Impact of maternal nutrition during pregnancy on pituitary gonadotrophin gene expression and ovarian development in growth-restricted and normally grown late gestation sheep fetuses.

Abstract

The influence of maternal nutrition during pregnancy on anterior pituitary gonadotrophin gene expression and ovarian development in sheep fetuses during late gestation was investigated. Embryos recovered from superovulated adult ewes that had been inseminated by a single sire were transferred, singly, into the uteri of adolescent recipients. After embryo transfer, adolescent ewes were offered a high or moderate amount of a complete diet. Pregnancies were terminated at day 131 +/- 0.6 of gestation and the fetal brain, anterior pituitary gland and gonads were collected. Gonadotrophin gene expression (LHbeta and FSHbeta subunits) in the fetal pituitary gland was examined using in situ hybridization. Ovarian follicular development was quantified in haematoxylin- and eosin-stained ovarian sections embedded in paraffin wax. Six dams that were offered a high nutrient intake carried normal-sized fetuses (weight within +/- 2 SD of mean weight for control fetuses from dams fed a moderate level of complete diet) and 13 dams carried growth-restricted fetuses (weight <or= +/- 2 SD of mean weight for control fetuses from dams fed a moderate level of complete diet). Mean placental masses in these groups were 354 +/- 24.5 and 230 +/- 21.1 g, respectively, compared with 442 +/- 54.3 g in the dams that were offered a moderate nutrient intake (n = 6). Growth-restricted fetuses from dams offered a high nutrient intake showed higher pituitary LHbeta mRNA expression (P < 0.05) than normal-sized fetuses from dams offered a moderate nutrient intake (252 +/- 21.6 and 172 +/- 23.6 nCi g(-1), respectively). FSHbeta mRNA expression was not influenced by growth status. Fewer follicles (primarily in the resting pool) were observed in the ovaries of both growth-restricted (P < 0.002) and normal-sized fetuses from dams offered a high nutrient intake (P < 0.01) compared with normal-sized fetuses from dams offered a moderate nutrient intake. Irrespective of nutritional treatment, the total number of follicles was positively associated with placental mass (P < 0.01). Thus, a high maternal nutrient intake during adolescent pregnancy had a negative influence on ovarian follicular development in fetuses as determined during late gestation.

Full text

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Introduction
Successful reproduction in adult life depends on appropriate
development of the reproductive axis, and much of the
structural and neuroendocrine development of the com-
ponents of the hypothalamic–pituitary–gonadal axis occurs
during prenatal life (Brooks et al., 1992; Caldani et al.,
1995; McNatty et al., 1995). In normally growing sheep
fetuses, LH and FSH gonadotrophs are present in the
anterior pituitary gland by day 70 and day 100 of gestation,
respectively (Thomas et al., 1993) and the fetal gonado-
trophin response to exogenous GnRH is highest after day
100 of gestation (term = 145 days; Mueller et al., 1981).
Thereafter, gonadotrophin concentrations decrease in
response to evolving feedback mechanisms from sex
steroids of both placental and gonadal origin (Gluckman
et al., 1983; Mesiano et al., 1991) in association with
increased central nervous system inhibition of GnRH
(Brooks et al., 1995). In the fetal ovary, the maximum
number of germ cells is present at day 75 of gestation
(McNatty et al., 1995). At this stage, the first primordial
follicles have formed and a large proportion of the germ
cells is lost or degenerates beyond this point (80% between
day 75 and day 90 when follicles are forming). Primary and
secondary follicles are present in the ovarian cortex of
sheep by day 100 and day 120 of gestation, respectively
(McNatty et al., 1995, 2000).
It is postulated that environmental factors, such as
maternal nutrition, which is known to influence the prenatal
growth trajectory and physiology of the major organ
systems (Robinson et al., 1999), may also have an adverse
effect on the prenatal development of the components of
the reproductive axis. Studies on animals and humans
Impact of maternal nutrition during pregnancy on pituitary
gonadotrophin gene expression and ovarian development in
growth-restricted and normally grown late gestation sheep fetuses
P. Da Silva1, R. P. Aitken1, S. M. Rhind2, P. A. Racey3
and J. M. Wallace1*
1Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK; 2Macaulay Land Use
Research Institute, Craigiebuckler, Aberdeen AB15 8QH, UK; and 3Department of Zoology,
University of Aberdeen, Aberdeen AB24 2TZ, UK
Reproduction (2002) 123, 769–777
Research
The influence of maternal nutrition during pregnancy on
anterior pituitary gonadotrophin gene expression and
ovarian development in sheep fetuses during late gestation
was investigated. Embryos recovered from superovulated
adult ewes that had been inseminated by a single sire were
transferred, singly, into the uteri of adolescent recipients.
