Cumulative dietary energy intake determines the onset of puberty in female rats.

Abstract

Laboratory animal diets for studies to determine the endocrine-disrupting potential of chemicals are under scrutiny because they can affect both assay control values and assay sensitivity. Although phytoestrogen content is important, we have previously shown that a phytoestrogen-rich diet and a phytoestrogen-free diet were equally uterotrophic to rats and advanced vaginal opening (VO) when compared with the standard diet RM1. Abolition of the effects by the gonadotrophin-releasing hormone antagonist Antarelix indicated that these effects were mediated through the hypothalamus-pituitary-reproductive organ axis. In the present study, we investigated the relationship between cumulative energy intake and sexual maturation in female rats. Infant formula (IF) at different concentrations and synthetic diets, with a wide range of metabolizable energy (ME) values, were used to modulate energy intake. Increasing energy intake was associated with an increase in uterine weight (absolute and adjusted for body weight) for both IF and the synthetic diets. In both cases, the increased uterine weight was directly proportional to energy intake. Body weight was unaffected by IF consumption but, in the case of the diets, was increased proportionally with energy consumption. Antarelix abolished the uterine weight increases with both formula and the diets, whereas body weight was unaffected. The mean day of VO was also advanced by high-ME diets and IF, whereas body weight at VO was unaffected. VO occurred at an energy intake of approximately 2,300 kJ/rat determined by measuring total food intake from weaning to VO, indicating that this cumulative energy intake was the trigger for puberty. ME is therefore a critical factor in the choice of diets for endocrine disruption studies.

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VOLUME 112 | NUMBER 15 | November 2004 • Environmental Health Perspectives
Research| Article
The choice of laboratory animal diet for
rodent studies to determine the endocrine-
disrupting potential of chemicals is currently
under intense scrutiny (Lawton 2003; Odum
et al. 2001; Owens et al. 2003; Owens and
Koëter 2003; Thigpen et al. 2003). This is
because the diet selected can affect both assay
control values and assay sensitivity; for exam-
ple, uterine weight in control animals needs
to be low to maximize the dynamic range of
the uterotrophic assay. One contributing fac-
tor is the phytoestrogen content of the diet.
Most of the commonly used laboratory ani-
mal diets are formulated with soy extracts,
which contain the isoflavones genistein
(GEN) and daidzein, and/or alfalfa (lucerne),
which contains coumestrol (Patisaul and
Whitten 1999). These phytoestrogens are
estrogenic to rodents, causing effects such as
increased uterine weight and advanced vagi-
nal opening (VO) in immature animals, simi-
lar to effects observed with xenobiotic
estrogens (Bickoff et al. 1962; Boettger-Tong
et al. 1998; Casanova et al. 1999; Medlock
et al. 1995; Thigpen et al. 1999; Tinwell
et al. 2000; Whitten et al. 1992).
An analysis conducted as part of the recent
Organisation for Economic Co-operation and
Development (OECD) evaluation of the
immature rat uterotrophic assay indicated that
isoflavone levels greater than 325–350 mg
GEN equivalents/kg diet should be avoided to
maintain optimal assay sensitivity and dynamic
range (Owens et al. 2003). The phytoestrogen
content of diets is not, however, the only factor
of importance. This is shown by our earlier
demonstration that the phytoestrogen-rich diet
Purina 5001 (Purina Mills, Inc., Richmond,
IN, USA) and the phytoestrogen-free diet
AIN-76A are equally uterotrophic to rodents,
compared with the standard diet RM1, and
that each is able to advance the mean day of
VO in rats, again compared with RM1
(Odum et al. 2001). Further, we showed that
coadministration of the gonadotrophin-
releasing hormone (GnRH) antagonist
Antarelix (ANT; Europeptides, Argenteuil,
France) abolished the uterotrophic activity of
both diets, indicating that these effects were
mediated at the level of the hypothalamus to
influence GnRH secretion (Odum et al.
2001). ANT is a synthetic peptide that was
shown to be a GnRH antagonist in several ani-
mal models, including suppression of ovula-
tion in rats and leutinizing hormone release in
rams (Deghenghi et al. 1993). In a related
series of experiments, we observed a correlation
between the quantity of infant formula (IF)
consumed by immature rats and mice and the
magnitude of the resultant uterotrophic effect
(Ashby et al. 2000). The uterotrophic effects
were independent of the phytoestrogen con-
tent of the IF because they were abolished by
inhibition of GnRH with ANT. In contrast,
the uterotrophic effect of the reference syn-
thetic estrogen diethylstilbestrol (DES) was
unaffected by ANT (Ashby et al. 2000).
These findings suggest that the type of food
consumed by female rodents could influence
the time of their puberty but that these influ-
ences were independent of phytoestrogen
intake at the levels present in the foods used
in these studies.
Energy intake is known to affect the onset
of puberty in mammals; for example, pigs and
rats with inadequate nutrition have retarded
sexual development (Frisch et al. 1975;
Kirkwood et al. 1987; Trentacoste et al.
