blood glucose concentrations were significantly affected by maternal feed intake independently of GH
treatment (Table 2).
GH treatment did not alter uterine or umbilical blood flow,
nor utero-placental uptake of oxygen, glucose, and lactate.
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MATERNAL GH TREATMENT IN SHEEP
TABLE 1. Effects of 10D maternal GH treatment on maternal and fetal plasma hormone concentrations
Days of treatment
Day 0
Day 3
Day 5
Day 7
Day 10
GHa,b
Control
GHc
Control
GH
Control
183 6 18 (11)
208 6 35 (10)
0.9 6 0.1 (11)
0.9 6 0.2 (10)
205 6 27 (11)
232 6 56 (9)
421 6 58 (11)
207 6 29 (10)
1.6 6 0.4 (11)
1.6 6 0.7 (10)
243 6 33 (11)
229 6 43 (10)
362 6 52 (11)
205 6 41 (8)
1.1 6 0.2 (11)
0.7 6 0.2 (8)
223 6 24 (11)
251 6 46 (10)
395 6 68 (9)
211 6 40 (10)
2.0 6 1.0 (9)
0.8 6 1.7 (10)
215 6 31 (8)
282 6 79 (10)
465 6 74 (9)
263 6 51 (9)
2.0 6 1.1 (8)
0.8 6 0.2 (9)
241 6 44 (8)
193 6 36 (9)
GHd
Control
GH
Control
GH
Control
81 6 8 (11)
82 6 10 (10)
0.8 6 0.3 (10)
0.8 6 0.2 (10)
11.1 6 1.5 (11)
14.8 6 1.8 (10)
92 6 12 (10)
84 6 16 (10)
0.5 6 0.1 (8)
0.5 6 0.2 (8)
9.8 6 1.1 (10)
13.8 6 1.8 (10)
94 6 12 (9)
82 6 10 (9)
0.5 6 0.1 (8)
0.6 6 0.2 (8)
9.2 6 1.0 (9)
13.6 6 2.4 (8)
89 6 9 (9)
92 6 16 (9)
0.5 6 0.1 (8)
0.5 6 0.2 (8)
12.3 6 1.6 (10)
13.9 6 2.6 (8)
84 6 13 (8)
110 6 13 (8)
0.7 6 0.2 (8)
0.8 6 0.2 (8)
10.6 6 1.5 (8)
17.8 6 3.2 (8)
Group
Maternal
IGF-1
Insulin
Placental
Lactogen
Fetal
IGF-1
Insulin
Placental
Lactogen
Values are mean 6 SE (n) in ng/ml.
Data are compared using multiple linear regression analysis, with treatment group, study day and daily ewe metabolisable energy intake
included as independent variables. There are no significant differences between groups on study day 0.
a
P , 0.001, c P , 0.05 for treatment 3 study day interaction.
b
P , 0.001, d P 5 0.05 for change over days of treatment.
TABLE 2. Effects of 10D maternal GH treatment on blood metabolite concentrations
Days of treatment
Group
Maternal
Glucose
Lactate
Amino
Nitrogen
Urea
Fatty acids
Oxygen
Fetal
Glucose
Lactate
Amino
Nitrogen
Urea
Fatty
Acids
Oxygen
Day 0
Day 3
Day 5
Day 7
Day 10
GH
Control
GH
Control
GH
Control
GHa,b
Control
GHc
Control
GH
Control
2.31 6 0.12 (11)
2.35 6 0.15 (10)
0.49 6 0.05 (11)
0.54 6 0.02 (10)
4.10 6 0.20 (11)
3.97 6 0.20 (10)
4.21 6 0.37 (11)
4.06 6 0.36 (10)
0.42 6 0.13 (11)
0.39 6 0.12 (10)
4.99 6 0.27 (11)
5.14 6 0.32 (10)
2.93 6 0.66 (11)
2.36 6 0.17 (9)
0.48 6 0.04 (11)
0.58 6 0.03 (9)
3.91 6 0.17 (11)
4.16 6 0.18 (9)
2.75 6 0.45 (11)
4.11 6 0.33 (9)
0.72 6 0.15 (11)
0.34 6 0.09 (9)
5.33 6 0.30 (11)