After embryo transfer, adolescent ewes were offered a
high or moderate amount of a complete diet. Pregnancies
were terminated at day 131 ? 0.6 of gestation and the
fetal brain, anterior pituitary gland and gonads were
collected. Gonadotrophin gene expression (LHβ and FSHβ
subunits) in the fetal pituitary gland was examined using in
situ hybridization. Ovarian follicular development was
quantified in haematoxylin- and eosin-stained ovarian
sections embedded in paraffin wax. Six dams that were
offered a high nutrient intake carried normal-sized fetuses
(weight within ? 2 SD of mean weight for control fetuses
from dams fed a moderate level of complete diet) and 13
dams carried growth-restricted fetuses (weight ? ? 2 SD
of mean weight for control fetuses from dams fed a
moderate level of complete diet). Mean placental masses
in these groups were 354 ? 24.5 and 230 ? 21.1 g,
respectively, compared with 442 ? 54.3 g in the dams that
were offered a moderate nutrient intake (n = 6). Growth-
restricted fetuses from dams offered a high nutrient intake
showed higher pituitary LHβ mRNA expression (P < 0.05)
than normal-sized fetuses from dams offered a moderate
nutrient intake (252 ? 21.6 and 172 ? 23.6 nCi g–1,
respectively). FSHβ mRNA expression was not influenced
by growth status. Fewer follicles (primarily in the resting
pool) were observed in the ovaries of both growth-
restricted (P < 0.002) and normal-sized fetuses from dams
offered a high nutrient intake (P < 0.01) compared with
normal-sized fetuses from dams offered a moderate
nutrient intake. Irrespective of nutritional treatment, the
total number of follicles was positively associated with
placental mass (P < 0.01). Thus, a high maternal nutrient
intake during adolescent pregnancy had a negative
influence on ovarian follicular development in fetuses as
determined during late gestation.
© 2002 Society for Reproduction and Fertility
1470-1626/2002
*Correspondence
Email: Jacqueline.Wallace@rri.sari.ac.uk

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indicate that impaired prenatal growth can subsequently
influence various aspects of postnatal reproductive develop-
ment, including age at puberty (Ibãnez et al., 2000a,b; Da
Silva et al., 2001), age at menopause (Cresswell et al., 1997)
and the incidence of male subfertility (Francois et al., 1997).
However, little is known about the effect of maternal
nutrient intake on the development of the reproductive axis
prenatally. A comparison of twin-bearing ewes that received
either a low (0.5 ? maintenance energy requirements) or
high (1.5 ? maintenance energy requirements) intake
during the first third of pregnancy revealed that germ cell
degeneration was retarded in the low nutrient intake group
at both day 47 and day 62 of gestation (Borwick et al.,
1997). These early effects appeared to be independent of
changes in placental mass or fetal weight.
In contrast, overnourishing the rapidly growing adolescent
sheep throughout pregnancy resulted in a profound
reduction in placental growth, which led to a significant
decrease in birth weight compared with moderately fed
adolescent ewes (Wallace et al., 1996). This paradoxical
situation arises because high maternal nutrient intake
promotes a major anabolic drive to maternal tissue synthesis
at the expense of the evolving nutrient requirements of the
gravid uterus (Wallace et al., 1997). The aim of the present
study, using this model, was to determine whether this form
of nutritionally mediated placental and fetal growth restriction
influences anterior pituitary gonadotrophin gene expression
and ovarian development of (approximately day 131)
female fetuses in late gestation. By this stage of gestation, the
normally growing fetus is considered to have achieved
80–85% of its final birth weight (Robinson, 1977) and the
components of the hypothalamic–pituitary–ovarian axis are
considered fully functional.
Materials and Methods
Animals and experimental design
All procedures were licensed under the UK Animals
(Scientific Procedures) Act of 1986. Embryos recovered on
day 4 after oestrus from superovulated adult ewes (Border
Leicester ? Scottish Blackface) that had been inseminated
by a single sire (Dorset Horn) were synchronously
transferred, singly, into the uteri of adolescent recipient
ewes (Dorset Horn ? Mule) as described by Wallace et al.