2001). Energy balance in mammals is con-
trolled by a series of complex central mecha-
nisms that allow adaptive responses to
situations of energy abundance or insuffi-
ciency. Two of the key hormones are leptin
and ghrelin, which act as signals at either end
of the spectrum (Zigman and Elmquist 2003).
Leptin is secreted by adipocytes in response to
increased food intake and energy balance. Its
action on the brain and peripheral tissues
results in activation of pathways suppressing
food intake and increasing energy expenditure
(Friedman and Halaas 1998). Ghrelin is
released from endocrine cells in the stomach
in response to decreased food intake and has
the opposite effect to leptin (Gualillo et al.
2003). A definitive role for leptin in the onset
of puberty has not yet been demonstrated
(Ahima et al. 1977; Cunningham et al. 1999),
but the importance of energy balance in sexual
development led us to consider whether the
effects described previously (Odum et al.
2001) were associated with the metabolizable
energy (ME) of the diets/formulas evaluated
and hence energy intake during the prepuber-
tal period. However, the range of the ME
densities of the diets used was small, and no
useful correlation was found (Odum et al.
2001). Subsequently, Thigpen et al. (2002,
2003) evaluated several proprietary rodent
diets containing phytoestrogens and with a
wider range of ME densities. They observed a
primary correlation of the phytoestrogen level
of the diet, and a secondary correlation of the
Address correspondence to J. Ashby, Syngenta
Central Toxicology Laboratory, Alderley Park,
Macclesfield, Cheshire, SK10 4TJ UK. Telephone:
44-0-1625-512833. Fax: 44-0-1625-590249.
E-mail: john.ashby@syngenta.com
The authors are employed by Syngenta and Harlan
Teklad.
Received 17 February 2004; accepted 21 July 2004.
Cumulative Dietary Energy Intake Determines the Onset of Puberty
in Female Rats
Jenny Odum,1Helen Tinwell,1Graham Tobin,2and John Ashby1
1Syngenta Central Toxicology Laboratory, Macclesfield, Cheshire, United Kingdom; 2Harlan Teklad UK, Bicester, Oxfordshire,
United Kingdom
Laboratory animal diets for studies to determine the endocrine-disrupting potential of chemicals
are under scrutiny because they can affect both assay control values and assay sensitivity. Although
phytoestrogen content is important, we have previously shown that a phytoestrogen-rich diet and a
phytoestrogen-free diet were equally uterotrophic to rats and advanced vaginal opening (VO) when
compared with the standard diet RM1. Abolition of the effects by the gonadotrophin-releasing hor-
mone antagonist Antarelix indicated that these effects were mediated through the hypothalamus–
pituitary–reproductive organ axis. In the present study, we investigated the relationship between
cumulative energy intake and sexual maturation in female rats. Infant formula (IF) at different con-
centrations and synthetic diets, with a wide range of metabolizable energy (ME) values, were used
to modulate energy intake. Increasing energy intake was associated with an increase in uterine
weight (absolute and adjusted for body weight) for both IF and the synthetic diets. In both cases,
the increased uterine weight was directly proportional to energy intake. Body weight was unaffected
by IF consumption but, in the case of the diets, was increased proportionally with energy consump-
tion. Antarelix abolished the uterine weight increases with both formula and the diets, whereas
body weight was unaffected. The mean day of VO was also advanced by high-ME diets and IF,
whereas body weight at VO was unaffected. VO occurred at an energy intake of approximately
2,300 kJ/rat determined by measuring total food intake from weaning to VO, indicating that this
cumulative energy intake was the trigger for puberty. ME is therefore a critical factor in the choice
of diets for endocrine disruption studies. Key words: energy intake, metabolizable energy, phyto-
estrogens, puberty, soy, uterotrophic assay. Environ Health Perspect 112:1472–1480 (2004).
doi:10.1289/ehp.7039 available via http://dx.doi.org/ [Online 21 July 2004]

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ME density of the diet, with the uterotrophic/
VO activity of the diet to immature mice.
However, food intake by the mice was not
monitored, and this precluded accurate assess-
ments of energy intake. Further, the analysis
was complicated by studying concomitant dif-
ferences in both dietary phytoestrogen levels
and dietary ME values.
In the present experiments, we have inves-
tigated the relationship between total (cumu-
lative) energy intake and sexual maturation in
female rats. Two types of dietary modification
were used. In one, IF (at different concentra-
tions) and sugar solutions were used to modu-
late metabolic energy intake. In the second,
open-formula synthetic phytoestrogen-free
diets, with a wide range of metabolizable
energies (8–22 kJ/g), were evaluated. Some
experiments were conducted in the presence
and absence of ANT to evaluate of the role of
the hypothalamus–pituitary–reproductive
organ axis on the effects observed.
Materials and Methods
Chemicals. DES (> 99% pure), glucose,
sucrose, and arachis oil (AO) were obtained
from Sigma Chemical Co. (Poole, Dorset,
UK). ANT was a gift from Europeptides, a
Division of Asta Medica (Argenteuil, France).
GEN was obtained from ChemService (West
Chester, PA, USA). Halothane anesthetic was
obtained from AstraZeneca (Alderley Park,
Cheshire, UK).