5.29 6 0.28 (9)
2.63 6 0.33 (11)
2.31 6 0.09 (10)
0.46 6 0.02 (11)
0.50 6 0.02 (10)
4.00 6 0.16 (11)
4.11 6 0.22 (10)
2.29 6 0.29 (11)
3.81 6 0.50 (10)
0.55 6 0.17 (11)
0.60 6 0.16 (10)
5.08 6 0.20 (10)
4.96 6 0.26 (10)
2.98 6 0.70 (8)
2.26 6 0.16 (10)
0.50 6 0.05 (8)
0.55 6 0.03 (10)
4.10 6 0.22 (8)
4.17 6 0.18 (10)
2.79 6 0.23 (8)
3.57 6 0.45 (10)
0.59 6 0.16 (9)
0.42 6 0.14 (10)
5.05 6 0.34 (8)
5.08 6 0.18 (10)
2.44 6 0.41 (7)
2.40 6 0.11 (10)
0.59 6 0.06 (7)
0.54 6 0.02 (10)
4.11 6 0.27 (7)
4.26 6 0.19 (10)
3.00 6 0.58 (7)
3.62 6 0.49 (10)
0.51 6 0.12 (9)
0.32 6 0.09 (10)
4.67 6 0.33 (6)
4.95 6 0.22 (10)
GHd
Control
GH
Control
GH
Control
GHa,b
Control
GHc
Control
GHb
Control
0.70 6 0.09 (11)
0.61 6 0.07 (10)
1.44 6 0.17 (11)
1.52 6 0.08 (10)
6.97 6 0.35 (11)
6.85 6 0.19 (10)
4.48 6 0.36 (11)
4.40 6 0.38 (10)
0.015 6 0.003 (9)
0.021 6 0.004 (9)
3.65 6 0.27 (11)
3.25 6 0.17 (10)
0.80 6 0.13 (9)
0.62 6 0.06 (10)
1.80 6 0.18 (9)
1.63 6 0.11 (10)
7.27 6 0.21 (9)
7.17 6 0.32 (10)
3.26 6 0.51 (9)
4.72 6 0.34 (10)
0.017 6 0.002 (8)
0.021 6 0.003 (10)
3.52 6 0.28 (9)
3.46 6 0.17 (10)
0.70 6 0.07 (10)
0.55 6 0.04 (10)
1.85 6 0.23 (10)
1.93 6 0.25 (10)
7.11 6 0.26 (10)
6.94 6 0.22 (10)
2.55 6 0.29 (10)
4.34 6 0.47 (10)
0.013 6 0.001 (9)
0.019 6 0.003 (9)
3.29 6 0.24 (10)
2.92 6 0.23 (9)
0.74 6 0.07 (10)
0.57 6 0.05 (8)
1.80 6 0.15 (9)
1.74 6 0.09 (8)
7.21 6 0.22 (10)
6.89 6 0.25 (8)
2.83 6 0.40 (10)
4.27 6 0.58 (8)
0.021 6 0.005 (9)
0.017 6 0.002 (7)
3.36 6 0.26 (10)
2.87 6 0.13 (8)
0.73 6 0.09 (7)
0.62 6 0.05 (10)
1.79 6 0.28 (6)
2.15 6 0.43 (10)
7.63 6 0.42 (7)
7.01 6 0.31 (10)
2.83 6 0.67 (7)
4.02 6 0.51 (10)
0.022 6 0.008 (7)
0.017 6 0.004 (9)
3.21 6 0.36 (6)
2.81 6 0.16 (10)
Values are mean 6 SE (n) in mmol/liter.
Data are compared using multiple linear regression analysis, with treatment group, study day and daily ewe metabolisable energy intake
included as independent variables. There are no significant differences between groups on study day 0.
a
P , 0.05 for treatment 3 study day interraction.
b
P , 0.01 for change over days of treatment.
c
P , 0.001, d P , 0.01 for effect of ewe energy intake.
Umbilical blood flow, fetal lactate uptake, and uterine and
fetal oxygen uptakes all rose over the study period in both
groups, with no differences between groups. Fetal protein
oxidation, measured by fetal urea production, increased 3fold in the GH treated group compared with 70% in controls
(P 5 0.07, Table 3). Fetal urea production and umbilical blood
flow were also affected by maternal feed intake independently of GH treatment (Table 3).