(1997). All animals were housed in individual pens under
natural lighting conditions at the Rowett Research Institute
(57?N, 2?W). The recipient ewes were approximately
7 months old and had a mean live weight of 45 kg at the
start of the study. Immediately after embryo transfer, the
adolescent ewes were allocated to one of two dietary
treatments, so that the two groups were similar with respect
to live weight, body condition score (Russel et al., 1969)
and ovulation rate at the time of embryo transfer. Care was
also taken to balance for donor embryo source where
possible. Recipient ewes were offered individually, a high
(H, n = 42) or a moderate (M, n = 32) amount of a complete
diet (Wallace et al., 1996). The aim of these studies was to
achieve rapid maternal growth rates throughout pregnancy
in the group offered a high nutrient intake and to maintain a
growth rate of 50–75 g day–1during the first 100 days of
pregnancy in the group offered a moderate nutrient intake.
The diet provided an estimated 10.2 MJ metabolizable
energy kg–1dry matter, 136.6 g crude protein kg–1dry
matter and had an average dry matter of 85.5%. All ewes
were offered their feed in two equal portions, at 08:00 h and
16:00 h each day. The daily feed refusal was weighed and
recorded before the feed at 08:00 h. Moderate nutrient
intake ewes were offered their experimental diets starting
immediately after embryo transfer, whereas the amount of
feed offered to high intake ewes was increased gradually
over a 2 week period until the amount of daily feed refusal
was approximately 15% of the total amount offered
(equivalent to ad libitum intakes). Ewes were weighed each
week and their body condition score was assessed at
monthly intervals throughout gestation. The amount of feed
offered was adjusted three times each week according to
both live weight changes and feed refusals. After day 100 of
gestation, the feed intake of the moderate nutrient intake
ewes was increased each week to maintain body condition
score during the final third of pregnancy and, hence, meet
the estimated increasing demands of the developing fetus
during late gestation.
At approximately day 50 of gestation, pregnancy was
confirmed by transabdominal ultrasonography. Pregnancies
were established and maintained in a total of 17 moderate
and 30 high nutrient intake dams.
Collection of tissue samples
Ewes were killed at day 131 ? 0.6 of gestation. On this
day, ewes received their morning feed, were weighed and
body condition scores were recorded; the ewes were then
killed by an i.v. administration of an overdose of sodium
pentobarbitone (20 ml Euthesate; 200 mg pentobarbitone
ml–1; Willows Francis Veterinary, Crawley) and exsanguina-
tion was performed by severing the main vessels of the
neck. The gravid uterus was removed immediately and
opened. The fetus was killed by intracardiac administration
of sodium pentobarbitone (5 ml Euthesate). The umbilical
cord was clamped and the fetus was removed, dried and
weighed. The head was detached within 4 min of fetal
death and the brain was collected and weighed. The
pituitary gland was removed from the base of the brain and
bisected in the coronal plane. Half of the anterior pituitary
gland was placed in tissue-tek embedding medium and
then snap-frozen in liquid nitrogen-chilled isopentane and
stored at –70?C until LHβ and FSHβ mRNA were quantified
by in situ hybridization.
Fetal gonads were dissected, weighed and fixed
immediately in Bouin’s fixative for 6 h before being
transferred to 70% (v/v) ethanol. Gonadal tissue was then
prepared for histology. The fetal liver was removed and
weighed. All whole placentomes were dissected and the
770P. Da Silva et al.

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total masses were recorded. The singleton fetuses from six
of 17 moderate nutrient intake dams and from 19 of 30 high
nutrient intake dams were female and form the basis of this
study.
In situ hybridization
In situ hybridization was performed on frozen sections
using a technique similar to that of Sirinathsinghji et al.
