Influence of estradiol on cortisol secretion in ovariectomized cynomolgus macaques (Macaca fascicularis).код для вставкиСкачать
American Journal of Primatology 60:17–22 (2003) BRIEF REPORT Influence of Estradiol on Cortisol Secretion in Ovariectomized Cynomolgus Macaques (Macaca fascicularis) R.C. STAVISKY1n, S.L. WATSON2, M.S. ANTHONY3, S.B. MANUCK4, M.R. ADAMS3, and J.R. KAPLAN3,5 1 Section of Neurobiology, School of Biological Sciences, University of Texas, Austin, Texas 2 Department of Psychology, University of Southern Mississippi, Hattiesburg, Mississippi 3 Department of Comparative Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 4 Department of Psychology, University of Pittsburgh, Pittsburgh, Pennsylvania 5 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 macaques Contract grant sponsor: NIH; Contract grant numbers: HL 45666; HL 40962; Contract grant sponsor: Novo Nordisk A/S, Målöv, Denmark. n Correspondence to: Ronda Stavisky, Section of Neurobiology, School of Biological Sciences, University of Texas, Austin, TX 78712. E-mail: firstname.lastname@example.org Received 22 February 2002; revision accepted 17 March 2003 DOI 10.1002/ajp.10076 Published online in Wiley InterScience (www.interscience.wiley.com). r 2003 Wiley-Liss, Inc. 18 / Stavisky et al. INTRODUCTION 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. METHODS Subjects 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. Design 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. Treatment 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., 1981]. 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. RESULTS 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 = 6.28, Po0.001) and estradiol-replaced animals (t = 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 55 Ovx 50 Est 45 40 35 30 25 20 3 months, 10.5 h 6 months 13.5h 9 months 13.5h 12 months 10.5h 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). DISCUSSION 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 = 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 = 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. 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