Animals. Alpk:APfSD (Wistar-derived)
rats, obtained from the AstraZeneca breeding
unit (Alderley Park, Macclesfield, UK), were
used in all studies. Studies were performed in
accordance with the U.K. Animals (Scientific
Procedures) Act (1986). Animal care and
procedures were carried out according to
in-house standards as described previously
(Odum et al. 2001).
In the uterotrophic assays with IF and glu-
cose, we used rats that were postnatal days
(PND)21–22 on arrival into the laboratory
(where birth is PND0). In the uterotrophic
assays with the synthetic diets, we used wean-
ling rats on PND18–19. This was because the
former studies were carried out using the speci-
fications described by Odum et al. (1997), and
the latter studies followed the specifications
required by the OECD evaluation of the
uterotrophic assay (Kanno et al. 2003a,
2003b). Control uterine blotted weights for
both series of studies were similar, generally
between 20 and 30 mg. The sexual maturation
study with IF was carried out in weanling rats
on PND21–22 on arrival in the laboratory,
whereas the study with the synthetic diets used
weanling rats that were PND18–19 on arrival.
To avoid confounding effects due to litter-
mates or initial body weights, the weanling rats
were taken from multiple litters and were ran-
domly allocated to groups such that the initial
group mean body weights were similar within
experiments. In all experiments, animals were
weaned on RM3 diet (Special Diet Services
Ltd., Witham, Essex, UK) in the breeding unit
and then fed the appropriate test diet upon
arrival at the laboratory and for the duration of
the assay. All solid diets, fluid diets, and drink-
ing water solutions were available ad libitum.
IF and sugar drinks. IF (Infasoy; Cow
and Gate, Trowbridge, Wiltshire, UK) was
purchased from several outlets in Cheshire
and Staffordshire (UK). It was prepared
according to manufacturer instructions using
sterile deionized water (considered as 100%
strength throughout). The basic constituents
are shown in Table 1. In one study, a dilute
solution (33% recommended strength) and a
more concentrated solution (200%) of IF
were used. A glucose (6.6% wt/vol) solution
in water was similarly prepared and evaluated.
All drinking water solutions were prepared
and replaced on a daily basis.
Diets. Two proprietary natural ingredient
diets, Rat and Mouse No. 3 (RM3) and Rat
and Mouse No. 1 (RM1) were supplied by
Special Diet Services Ltd. (Witham, Essex,
UK). RM1 has been consistently used as the
standard diet in our postweaning studies since
1997 (Odum et al. 2001). A series of open-
formula synthetic diets with a range of MEs
(diets A–E) were produced by Harlan Teklad
UK (Bicester, Oxfordshire, UK) and were
based on AIN-76A (Knapka 1983). The con-
stituents and proportions for all diets used, as
well as the unique Harlan Teklad reference
numbers for diets A–E, are listed in Table 1.
AIN-76A and RM1 were included to provide
links to our previous findings (Odum et al.
2001). In order to derive a wide range of ME
densities, a base diet (designated diet B) was
created. Diets with increasing ME densities
were then achieved by substituting increasing
proportions of lard for cellulose (diets C–E).
A diet with an additional decrease in ME
(diet A) was obtained by reducing the propor-
tion of sucrose and maltodextrin. All diets
were prepared as pellets.
We estimated ME densities of the syn-
thetic diets using the values for protein, fat,
and carbohydrate given by Blaxter (1989).
The figure for casein protein takes into
account the fact that it contains 10% mois-
ture and 1% fat. The energy in the minerals
and vitamins was derived from the excipients.
Protein, vitamins, and fatty acids were main-
tained at a constant level in all the diets. The
diets lowest in fat contained sufficient essen-
tial fatty acids to meet normal dietary require-
ments. The values for RM1 and IF were as
reported by the manufacturer (Table 1). Total
ME intake over the duration of the studies
was calculated from solid and liquid food
Article|Energy intake determines puberty onset in female rats
Environmental Health Perspectives • VOLUME 112 | NUMBER 15 | November 2004
1473
Table 1. Composition and ME content of the diets.