(1990). All chemicals were obtained from Sigma-Aldrich
Co. (Poole) unless specified otherwise. Sense and antisense
oligonucleotide probes (45 bases) were used (antisense;
5’CAGATGCTGGTGGTGAAAGTGATACAGACAGGGCA-
GGCCTCCTTC3’, complementary to bases 124–168 of the
ovine LHβ gene; D’Angelo-Bernard et al., 1990; EMBL
accession no. X52488; and 5’GTGACATTCAGTGGCTACT-
GGGTACGTGTA CAGGGAGTCTGCATG3’, complemen-
tary to bases 314–358 of the ovine FSHβ gene; Mountford et
al., 1989; EMBL accession no. X15493; Oswel DNA service,
Southampton). Probes were labelled with [35S]deoxyadeno-
sine 5’-(alpha-thio)triphosphate (dATP; 1300 Ci mmol–1;
NEN, Life Science Products, Hounslow) using terminal
deoxynucleotidyl transferase (TdT; Roche, Lewes) in a
reaction mixture containing
(10 ng µl–1), 4 µl diethyl pyrocarbonate (DEPC)-treated
water, 2 µl of 5 ? TdT buffer, 1 µl CoCl2, 1 µl [35S]dATP
and 1 µl TdT, and incubated at 37?C for 1 h. The reaction
was terminated by adding 40 µl DEPC-treated water.
After centrifugation through a Sephadex (G50) column at
1200 g for 10 min, 2 µl (1 mol dithiothreitol (DTT) l–1) was
administered to prevent RNAase degradation. Sections from
the same pituitary glands were probed with antisense and
sense probes, simultaneously.
Coronal pituitary sections (10 µm) from 11 growth-
restricted and five normally developed fetuses (from high
nutrient intake and moderate nutrient intake ewes, respec-
tively) were cut on a cryostat and mounted onto poly-L-
lysine-coated slides. One pituitary gland from the moderate
nutrient intake group was not included because it was
damaged during dissection from the fetus. A decision was
taken to compare the pituitary glands of growth-restricted
fetuses from the high nutrient intake group with the
normally grown fetuses from the moderate nutrient intake
group because this number of slides (sense and antisense)
could be processed in a single analysis. Slides were then
fixed in 4% (w/v) paraformaldehyde in DEPC-treated
phosphate-PBS at 4?C for 5 min, washed twice in PBS and
dehydrated in a series of alcohol. Slides were removed
from 95% alcohol and air-dried at room temperature. The
labelled probe (1 µl at 150000 c.p.m. per slide) was mixed
with 100 µl hybridization buffer per slide. The hybridiza-
tion buffer contained 50% (v/v) deionized formamide, 4 ?
saline sodium citrate (SSC), 25 mmol sodium phosphate l–1,
pH 7, 1 mmol sodium pyrophosphate l–1, 0.5 ? Denhardt’s
solution, 200 µg salmon sperm DNA ml–1(final), 100 µg
polyadenylic acid ml–1(final), 120 µg sodium heparin ml–1,
10% (w/v) dextran sulphate and 20 mmol DTT l–1. Slides were
1 µl oligonucleotide
hybridized overnight at 42?C with the radiolabelled
oligonucleotide probe in hybridization buffer. After hy-
bridization, slides were washed in 1 ? SSC–0.5% (w/v)
β-mercaptoethanol at room temperature for 20 min on
a rocking platform and in 1 ? SSC–0.5% (w/v) β-
mercaptoethanol at 55?C for 35 min in a shaking
hybridization oven. The slides were then washed twice at
room temperature in 1.0 ? SSC, and then once each in
0.1 ? SSC, 70% (v/v) ethanol and 95% (v/v) ethanol.
Quantification of mRNA expression
Sections were air-dried and exposed to autoradiography
film (Hyperfilm beta-max; Amersham International plc,
Little Chalfont) at room temperature together with an
autoradiographic [14C]micro-scale of 40–1070 nCi g–1
(Amersham International plc). At the end of the 5 day
exposure period, the autoradiograph film was developed
and fixed using standard methods (Kodak developer/fixer
GBX). This exposure time was selected as it allowed the
weakest autoradiographic images to be quantified without
the strongest images saturating the film. An image analysis
system (Image Pro-Plus; Media Cybernetics, MD) was used
to convert tissue equivalent radioactivity levels of scanned
autoradiographic films (Umax Scanner; Astra 1220S,
Dusseldorf) to absorbance of autoradiographs on the basis
of the autoradiographic micro-scale. Average absorbance
readings were taken from three sections per pituitary gland
and mean absorbance values for each animal were then
calculated.
Slides exposed previously to autoradiography film were
coated with autoradiography silver emulsion (Hypercoat
LM-1 emulsion; Amersham International plc). The emulsion-
coated slides were stored in a light-tight box with silica gel
at 4?C for 7 days. Slides were then developed and fixed
with Kodak EDF/EDP photochemicals (Anachem, Luton)
and counterstained with Mayer’s haematoxylin solution.