Maternal GH treatment increased placental capacity for
simple diffusion as measured by a 70% rise in [14C]urea
clearance compared with a 20% rise in controls (P , 0.01,
Table 3). Placental capacity for facilitated diffusion, mea-
MATERNAL GH TREATMENT IN SHEEP
5355
TABLE 3. Effects of 10D maternal GH treatment on blood flow, placental clearances and metabolite uptakes
Days of Treatment
Day 0
Day 5
GH
Control
GHa,b
Control
1657 6 251 (11)
1434 6 146 (9)
800 6 116 (10)
719 6 86 (8)
1781 6 220 (11)
1638 6 156 (9)
824 6 70 (10)
694 6 51 (8)
1419 6 124 (5)
1520 6 140 (8)
812 6 168 (5)
1060 6 229 (7)
GHc,d
Control
GHd,e
Control
61.6 6 5.7 (11)
57.4 6 3.8 (10)
79.4 6 8.5 (11)
60.8 6 4.5 (10)
83.7 6 9.9 (10)
58.0 6 7.5 (10)
79.4 6 9.2 (10)
65.8 6 6.8 (10)
104.6 6 18.4 (5)
68.6 6 9.2 (10)
100.6 6 20.0 (5)
81.5 6 14.4 (10)
GHd,e,f
Control
14.2 6 3.4 (10)
21.5 6 5.8 (10)
31.9 6 8.4 (10)
29.1 6 7.3 (10)
53.8 6 7.4 (4)
37.2 6 12.9 (9)
GH
Control
GH
Control
GH
Control
362 6 46 (11)
297 6 29 (9)
98 6 18 (10)
93 6 14 (8)
251 6 47 (10)
199 6 36 (7)
316 6 76 (11)
304 6 21 (9)
115 6 15 (9)
100 6 17 (8)
162 6 45 (8)
209 6 24 (6)
226 6 71 (5)
312 6 39 (8)
93 6 23 (5)
147 6 39 (7)
77 6 46 (4)
222 6 58 (5)
GH
Control
GHa
Control
GH
Control
72 6 24 (11)
47 6 23 (9)
65 6 16 (10)
91 6 16 (8)
2146 6 27 (10)
2138 6 17 (7)
122 6 32 (11)
93 6 26 (9)
94 6 19 (10)
95 6 18 (8)
2217 6 33 (10)
2205 6 25 (7)
34 6 21 (5)
57 6 16 (8)
103 6 30 (5)
110 6 21 (7)
2156 6 106 (4)
2175 6 59 (5)
GHg
Control
GHg
Control
GH
Control
2022 6 338 (11)
2032 6 240 (9)
1102 6 105 (10)
1039 6 153 (8)
1028 6 350 (10)
1028 6 357 (7)
2297 6 294 (10)
2190 6 203 (9)
1297 6 78 (9)
1151 6 148 (8)
1147 6 318 (8)
987 6 266 (7)
1862 6 102 (5)
1990 6 143 (8)
1240 6 182 (5)
1526 6 192 (7)
534 6 192 (4)
642 6 84 (5)
Group
Blood flows (ml/min)
Uterine
Umbilical
Placental clearance (ml/min)
14
C-Urea
30 MG
Fetal urea production (mmol/min)
Glucose uptake (mmol/min)
Uterine
Fetal
Placental
Lactate uptake (mmol/min)
Uterine
Fetal
Placental
Oxygen uptake (mmol/min)
Uterine
Fetal
Placental
Day 10
Values are mean 6 SE (n).
Data are compared using multiple linear regression analysis, with treatment group, study day and daily ewe metabolisable energy intake
included as independent variables. There are no significant differences between groups on study day 0.
a
P , 0.05, g P , 0.01, d P , 0.001 for change over days of treatment.
b
P , 0.05, f P , 0.01 for effect of ewe energy intake.
e
P 5 0.07, c P , 0.01 for treatment 3 study day interraction.
sured by 3-O-[methyl-3H]glucose clearance, increased in both
groups over the study period but the trend toward an increase with maternal GH treatment did not reach significance
(P 5 0.07, Table 3).
Fetuses of GH treated and control ewes had similar rates
of growth over the study period, as indicated by similar
rates of girth increment measured by growth catheters
(Table 4). At post mortem there was no difference between
groups in fetal weight (4261 6 186 g in GH treated vs.
4278 6 291 g in controls), crown-rump length (47.6 6 0.8
vs. 46.0 6 1.6 cm), girth (35.0 6 0.6 vs. 34.7 6 1.0 cm),
placental weight (441 6 36 vs. 411 6 42 g), uterine weight
(683 6 45 vs. 668 6 36 g), amniotic fluid volume (1495 6
500 vs. 1116 6 249 ml) or weights of any individual fetal
organs (data not shown).