Diets A–E (%)
B
01364
20
32.5
15
25
2.5
3.5
1
0.3
0.2
0.001
20
RM1a
IF (Infasoy)b
AIN-76AACDE
Constituent
Wheat/barley/wheat
middlings
Soybean meal
Whey powder
Soy oil
Minerals
Vitamins
Amino acids
g/100 g
88.5
Constituent
Glucose syrup
Carbohydrates
Vegetable oils
Fat
Soy protein isolate
Minerals
Vitamins
g/100 mL
NS
6.7
NS
3.6
NS
0.4
Constituent
Casein
Sucrose
Corn starch
Cellulose
Corn oil
Minerals
Vitamins
DL-Methionine
Choline
Ethoxyquin
Total protein
content (% wt/wt)
Total MEd
(kJ/g diet)
g/100 g
20
50
15
5
5
3.5
1
0.3
0.2
0.001
20
Constituent
Casein
Sucrose
Maltodextrin
Cellulose
Lard
Minerals
Vitamins
DL-Methionine
Choline
Ethoxyquin
Total protein
(% wt/wt)
Total MEd
(kJ/g diet)
02171c
20
17.5
5
50
2.5
3.5
1
0.3
0.2
0.001
20
01365
20
32.5
15
13.75
13.75
3.5
1
0.3
0.2
0.001
20
02332
20
32.5
15
2.5
25
3.5
1
0.3
0.2
0.001
20
01366
20
27.5
15
0
32.5
3.5
1
0.3
0.2
0.001
20
6.0
2.5
0.5
2.5
Total protein content
(% wt/wt)
Total ME
(kJ/g diet)
14.7Total protein
content (% wt/vol)
Total ME
(kJ/g diet)
1.8
10.9 2.815.7 8.212.1 16.220.3 22.3
aAll values for RM1 are as stated on the manufacturer’s data sheet. bMajor constituents as stated on the Infasoy packaging; the quantities of glucose syrup, vegetable oils, and soy pro-
tein isolate were not specified (NS), but proportions of carbohydrates, fat, and protein were given. cUnique Harlan Teklad reference numbers of the synthetic diets. dME was calculated
using the following values (kJ/g constituent): casein, 16 kJ/g; sucrose, 16 kJ/g; corn starch, 16 kJ/g; maltodextrin, 16 kJ/g; cellulose, 0.3 kJ/g; corn oil, 37 kJ/g; lard, 37 kJ/g; minerals,
1.9 kJ/g; vitamins, 15.7 kJ/g; DL-methionine, 17 kJ/g; choline, 0 kJ/g; ethoxyquin, 0 kJ/g. The composition of the synthetic diets A–E was based on that of AIN-76A such that the protein
content was identical but the carbohydrate and fat content were adjusted to give varying total ME values.

Page 3

consumption data and the ME content of the
diets and drinks.
Analysis of diets for phytoestrogens. We
analyzed IF for daidzein and GEN content
using the method described previously by
Odum et al. (2001) and Owens et al. (2003).
The limits of detection were 0.1 µg/g diet.
The phytoestrogen aglycone contents of diet B
(as representative of the phytoestrogen-free
synthetic diets A–E) and RM1 were deter-
mined as described in detail by Wiseman et al.
(2002). Portions of the diet (200 mg) were
extracted by shaking with aqueous methanol
at 60°C for 1 hr. The extracts were defatted
with hexane and hydrolyzed to the aglycones
with dilute hydrochloric acid. The aglycones
were then extracted with ether. Daidzein,
GEN, glycitein, and coumestrol were detected
and quantified against reference samples by
liquid chromatography coupled with mass
spectroscopy. Data were adjusted for extrac-
tion efficiency. Quality control was deter-
mined by the concurrent analysis of a soy flour
of known daidzein and GEN content, and
results were < 9% different from those
expected. The limit of detection was 0.05 µg/g
diet for daidzein, GEN, and glycitein and
0.1 µg/g diet for coumestrol.
Animal studies. In all experiments, wean-
ling rats were fed IF or synthetic diets upon
arrival in the laboratory. Uterotrophic assays
were based on the protocol described by
Kanno et al. (2003a, 2003b) where the basic
end point is uterine weight. In the sexual
maturation studies, dietary modulation con-
tinued from weaning to postpuberty, and end
points related to puberty (e.g., VO) were
monitored. A scheme of the experiments and
the hypotheses that they were designed to
address are shown in Table 2.
Uterotrophic assays. Uterotrophic assays
were conducted using IF at different concen-
trations selected to provide a concentration-
dependent response (experiments 1 and 2). In
experiment 3, we administered a 6.6% glu-
cose solution, either alone or in addition to
coadministration of 5 mg/kg body weight
GEN. In these experiments, the normal drink-
ing water supply was replaced with either IF or
glucose solutions. RM1 was provided as an
optional solid food. Control rats were fed
RM1 and water. Three uterotrophic assays
were conducted using the pelleted synthetic
diets (experiment 4 of 4 days’ duration and
experiments 5 and 6 of 6 days’ duration).
Control rats were fed RM1. Rats were housed
up to five per cage. Food and fluid were avail-
able ad libitum and monitored (by cage) daily.
In experiments 1, 4, and 6, the GnRH
antagonist ANT was coadministered at a dose
of 300 µg/kg/day by subcutaneous (sc) injec-
tion (dosing volume, 1.5 mL/kg; Odum et al.
2001). In experiment 3, GEN was coadminis-
tered orally at 5 mg/kg/day in AO (dosing vol-
ume, 5 mL/kg). DES was used as a positive
control in all studies. In experiments 1–3,
DES was administered in the drinking water
at either 10 or 20 µg/L, starting on the day of
arrival of the rats at the laboratory and contin-
uing throughout the experiment. In experi-
ments 4–6, it was administered by sc injection
at 5 µg/kg/day with a dosing volume of
1.5 mL/kg. Some animals received both DES
and ANT or DES and AO, administered by
two successive sc injections made within
5 min of each other. Rats administered DES
were fed RM1. Control animals received vehi-
cle only. Dosing of compounds (by sc or oral
routes) commenced on the day after the rats
had been placed on the test diets and contin-
ued daily. Animals were killed by an overdose
of halothane 24 hr after the final chemical
administration. Uteri were removed, blotted,
and weighed as described previously (Odum
et al. 1997).