Slides were dehydrated in a series of ethanol and a coverslip
mounted with DPX mountant (BDH Chemicals Ltd, Poole)
to produce visible silver grains over the sites of
hybridization. The number of silver grains produced was
observed and photographed under dark-field illumination
using an Olympus BH-2 microscope (? 40 objective) with
Image Pro Plus software.
Ovarian histology and morphometry
The right ovary was dehydrated in a series of ethanol,
cleared in xylene and embedded in paraffin wax. The ovary
was then bisected transversely and both planar transections
were embedded in the same paraffin wax block. Serial
sections were cut at 5 µm intervals and then stained with
haematoxylin and eosin for examination of germ cell
development. An average of 25 ovarian sections were
examined per fetus. Stained ovarian sections were examined
using a light microscope (Wang Biomedical, Microscopy
Supplies and Consultants Ltd, Fife) with a 1 cm2ocular
graticule (21 mm; Agar Scientific, Stanstead) as follows. The
Growth restriction and fetal pituitary–ovarian development771

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width of the cortical tissue was measured at ? 100 magni-
fication. Thereafter, four standard graticule areas were
selected for each ovary and the number of germ cells was
counted in a standardized manner between sections at a
magnification of ? 250. Ovarian structures were classified
as: oocytes (germ cells devoid of follicular cells and
undergoing meiosis); primordial follicles (germ cells
surrounded by flattened follicular cells); primary follicles
(enlarged oocyte completely surrounded by one to two
layers of cuboidal follicular cells); and secondary follicles
(enlarged oocyte surrounded by two or more concentric
layers of cuboidal cells), as described by Smith et al. (1994).
The total number of follicles was estimated by first counting,
and then averaging, the number of primordial, primary and
secondary follicles for each graticule area in each cut
section. Subsequently, an index of the total number of
follicles was calculated by adding the results from the four
graticule areas.
Statistical analyses
Fetal weight at necropsy was adjusted to a standard
gestational age of 137 days (equivalent to the age of the
oldest fetus studied) using the formula: adjusted fetal weight
= fetal weight at necropsy (g) ? 1.01305 for each day of
gestation. Fetuses from the high nutrient intake group were
characterized as small or growth-restricted if fetal weight
was ? the mean for moderate group fetuses minus two
times the SD (Robinson et al., 1979). Thus, in this study, for
this genotype a female fetus was considered to be growth-
restricted if its adjusted weight was < 4086 g. Consequently,
13 female fetuses derived from high nutrient intake dams
were designated as growth-restricted and six as normal-
sized. Maternal live weight and body condition changes
and gene expression data were analysed by Student’s t test.
Pregnancy outcome and ovarian follicular data were
examined by ANOVA, after which specific treatment
comparisons were made by t test using pooled SD.
Correlation analysis was carried out by Pearson’s product
moment test where appropriate.
Results
Maternal live weight and body condition
At embryo transfer, the mean live weight and body
condition scores for ewes that conceived and carried female
fetuses were 47.1 ? 0.88 and 45.1 ? 0.68 kg, and
2.3 ? 0.05 and 2.2 ? 0.03 score units in the moderate and
high nutrient intake groups, respectively. At approximately
day 131 of gestation when the ewes were killed, mean
live weights and body condition scores of ewes in the
high and moderate nutrient groups were 73.4 ? 1.87 and
54.2 ? 1.95 kg (P < 0.001), and 3.3 ? 0.06 and 2.3 ? 0.09
score units (P < 0.001), respectively. The mean live weight
gain during the first 100 days of gestation for the high and
moderate nutrient intake groups was 306 ? 19.6 and
68 ? 10.2 g day–1, respectively (P < 0.001).
Placental and fetal growth
Mean gestational age at necropsy and morphometric
data relating to pregnancy outcome in high and moderate
nutrient intake group pregnancies are shown (Table 1). High
nutrient intake ewes were divided into those carrying
normal-sized fetuses and those carrying growth-restricted
fetuses as detailed above. Total placentome mass in the
high nutrient intake dams with growth-restricted fetuses was
48 and 35% lower than that in the moderate (P < 0.001)
and high nutrient intake groups (P = 0.01) with normal-
sized fetuses, respectively. Similarly, fetal liver masses were
lower (P < 0.01) in the growth-restricted fetuses than in both
groups of normal-sized fetuses (Table 1). Although fetal
772P. Da Silva et al.
