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Influence of estradiol on cortisol secretion in ovariectomized cynomolgus macaques (Macaca fascicularis).

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American Journal of Primatology 60:17–22 (2003)
Influence of Estradiol on Cortisol Secretion in
Ovariectomized Cynomolgus Macaques (Macaca
and J.R. KAPLAN3,5
Section of Neurobiology, School of Biological Sciences, University of Texas, Austin, Texas
Department of Psychology, University of Southern Mississippi, Hattiesburg, Mississippi
Department of Comparative Medicine, Wake Forest University School of Medicine,
Winston-Salem, North Carolina
Department of Psychology, University of Pittsburgh, Pittsburgh, Pennsylvania
Department of Anthropology, Wake Forest University, Winston-Salem, North Carolina
In an investigation of cortisol secretion in fully mature, ovariectomized
cynomolgus monkeys (Macaca fascicularis), we compared monkeys that
were given either placebo (OVX, n = 26) or 17b estradiol (E2 ) (EST, n =
26) in a daily oral dose. Serum cortisol concentrations were measured
prior to the experimental manipulation and 3, 6, 9, and 12 months
following initiation of treatment. Pretreatment cortisol values did not
differ between groups. Assessment of the treatment period values
revealed that cortisol concentrations were significantly higher (E10%)
in the EST than in the OVX monkeys. Cortisol also varied significantly
across periods of sampling. This time-dependent variation was attributable to elevations in months 6 and 9 (when daylight was generally long),
relative to months 3 and 12 (when daylight was relatively short). The
modest stimulatory effect of estrogen on corticosteroid production
observed in this study is consistent with what has been seen in women,
and contrasts with the more robust effects observed in New World
monkeys. The possible relationship between season and cortisol secretion
observed here has not been previously described in monkeys. Am. J.
Primatol. 60:17–22, 2003.
r 2003 Wiley-Liss, Inc.
Key words: reproductive condition; ovariectomy; cortisol; female
Contract grant sponsor: NIH; Contract grant numbers: HL 45666; HL 40962; Contract grant
sponsor: Novo Nordisk A/S, Målöv, Denmark.
Correspondence to: Ronda Stavisky, Section of Neurobiology, School of Biological Sciences,
University of Texas, Austin, TX 78712. E-mail:
Received 22 February 2002; revision accepted 17 March 2003
DOI 10.1002/ajp.10076
Published online in Wiley InterScience (
2003 Wiley-Liss, Inc.
18 / Stavisky et al.
The activation of the hypothalamic-pituitary-adrenocortical (HPA) axis and
the subsequent release of cortisol in response to physical or emotional stress
comprise one of the most robust observations in socioendocrinology [Tsigos &
Chrousos, 2002]. However, the relationship between the HPA axis and the
hypothalamic-pituitary-gonadal (HPG) system is not unidirectional. In primates,
for example, an extensive literature indicates that ovarian hormones, particularly
estrogen, stimulate HPA activity as measured by increases in cortisol secretion.
New World monkeys seem particularly responsive to estrogenic stimulation
of the HPA axis. In many of these species, hyperestrogenic states, such as
pregnancy or the ovulatory portion of the menstrual cycle, are accompanied by
large increases in circulating concentrations of cortisol. Conversely, ovariectomy
(a hypoestrogenic state) reduces cortisol secretion [Coe et al., 1986; Saltzman
et al., 1998]. Both estrogenic stimulation and ovariectomy appear to exert more
modest effects in Old World monkeys, especially macaques [Smith & Norman,
1987; Xiao et al., 1994]. Similarly, studies in women indicate that estrogen
supplementation (e.g., hormone replacement therapy) moderately stimulates
corticosteroid production [Fonseca et al., 2001], while estrogen deficiency (due to
menopause or ovariectomy) has no consistent effect [Fonseca et al., 2001].
Although the aggregate evidence from women and monkeys suggests that
ovarian hormones exert a stimulatory effect on the primate HPA axis, the
persistence and strength of these effects remain uncertain. Investigations
involving women are generally small and short-term, and in any event the
effects of aging are not easily disentangled from those of hormone exposure.
Among nonhuman primates, study design varies widely. Most studies of
macaques have focused on individually housed or heavily instrumented animals
[Smith & Norman, 1987; Xiao et al., 1994], while those involving New World
monkeys have generally employed social housing and heterosexual grouping [Coe
et al., 1986; Saltzman et al., 1998]. As with studies of women, treatment periods
are generally short and treatment groups are relatively small.