Sexual maturation studies. Weanling rats
were provided with IF solutions, in place of
drinking water supply, and RM1 diet in
experiments 7 and 8. In experiment 9, wean-
ling rats were fed either diet B or diet D
instead of RM1. Control animals were fed
RM1 in all experiments. Experiment 7 also
contained a group of “heavy” control animals
consisting of a group of the heaviest animals
selected from the required weight range.
Consequently, in the sexual maturation stud-
ies, the initial weights of the standard RM1
control group and the IF group were similar,
whereas the weight of the “heavy control
group” was greater. DES (30 µg/L in the
drinking water) was administered in experi-
ment 9 as a positive control with RM1 as the
diet. This concentration of DES has previ-
ously been shown to decrease the mean age at
VO by 7 days in the absence of changes in
body weight (Odum et al. 2002). All diets and
drinking water were available ad libitum. Rats
in experiment 7 were housed singly, whereas
rats in experiments 8 and 9 were housed in
groups of five. Food and fluid consumption
Article|Odum et al.
1474
VOLUME 112 | NUMBER 15 | November 2004 • Environmental Health Perspectives
Table 2. Experimental scheme and hypotheses.
Experiment
Uterotrophic studies
Experiment 1
HypothesisTreatmentDuration
IF consumption increases uterine weight
ANT antagonizes IF-induced uterine weight
increase
ANT does not antagonize DES-induced uterine
weight increasec
IF-induced uterine weight increase is dependent
on IF concentration
Glucose and GEN increase uterine weight
Consumption of synthetic diets with higher ME
than RM1 increases uterine weight over 4 days
ANT antagonizes synthetic diet-induced uterine
weight increase
ANT does not antagonize DES-induced uterine
weight increase
Consumption of synthetic diets with higher ME
than RM1 gives greater uterine weight
increase over 6 days
Consumption of synthetic diets with low–high
ME range shows correlation of ME with
uterine weight
ANT antagonizes synthetic diet-induced uterine
weight increase
ANT does not antagonize DES-induced uterine
weight increase
Sexual maturation studies
Experiment 7 IF consumption reduces age at VO
Age-matched heavy controls have earlier VO
Experiment 8 IF consumption reduces age at VO and age at
first and second estrus
Energy intake after weaning determines age
at VO
Experiment 9 Consumption of synthetic diets with higher
ME than RM1 reduces age and body weight
at VO
Energy intake after weaning determines age
at VO
DES treatment reduces age and body weight
at VOd
Experiment 9 Consumption of synthetic diets affects organ
weight
IF, ANT,aDESb
4 days (PND21–25)
Experiment 2IF (33–200%), DES4 days (PND21–25)
Experiment 3
Experiment 4
Glucose, GEN, DES
Synthetic diets,
ANT, DES
4 days (PND21–25)
4 days (PND18–22)
Experiment 5Synthetic diets, DES6 days (PND18–24)
Experiment 6Synthetic diets,
ANT, DES
6 days (PND18–24)
IF 20 days (PND21–41)
IF 97 days (PND21–118)
Synthetic diets,
ANT, DES
23 days (PND18–41)
Synthetic diets,
DES
23 days (PND18–41)
aANT is a GnRH antagonist used to determine whether GnRH mediates uterine weight increases. bDES was used through-
out as a positive control. cAs demonstrated previously (Ashby et al. 2000). dAs demonstrated previously (Odum et al. 2002).

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were monitored daily. VO was monitored
daily from PND21, and individual body
weights on the day of VO were recorded.
The age at first and second estrus were
determined in experiment 8 by analysis of daily
vaginal smears that were taken from the day of
VO to PND65. First estrus was defined as the
first day on which only cornified epithelial cells
were observed on the vaginal smear. Second
estrus was defined as the day on which a smear
indicating estrus fell within a run of smears
clearly showing the correct cyclic sequence of
proestrus, estrus, metestrus, and diestrus.
Animals were killed on PND41, when all
animals had open vaginas (experiments 7
and 9), or PND118, after second estrus
(experiment 8). Liver, kidney, and uterine
weights were determined at necropsy.
Statistical methods. For uterotrophic
assays, we analyzed uterine weights by covari-
ance with the terminal body weights. Terminal
body weights were analyzed by covariance with
initial body weights. Differences from control
values (RM1 or RM1 with AO, as appropriate)
were assessed statistically using a two-sided
Student’s t-test based on the error mean square
from the analysis of covariance (ANCOVA).
Relationships between energy intake and body
or uterine weight were analyzed by linear
regression.
For sexual maturation studies, analysis of
variance (ANOVA) was carried out on body
weights, food consumption, and organ weights.
Organ weights were also analyzed by covariance
with the terminal body weights (Shirley 1996).