Table 1. Gestational age at necropsy, placental mass and fetal weight, and fetal brain and liver mass in relation to maternal nutrient intake
and growth status in late gestation sheep fetuses
Maternal nutrient intake
High ModerateHigh
Fetal growth status
Restricted (H–R)
Significance level
M versus H–N H-R versus H–N Normal (M)Normal (H–N) M versus H–R
Number of pregnancies
Gestational age (days)
Total placentome mass (g)
Fetal weight (g)
Adjusted fetal weight: day
137 of gestation (g)
Fetal brain mass (g)
Fetal liver mass (g)
Fetal brain:liver mass
6 136
132 ? 1.7
442 ? 54.3
4300 ? 155.7
4599 ? 104.6
131 ? 0.8
230 ? 21.1
2963 ? 248.6
3213 ? 269.8
131 ? 1.3
354 ? 24.5
4065 ? 145.1
4428 ? 130.3
ns ns
ns
ns
ns
ns
P < 0.001
P < 0.001
P < 0.002
P = 0.01
P < 0.01
P < 0.01
41.6 ? 1.70
127.5 ? 7.12
0.33 ? 0.018
36.4 ? 1.35
81.9 ? 8.06
0.43 ? 0.030
40.1 ? 0.82
123.4 ? 8.52
0.33 ? 0.020
P < 0.02
P < 0.002
P < 0.02
ns
ns
ns
ns
P < 0.01
P < 0.02
Values are mean ? SEM.
M: moderate; H–R: high–restricted; H–N: high–normal; ns: not significant.

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brain mass was lower (P < 0.02) in the growth-restricted
fetuses from high nutrient intake dams when compared with
the normal-sized fetuses from the moderate nutrient intake
group, the brain mass to liver mass ratio was higher in
growth-restricted fetuses when compared with both groups
of normal-sized fetuses (P < 0.02). Irrespective of nutritional
treatment, fetal weight at autopsy was positively correlated
with total placental mass (r = 0.793, n = 25, P < 0.001). A
similar relationship was observed between total placentome
mass and the mass of the fetal brain (r = 0.712, n = 25,
P < 0.001) and liver (r = 0.883, n = 25, P < 0.001).
Fetal pituitary LHβ and FSHβ gene expression
The amount of LHβ mRNA in the fetal pituitary gland was
significantly higher (P < 0.05) in growth-restricted fetuses
from high nutrient intake dams compared with normally
grown female fetuses from moderate nutrient intake dams
(Table 2). Irrespective of growth status, LH mRNA expression
was negatively correlated with total placentome mass
(r = –0.535, n = 16, P < 0.05). Film autoradiographs and
dark-field photomicrographs of representative fetal pituitary
sections hybridized to the LHβ probe are illustrated (Fig. 1).
There was no significant effect of maternal nutritional
treatment on fetal pituitary FSHβmRNA expression (Table 2).
Ovarian development
The ovarian sections of the two most severely growth-
restricted fetuses were difficult to cut and were of poor
quality after staining. Although these ovaries were virtually
devoid of follicles of any type, the data were not included in
the overall group mean, as insufficient high quality sections
for accurate quantification were obtained. The weights of
these two fetuses were 1217 and 1460 g, respectively.
Neither ovarian mass nor the width of the cortical layer
was influenced by maternal nutritional treatment or fetal
growth status (Table 3). In contrast, the total number of
ovarian follicles (per four graticule areas) was lower in both
growth-restricted (P < 0.002) and normal-sized fetuses
Growth restriction and fetal pituitary–ovarian development773
(A)(B)
(a)
(b)
I II
Fig. 1. (A) Film autoradiographs (I: antisense; II: sense) and (B) dark field photomicrographs (? 400 magnifi-
cation) of fetal anterior pituitary gland sections hybridized to the LHβ probe in a representative (a) growth-
restricted and (b) normally grown female sheep fetus.
Table 2. LHβ and FSHβ mRNA concentrations in the anterior pituitary gland of normally grown and
growth-restricted late gestation female sheep fetuses
Maternal nutrient intake
ModerateHigh
Fetal growth status
NormalRestricted Significance level
Number of fetuses
LHβ mRNA (nCi g–1)
(range)
FSHβ mRNA (nCi g–1)
(range)
5 11
172 ? 23.6
(118–238)
291 ? 52.3
(160–444)
252 ? 21.6
(136–345)
250 ? 16.6
(164–331)
P < 0.05
ns
Values are mean ? SEM.
ns: not significant.