Discussion
In this study, maternal GH treatment increased placental capacity for simple diffusion but did not alter fetal or
placental growth. To our knowledge, this is the first demonstration that any maternal hormone treatment is able to
alter placental transport capacity. The effect was not due
to a generalized increase in placental size, at least as measured by placental weight. Furthermore, because the effect
on simple diffusion was greater than that on facilitated
diffusion, it seems likely that GH treatment altered placental surface area or membrane thickness rather than
activity of placental carrier proteins such as glucose
transporters.
The mechanism for this effect of GH on placental function
is not known. We have previously shown that short-term
infusion of IGF-I into the maternal or fetal circulations alters
placental metabolism, especially of lactate, suggesting that
placental function can be influenced by maternal and fetal
endocrine status (16, 17). In those studies, maternal IGF-I
infusions sufficient to increase maternal plasma IGF-I levels
3-fold for 3 h approximately doubled placental lactate production and fetal lactate uptake. However, a similar meta-
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MATERNAL GH TREATMENT IN SHEEP
TABLE 4. Effects of 10D maternal GH treatment on ewe weight, condition score, food intake and fetal growth rate
Before surgery
Ewe weight (Kg)
GH
Control
Days 23– 0
Days 3– 6
Days 7–10
57.2 6 2.1 (11)
53.1 6 4.2 (10)
59.2 6 1.9 (11)
53.9 6 4.2 (10)
59.2 6 2.1 (11)
55.5 6 3.7 (10)
58.6 6 2.4 (10)
56.3 6 4.1 (10)
Body condition score
GH
Control
3.8 6 0.3 (8)
3.9 6 0.4 (8)
3.3 6 0.2 (10)
3.4 6 0.3 (10)
3.1 6 0.2 (11)
3.3 6 0.3 (10)
3.0 6 0.2 (10)
3.4 6 0.3 (8)
Energy intake (MJ/day)
GH
Control
9.5 6 1.6 (9)
8.8 6 1.4 (10)
11.4 6 1.9 (11)
11.2 6 1.5 (10)
9.3 6 1.7 (11)
11.1 6 2.0 (10)
9.6 6 2.0 (11)
12.1 6 1.4 (10)
Protein intake (g/day)
GH
Control
117 6 20 (9)
109 6 18 (10)
140 6 23 (11)
138 6 19 (10)
113 6 21 (11)
136 6 26 (10)
117 6 25 (11)
148 6 18 (10)
2.84 6 0.37 (11)
2.57 6 0.43 (10)
2.30 6 0.54 (11)
2.33 6 0.51 (10)
2.40 6 0.43 (11)
2.32 6 1.50 (10)
Fetal girth increment (mm/day)
GH
Control
Values are mean 6 SE (n).
There are no significant differences between groups.
bolic effect was not seen in this study despite elevation in
maternal IGF-I, albeit over a much longer time course.
We had hypothesized that maternal GH therapy, by increasing maternal and fetal substrate supply and circulating
IGF-I concentrations, would increase fetal growth. Although
the treatment did increase placental diffusion capacity, we
found no evidence for enhanced fetal growth. This was despite our direct measurement of fetal growth by longitudinal
assessment of fetal girth increment as well as indirect measurements by assessment of fetal and organ sizes at postmortem. There are a number of possible reasons for this.
Firstly, pregnancy is associated with relative GH resistance. Although maternal GH treatment did increase maternal IGF-I concentrations as expected, we did not achieve a
significant rise in maternal blood glucose, and hence we did
not see the postulated rise in fetal blood glucose and plasma
IGF-I concentrations. GH treatment in the nonpregnant state
increases blood glucose and fatty acid concentrations by
inhibition of the effects of insulin leading to reduced peripheral glucose uptake and increased lipolysis (34). Pregnancy
is associated with a state of relative insulin resistance at least
partly due to high circulating concentrations of placental
lactogen and GH of placental origin (35). This is suggested
as the likely cause of resistance to the metabolic effects of the
exogenous GH reported in pregnant rats (9). Thus, we may
have found no increase in fetal growth because pregnancy
associated GH resistance meant that GH treatment did not
sufficiently increase the availability of substrates such as
glucose and fatty acids to the fetus.
Secondly, maternal GH treatment may have actually reduced the availability to the fetus of substrates that are potentially limiting to fetal growth. GH has anabolic effects on
protein metabolism, increasing amino acid uptake and protein synthesis while reducing urea synthesis (36). This effect
is reflected in our finding of a marked reduction in maternal
blood urea concentrations. The fall in fetal urea concentrations is most likely to be secondary to the fall in maternal
concentrations because urea produced by the fetus is excreted to the mother across the placenta by simple diffusion
down a concentration gradient. Despite this, GH treatment
was associated with a trend toward an increase in fetal urea
production, a direct measure of fetal protein oxidation.