VO was analyzed by Fisher’s exact test on the
proportions of animals recorded each day with
VO and by ANOVA for the observed days of
VO and body weights at the time of VO.
Differences from control values in all cases
were assessed statistically using a two-sided
Student’s t-test based on the error mean square
from the ANOVA or ANCOVA. Analyses
were carried out with SAS software (Version 8;
SAS Institute, Inc., Cary, NC, USA).
Results
Diet analyses. The synthetic diets A–E were
free from daidzein, GEN, glycitein, and
coumestrol. RM1 contained low levels of the
phytoestrogens daidzein, GEN, and glycitein
(11, 9, and 2 µg/g diet, respectively) and non-
detectable levels of coumestrol. IF contained
45.7 µg daidzein and 133.4 µg GEN per gram
dry formula (glycitein and coumestrol were
not analyzed). ME values for the diets and IF
are shown in Table 1.
Uterotrophic assays. In experiments 1
and 2 (Table 3), IF gave a positive uterotrophic
response except when 33% IF was used
(experiment 2, Table 3). All increases in uter-
ine weight (compared with RM1 controls)
occurred without significant effects on final
body weights, except for the 200% IF group
(Table 3). Energy intake was also increased
above the RM1 controls in animals consuming
IF. In experiment 2, uterine weight was
increased proportionally with increasing IF
concentration and energy intake (Figure 1).
Coadministration of the GnRH antagonist
ANT abolished the uterine weight increases
induced by IF but did not affect the response
given by DES (experiment 1, Table 3;
Figure 2).
Administration of a solution of glucose to
rats in 4-day uterotrophic assays (experiment 3,
Table 3) had no effect on uterine weight. The
concentrations were chosen based on the pres-
ence of 6.6% glucose in IF. Uterine weight was
also unaffected by GEN at 5 mg/kg/day; this
was the calculated daily intake of isoflavone in
human infants consuming IF (at 100% con-
centration). The lack of effect is as expected
from the dose response of GEN in the
uterotrophic assay (Kanno et al. 2003a).
The results of the uterotrophic assays with
the synthetic diets are shown in Tables 4
and 5. In all cases, uterine wet weight increased
as energy intake increased in animals fed the
synthetic diets. Body weights also increased,
but uterine weights adjusted for covariance
with terminal body weights were still increased.
In experiment 4, rats were fed diets B–D
and AIN-76A for 4 days, and the energy con-
tent of the synthetic diets ranged from 12.1 to
20.3 kJ/g. Absolute and adjusted uterine weight
was significantly increased from 21 mg (RM1
control) to a maximum of approximately
35 mg by all the synthetic diets. The increase
was abolished by coadministration of the
GnRH antagonist ANT, when all diet groups
attained absolute uterine weights of 17–19 mg
(Table 4, Figure 2). Coadministration of ANT
to the DES group had no effect on uterine
weight (Figure 2). In experiment 5, the dura-
tion of the experiment was increased to 6 days
in an attempt to enhance the sensitivity of the
assay. Absolute and adjusted uterine weights
for rats consuming diets B–D were signifi-
cantly increased to a maximum of approxi-
mately 48 mg, and energy consumption was
increased concomitantly (Table 5). In experi-
ment 6, two more diets (diets A and E) were
evaluated, expanding the ME range to
Article|Energy intake determines puberty onset in female rats
Environmental Health Perspectives • VOLUME 112 | NUMBER 15 | November 2004
1475
Table 3. Immature rat uterotrophic assays with IF and sugar drinks (experiments 1–3).
Total energy
intake (kJ)a/rat
Percent energy
intake as drink
Absolute uterine
blotted weight (mg)b
Adjusted uterine
blotted weight (mg)c
Final body
weight (g)b
Experiment/treatment
Experiment 1
RM1
RM1, IF 100%
RM1, DES 10 µg/L
RM1, ANT
RM1, IF 100%, ANT
RM1, DES 10 µg/L, ANT
Experiment 2
RM1
RM1, IF 33%
RM1, IF 100%
RM1, IF 200%
RM1, DES 10 µg/L
Experiment 3
RM1, AO
RM1, glucose 6.6%
RM1, GEN 5 mg/kg/day
RM1, glucose 6.6%,
GEN 5 mg/kg/day
RM1, DES 20 µg/L
No.
238
416
243
237
431
254
020.7 ± 2.8
29.9 ± 4.9**
41.8 ± 11.9**
16.3 ± 1.5
16.9 ± 1.1
39.7 ± 13.8#
20.4
29.8**
41.6**
16.0
16.7
39.5#
52.3 ± 4.5
53.8 ± 3.3
53.8 ± 4.1
52.7 ± 4.1
56.1 ± 3.7
52.8 ± 4.4
10
10
10
10
10
10
88
0
0
89
0
311
341
464
547
321
0 27.3 ± 4.6
31.5 ± 5.2
40.7 ± 11.6**
44.7 ± 16.2**
40.7 ± 6.0**
28.0
32.4
40.5**
43.3**
39.3**
62.1 ± 5.4
61.8 ± 5.8
63.3 ± 5.2
64.9 ± 5.3*
65.0 ± 4.5
9
22
62
75
0
10
10
10
5
222
333
ND
253
0 22.2 ± 5.8
22.0 ± 6.0
21.3 ± 2.4
23.9 ± 5.9
23.3
22.7
21.1
24.8
54.5 ± 6.4
55.2 ± 7.2
56.6 ± 7.4
54.8 ± 7.3
9
9
9
9
19.6
0
22.6
239073.9 ± 15.3** 73.0**57.8 ± 6.49
ND, not determined. DES was administered in drinking water.