To further investigate the influence of ovarian function on HPA activity in
Old World monkeys, we evaluated cortisol levels in 52 socially housed cynomolgus
macaques (Macaca fascicularis) that were ovariectomized either with or without
estrogen replacement. These animals were part of a larger study evaluating the
effects of various hormone treatments on bone density and cardiovascular
disease. The current study was thus opportunistic, and took advantage of cortisol
samples that had been collected to assess stress levels. These data, collected both
before treatment and over a 1-year experimental period, allowed us to determine
whether cortisol concentrations differ between ovariectomized, estrogen-replaced
animals and their ovariectomized, nonreplaced controls.
The subjects were 52 female cynomolgus monkeys housed in 10 social groups,
with five or six animals per pen. The pens measured 2 3.2 2.5 m and had
indoor/outdoor access. The animals were imported from Indonesia (Institut
Pertanian Bogor, Bogor, Indonesia). Although their reproductive histories and
exact ages were unknown, all of the animals exhibited radiographic evidence of
epiphyseal closure, which occurs at approximately 9 years of age in this species
[Jayo et al., 1994]. All experimental procedures were done in accordance with
Cortisol Secretion in Female Macaques / 19
state and federal guidelines, and with the approval of our institution’s animal
care and use committee. The Wake Forest University School of Medicine is fully
accredited by the Association for Assessment and Accreditation of Laboratory
Animal Care International.
Eight months after the groups were formed, the animals were divided into
the two treatment conditions focused on in this report: 1) ovariectomized,
placebo-treated (OVX, n=26); and 2) ovariectomized, E2-treated (EST, n=26). All
of the animals in a pen were subjected to the same reproductive manipulation,
and social group composition remained consistent throughout the study. As part
of the larger experimental design, the monkeys consumed a diet relatively high in
fat and cholesterol, which was designed to mimic the diet typically consumed by
North Americans.
Following training, all animals were orally dosed with a flavored placebo
solution on a daily basis. The EST animals received the placebo solution, to which
0.016 mg/kg of E2 had been added. The treatment resulted in mean plasma E2
concentrations of 122.45 pg/ml 7 SEM when averaged across all animals and all
samples in the EST condition. In contrast, the plasma E2 concentrations of the
OVX animals averaged o10 pg/ml.
Blood Collection
At each sample point, serum was collected via femoral puncture over a period
of 2 days. The sampling order was reversed at each time point. For collection of
samples, the animals entered a squeeze cage from their group pens and were
immediately administered ketamine HCl (10 mg/ml, IM), which resulted in rapid
sedation. Previous studies have shown that ketamine does not alter cortisol
concentrations in this species [Castro et al., 1981]. Blood was collected within 4
min after injection of ketamine HCl to reduce the possibility that cortisol levels
would reflect anesthesia effects rather than baseline circulating values. Blood
samples were collected between 09:00 and 11:00 hr (which corresponded to the
regular dosing time).
Cortisol Radioimmunoassay
Cortisol concentrations were determined using a commercial radioimmunoassay kit (Diagnostics Products Corp., Los Angeles, CA). Interassay precision
was 7.99% at 27 mg/dl (60 assays) and 13.20% at 60 mg/dl (55 assays). The
intraassay precision was 6.07%. The range of assay sensitivity was 0.50–50 mg/dl
at 25 ml (normal dose) and 0.83–83.33 mg/dl at 15 ml (normal dose for monkeys). All
of the assays were previously validated for cynomolgus macaques [Castro et al.,
Estradiol Radioimmunoassay
Serum estradiol was analyzed by a modification of commercially available
reagents (Diagnostic Products Corp., Los Angeles, CA) as previously described
[Wilson et al., 1988]. The kit was modified for use in monkeys, using blank
monkey serum (ovx pool or tested low male pool) as diluents for standards made
20 / Stavisky et al.
up in ethanol. The interassay precision was 12.09% at 276 pg/ml (20 assays) and
19.77% at 90.51 pg/ml (20 assays). The intraassay CV was 7.73%. The range of
assay sensitivity was 5–1,000 pg/ml at 200 ml (normal dose).
Statistical Analyses
A one-factor (ConditionOVX,EST) analysis of variance (ANOVA) was used to
test for the presence of significant pretreatment differences in cortisol secretion.
Subsequently, a ConditionOVX,EST TimeS1,S2,S3,S4 ANOVA with repeated
measures was used to assess the effect of hormone treatment on ovariectomized
animals across the 1-year period of study. Tukey’s HSD was then used as to test
for pairwise differences among sample periods. A two-tailed significance level of
0.05 was applied for all evaluations.
Cortisol values were normally distributed. Baseline cortisol concentrations
did not differ across conditions prior to initiation of treatment (OVX: 51.1 7 2.7
SEM mg/dl; EST: 50.1 7 1.6 mg/dl; F[1,50] = 0.13, P = 0.72). Mean values for all the
treatment conditions as seen at 3, 6, 9, and 12 months are depicted in Fig. 1.