Amino acids are normally taken up across the placenta by the
late gestation fetal sheep in considerable excess of requirements for tissue accretion (37) and may normally account for
some 20 –30% of fetal oxidative substrate requirements (32).
However, some amino acids are taken up at rates very close
to those required for tissue accretion, and the margin of
safety of these amino acids for fetal growth is very small (37).
Maternal GH treatment, by increasing maternal tissue amino
acid uptake, may have reduced the availability of some
amino acids to the fetus. This could in turn limit overall
amino acid incorporation into protein in the fetus and result
in their disposal by oxidation to lead to the observed increase
in fetal urea production.
This explanation is consistent with previous observations that GH treatment of pregnant rats may inhibit fetal
growth if the dams are poorly nourished (9). In contrast,
treatment of pregnant rats with GH antibody inhibited
maternal muscle protein synthesis and increased fetal
weight (38), suggesting that inhibition of maternal GH
effects on maternal protein accretion improved fetal substrate supply and hence fetal growth. Furthermore, we
have shown that maternal IGF-I infusion reduces the circulating concentration of essential amino acids in the fetal
circulation, as well as lowering both essential and nonessential amino acid concentrations in the maternal circulation (39). This would be consistent with the reduction in
the supply of amino acids to the fetus that we postulate
under the circumstances of the present study.
Thirdly, any effect of GH therapy on fetal growth and
metabolism may have been overshadowed in our studies by
the powerful effects of maternal nutrition on all the parameters of interest. Although all ewes in this study were considered to be in good condition and were fed ad libitum, their
feed intake varied widely both between animals and from
day to day in the same animal. In ruminants, maternal feed
intake directly influences the circulating concentrations of
glucose and fatty acids, and hence of insulin and IGF-I. This
influence is reflected in the independent effect of feed intake
MATERNAL GH TREATMENT IN SHEEP
on several of these parameters in our study in multivariate
analysis. This wide variation in food intake may have been
large enough to obscure any relatively small effects of GH
treatment.
A number of studies of the effects of maternal GH therapy
on fetal growth have previously been reported, with variable
results. Zamenhof and colleagues (10) reported that administration of large doses (3 mg/day) of bovine GH to pregnant
rats from days 7 to 20 of gestation increased the brain but not
body weight of the offspring. Clendinnen and Eayrs (11)
used a more highly purified preparation of bovine GH from
days 7 to 19 and found increased birthweight as well as
altered brain structure and function in the offspring. Similar
findings were obtained using doses of 0.1–3 mg/day of purified porcine GH from days 7 to 20 (12). More recently,
Gargosky and colleagues (13) used recombinant human GH
administered by osmotic minipump at 2.4 mg/kgzday from
day 11 but failed to show any effect of this treatment on
maternal IGF-I concentrations or fetal or placental size on
day 21. Treatment of pregnant pigs with purified porcine GH
from days 28 to 40 of gestation also increased fetal length but
not weight (14). Treatment of pregnant sheep with GRF from
days 130 to 140 of gestation increased lamb weight and cord
blood IGF-II but not IGF-I concentrations (15). These differing findings may in part be a result of differing maternal
nutritional planes and ages (38), resulting in differing effects
on nutrient partitioning between mother and fetus, as well as
different GH preparations and dose regimens. We are not
aware of any previous studies of maternal GH administration in large, chronically catheterized animals that allow in
vivo evaluation of the metabolic and endocrine changes in
mother and fetus during treatment to clarify these effects.
We conclude that maternal GH therapy in late gestation
pregnant sheep increases placental diffusion capacity but
does not increase fetal growth. This suggests that placental
diffusion capacity is not limiting on fetal growth under these
circumstances. Although a rise in maternal IGF-I concentrations may be expected to increase placental substrate uptake,
the anabolic effects of GH in the mother may counteract any
potential benefit to the fetus by reducing fetal supply of
limiting substrates such as amino acids. It seems likely, therefore, that enhancement of fetal growth will require enhanced
substrate supply as well as an appropriate endocrine milieu
and placental transport capacity.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
Acknowledgments
25.
We would like to thank Dr. B. Breier for the hormone assays. Fiona
Ffolliott-Powell, Christine Gibson, Sam Rossenrode, and Eric
Thorstensen provided excellent technical assistance. Dr. D. Chaleff of
American Cyanamid provided the GH for these studies.
26.
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