aTotal energy intake was calculated from the total amount of liquid and solid food consumed per rat over the duration of the study and their MEs. The ME value for RM1 was taken from
the manufacturer’s data sheet; the ME value of IF was taken from information supplied by the manufacturer and adjusted for concentration where necessary; and the ME value of
16 kJ/g for glucose/sucrose was adjusted for concentration. bMean ± SD. cUterine weights adjusted for covariance with terminal body weights. *p < 0.05 and **p < 0.01 compared with
RM1 or RM1/AO control. #p < 0.01 compared with RM1/ANT control.

Page 5

8.2–22.3 kJ/g. Diets were also fed for 6 days.
The body weight curves (Figure 3) display a
clear relationship between increasing energy
intake and body weight, with body weights of
animals fed diets B, C, D, and E being signifi-
cantly increased relative to the RM1 control.
Total energy intake over the 6 days was pro-
portional to the ME of the diets (Figure 4).
Coadministration of ANT had no effect on
body weight (Table 5). Absolute and adjusted
uterine weights were again significantly
increased in animals fed the synthetic diets
B–E compared with those fed RM1. In ani-
mals receiving diet A, with the lowest ME,
absolute uterine weight was not significantly
increased, but the increase in adjusted uterine
weight was significant. A plateau was reached at
absolute uterine weights of approximately
50–55 mg, suggesting that this may be the limit
of prepubertal stimulation of uterine growth by
manipulation of energy intake (Table 5). ANT
again abolished the increases in uterine growth,
reducing all absolute uterine weights to
16–19 mg (Table 5). The relationship between
energy intake and either final body weight,
absolute uterine weight, or uterine weight
adjusted for body weight, for the data from
experiment 6 (Table 5), was analyzed by linear
regression (Figure 5).
In the uterotrophic assays with synthetic
diets, the SDs for uterine weights were gener-
ally at least double those obtained with RM1.
The reason for this is not clear. When ANT
was coadministered, the SDs for uterine weight
were smaller and less variable. We carried out
an experiment in which rats were fed diet D
under conditions of both single and group
housing—in case competition for food within
the cage was a factor—but SDs in both cases
were similarly large (data not shown). We also
attempted to reduce the uterine weight of rats
fed diet D to that of rats fed diet B by restrict-
ing food (and therefore caloric) intake. The
restriction achieved, however, was only partial
because the animals ate their allocated amount
of diet D so quickly that they would have been
without food for long periods of the night.
This was considered to be unacceptable for our
animal license, and therefore the rats were
given more food. A total energy intake of
Article|Odum et al.
1476
VOLUME 112 | NUMBER 15 | November 2004 • Environmental Health Perspectives
Figure 1. Total energy intake for rats drinking IF
(33%, 100%, or 200% solutions) shown plotted
against the increase in uterine weight above con-
trol levels (RM1 diet and water; all animals had
access to RM1 diet). R2= 0.99, p < 0.01. Data are
based on experiment 2 (Tables 2 and 3).
aUterine weight increase not significant. **Uterine weight
increase significant at p< 0.01
Figure 2. The effect of ANT (0.3 mg/kg/day, sc) on
adjusted blotted uterine weights of rats fed IF or
synthetic diets or dosed with DES [10 µg/L in drink-
ing water (experiment 1) or 5 µg/kg/day sc (experi-
ment 4)] in 4-day immature rat uterotrophic assays
(Tables 3 and 4, respectively). Values are ANCOVA-
adjusted means.
**p < 0.01 compared with RM1 control. #p < 0.01 compared
with RM1/ANT control.
18
16
14
12
10
8
6
4
2
0
0 1020 304050 60 7080
Adjusted uterine blotted weight
of test – adjusted uterine
blotted weight of control (mg)
Percent energy intake as drink
33% IFa
100% IF**
200% IF**
140
120
100
80
60
40
20
0
RM1
Adjusted uterine
blotted weight (mg)
IF
100%
RM1/
DES
10 µg/L
RM1 Diet B AIN-
76A
Diet C Diet D RM1/
DES
5 µg/L
Experiment 4
**
**
**
**
**
**
**
– Ant
+ Ant
#
#
Experiment 1
Table 5. Immature rat uterotrophic assays (6 days’ duration) using synthetic diets of different ME content
(experiments 5 and 6).