Pretreatment values were significantly higher than those observed following
ovariectomy (at 3 months) in both placebo-treated (t[25] = 6.28, Po0.001) and
estradiol-replaced animals (t[25] = 6.28, Po0.001). The repeated-measures
ANOVA applied to the treatment period values revealed significant effects for
Condition (F[1,50] = 10.53, P = 0.002) and Time (F[3,150] = 10.11, Po0.001), with
no significant interaction between these factors (F[3,150]= 1.61, NS). Hence,
cortisol values in the EST condition were modestly (but significantly) higher than
in the OVX condition (Fig. 1). Regarding the Time effect, pairwise comparisons
revealed that across all treatment conditions, cortisol concentrations sampled
during months 6 and 9 were significantly higher than those collected in months 3
and 12 (Po0.01). Although the interpretation of this pattern is unclear, it is
3 months,
10.5 h
6 months
9 months
12 months
Fig. 1. Cortisol secretion mg/dl (7 SEM) across time and treatment condition.
Cortisol Secretion in Female Macaques / 21
consistent with an effect of seasonality, as cortisol was higher when daylight
hours were long (month 6: 13.5 7 0.07 (SEM) hr; month 9: 13.7 7 0.06 hr) than
when daylight was short (month 3: 10.5 7 0.05 hr; month 12: 11.0 7 0.06 hr).
The principal finding reported here is that exogenous estradiol, provided to
ovariectomized macaques noncyclically but in physiologic concentrations,
modestly increased circulating cortisol in comparison to that observed in
untreated, ovariectomized controls. A further observation was that cortisol
secretion varied significantly across time irrespective of hormone condition, an
effect that seemed to occur in relation to the number of daylight hours available
at each sample period.
The observation that chronic exposure to estradiol in physiological
concentrations increased circulating cortisol concentrations is generally consistent with prior studies in nonhuman primates and women. Furthermore, the data
support the prior observation that these effects are less pronounced in women
and macaques than in New World monkeys [Coe et al., 1986; Xiao et al., 1994;
Fonseca et al., 2001]. In this regard, numerous investigators have suggested that
cortisol responses are significantly exaggerated in New World as compared to Old
World monkeys [Chrousos et al., 1982].
Somewhat puzzling was the steep decline in cortisol observed across all
animals (estrogen-replaced or not) from the preexperimental period to the
treatment period, suggesting that ovariectomy may have influenced cortisol
secretion. In this regard, data obtained from an additional set of animals (not
included in the current report) are relevant. These monkeys were 27
reproductively intact females that were equivalent in age to the OVX and EST
animals, housed in the same location, and sampled for cortisol on the same dates
(albeit without regard to menstrual cycle stage). These reproductively intact
animals also exhibited a significant decline in cortisol across the same two
sampling periods (pretreatment: 51.8 7 1.12 ng/ml; 3 months: 37.8 7 1.7 ng/ml;
t[26] = 6.67, Po0.001). The observation that cortisol declined similarly
irrespective of reproductive condition or treatment suggests a common mediating
factor–perhaps a further accommodation to social housing or daily oral dosing.
In addition to the foregoing results, the current findings suggest that cortisol
secretion was greater during sample periods characterized by increased daylight.
Notably, the reproductively intact animals referred to above exhibited a similar
pattern of response (months 6 and 9: 43.9 7 1.3 ng/ml; months 3 and 12: 39.1 ng/
ml 7 1.4 ng/ml; t[26] = 4.19, Po0.001). Because the monkeys lived in pens with
outdoor exposure, it is not clear whether this effect was related to daylight or to
some other factor (such as average outside temperature) that is associated with
daylight. However, photoperiod itself has been documented to have an effect on
cortisol secretion in mouse lemurs (Microcebus murinus), with increases in
circulating cortisol observed during long photoperiods compared to short [Genin
& Perret, 2000]. This result is similar to that seen in the current study.
Finally, we note that this study is limited by the relatively low frequency of
sampling, as well as by the sole focus on endogenous (basal) cortisol. Both features
relate to the opportunistic nature of the investigation, which took advantage of an
experiment designed for other reasons. Despite its limitations, we believe that the
current investigation, by virtue of its size and relatively long period of treatment,
adds to the literature suggesting that E2 is an important regulator of adrenal
22 / Stavisky et al.
steroidogenesis in female macaques. The study also identifies seasonal factors as a
possible independent modulator of HPA activity.
The authors thank Elizabeth Nicks, Dewayne Cairnes, and Melissa Ayers for
technical assistance. We also thank the staff of the Endocrine Core Lab at Yerkes
Primate Research Center, especially Susie Lackey and Dr. Mark E. Wilson.
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cynomolgus, estradiol, fascicularis, macaque, secretion, macaca, cortisol, influence, ovariectomized
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