Diet ME
intake
(kJ/g diet)a
Total energy
intake
(kJ)b/rat
Absolute
uterine blotted
weight (mg)c
Adjusted
uterine blotted
weight (mg)d
Final body
weight (g)c
Experiment/treatment
Experiment 5
RM1
Diet B
AIN-76A
Diet C
Diet D
RM1/AO
RM1/DES 5 µg/kg
Experiment 6
RM1/AO
Diet A/AO
Diet B/AO
Diet C/AO
Diet D/AO
Diet E/AO
RM1/DES 5 µg/kg
RM1/ANT
Diet A/ANT
Diet B/ANT
Diet C/ANT
Diet D/ANT
Diet E/ANT
RM1/DES 5 µg/kg/ANT
No.
10.9
12.1
15.7
16.2
20.3
10.9
10.9
485
483
696
666
907
471
530
27.6 ± 3.1
36.6 ± 6.7*
45.0 ± 12.4**
44.7 ± 7.2**
47.5 ± 8.3**
30.4 ± 4.0
131.2 ± 18.0**
31.0
40.2**
42.1**
42.9**
42.0**
38.1
133.9**
57.7 ± 6.5
57.5 ± 5.1
66.4 ± 5.1**
64.9 ± 5.8**
70.0 ± 6.4**
51.8 ± 8.2**
60.3 ± 5.6**
10
10
10
10
10
10
4
10.9
8.2
12.1
16.2
20.3
22.3
10.9
10.9
8.2
12.1
16.2
20.3
22.3
10.9
520
426
555
897
1,010
481
434
493
408
816
314
991
1,131
465
26.4 ± 5.3
33.2 ± 6.7
39.7 ± 7.8**
50.8 ± 16.2**
55.6 ± 18.6**
50.9 ± 15.4**
122.4 ± 17.2**
16.1 ± 1.2
17.4 ± 1.5
18.6 ± 2.1
19.2 ± 1.5
18.4 ± 1.9
19.1 ± 2.6
153 ± 20.8#
29.3
36.9*
39.8**
49.0**
51.9**
48.0**
124.6**
20.1
22.6
21.5
14.9
15.6
14.7
154.5#
62.3 ± 5.4
61.0 ± 6.8
66.5 ± 7.0*
69.3 ± 5.0**
72.2 ± 5.5**
71.0 ± 4.1**
60.7 ± 6.3
60.6 ± 4.6
58.9 ± 5.6#
62.3 ± 8.6
72.9 ± 6.3#
70.9 ± 5.1#
73.1 ± 6.9#
58.7 ± 2.7
10
10
10
10
10
10
4
10
10
10
10
10
10
4
DES was administered subcutaneously.
aThe ME value for RM1 was taken from the manufacturer’s data sheet. bTotal energy intake was calculated as the prod-
uct of the total amount of food consumed per rat over the duration of the study and the ME of the diet. cMean ± SD.
dUterine weights adjusted for covariance with terminal body weights. *p < 0.05 and **p < 0.01 compared with RM1 or
RM1/AO control. #p < 0.01 compared with RM1/ANT control.
Table 4. Immature rat uterotrophic assays (4 days’ duration) using synthetic diets of different ME content
(experiment 4).
Diet ME
intake
(kJ/g diet)a
10.9
12.1
15.7
16.2
20.3
10.9
10.9
12.1
15.7
16.2
20.3
10.9
Total Absolute
uterine blotted
weight (mg)c
21.4 ± 3.2
29.2 ± 7.4**
35.8 ± 6.4**
34.7 ± 9.1**
34.4 ± 5.6**
105.1 ± 3.4**
17.1 ± 1.9
17.3 ± 2.4
17.8 ± 1.2
18.1 ± 2.0
18.8 ± 2.2
119.2 ± 7.3#
Adjusted
uterine blotted
weight (mg)d
22.4
30.7**
34.9**
34.5**
32.5**
106.1**
18.5
18.8
17.0
18.3
16.9
120.6#
energy intake
(kJ)b/rat
222
243
325
316
218
434
222
227
336
314
422
232
Final body
weight (g)c
51.2 ± 7.2
50.0 ± 8.0
55.8 ± 8.0**
54.0 ± 7.8**
58.2 ± 7.6**
51.3 ± 6.6
50.0 ± 6.8
49.8 ± 7.8
55.6 ± 6.4#
53.2 ± 8.3#
58.3 ± 6.5#
49.6 ± 7.5
Treatment
RM1/AO
Diet B/AO
AIN-76A /AO
Diet C/AO
Diet D/AO
RM1/DES 5 µg/kg
RM1/ANT
Diet B/ANT
AIN-76A /ANT
Diet C/ANT
Diet D/ANT
RM1/DES 5 µg/kg/ANT
No.
10
10
10
10
10
4
10
10
10
10
10
4
ND, not determined. DES was administered sc.
aThe ME value for RM1 was taken from the manufacturer’s data sheet. bTotal energy intake was calculated as the prod-
uct of the total amount of food consumed per rat over the duration of the study and the ME of the diet. cMean ± SD.
dUterine weights adjusted for covariance with terminal body weights. **p < 0.01 compared with RM1 or RM1/AO control.
#p < 0.01 compared with RM1/ANT control.