The behavioral neuroendocrinology of dopamine systems in differently reared juvenile male rhesus monkeys (Macaca mulatta).

Dopamine (DA) is a critical neuromodulator of behavior. With propensities for addiction, hyper-activity, cognitive impairment, aggression, and social subordinance, monkeys enduring early maternal deprivation evoke human disorders involving dopaminergic dysfunction. To examine whether DA system alterations shape the behavioral correlates of adverse rearing, male monkeys (Macaca mulatta) were either mother-reared (MR: N = 6), or separated from their mothers at birth and nursery-reared (NR: N = 6). Behavior was assessed during 20-minute observations of subjects interacting with same- or differently-reared peers. Cerebrospinal fluid (CSF) biogenic amines, and serum testosterone (T), cortisol (CORT), and prolactin (PRL) were collected before and after pharmacologic challenge with saline or the DA receptor-2 (DRD2) antagonist Raclopride (RAC). Neuropeptide correlations observed in MR were non-existent in NR monkeys. Compared to MR, NR showed reduced DA tone; higher basal serum T; and lower CSF serotonin (5-HT). RAC increased PRL, T and CORT, but the magnitude of responses varied as a function of rearing. Levels of PRL significantly increased following RAC in MR, but not NR. Elevations in T following RAC were only significant among MR. Contrastingly, the net change (RAC CORT - saline CORT) in CORT was greater in NR than MR. Finally, observations conducted during the juvenile phase in a novel play-arena revealed more aggressive, self-injurious, and repetitive behaviors, which negatively correlated with indexes of dopaminergic tone in NR monkeys. In conclusion, early maternal deprivation alters brain DA systems, and thus may be associated with characteristic cognitive, social, and addiction outcomes.

Measurement of testosterone in infant fecal samples.

OBJECTIVES: This study reports the validation of a noninvasive method for repeated assessment of testosterone from infant fecal samples. METHODS: Fecal samples were collected from cotton diaper liners, subjected to methanol extraction, and assayed using a modified commercial testosterone RIA kit. RESULTS: Method validity was supported by a recovery near 100%, a sensitivity of 1.23 pg/ml, and inter- and intra-assay coefficients of variations less than 10 and 15%, respectively. Testosterone was detected in all samples from male and female infants aged 2 weeks to 15 months. CONCLUSIONS: Fecal assessment is supported as a novel, non-invasive tool for studying testosterone during early human development.

Birth season glucocorticoids are related to the presence of infants in sifaka (Propithecus verreauxi).

The responses of plural breeding mammals to environmental stressors are little understood in free-ranging populations, but recent studies of singular breeders suggest that ecological factors and social milieu influence the variable physiological stress responses observed among individuals. Our previous studies examining faecal glucocorticoid (fGC)-behaviour interactions in plural breeding male sifaka (Propithecus verreauxi) show that fGC elevations coincide with specific dispersal events, particularly the eviction of subordinates by resident alpha males. This study examined the utility of fGC assays for assessing the stress responses of male sifaka to demographic changes in the population during the birth season. Analyses were based on 889 faecal samples collected over five birth seasons from 124 adult males residing in 55 different groups at Beza Mahafaly, Madagascar. Results show that fGC levels in males are unrelated to age, residence, group stability or rank, but are significantly and positively correlated with the presence of infants, annual elevations in weekly mean fGC concentrations in males paralleling increasing numbers of infants born annually in the population. These data are the first to show that in seasonal plural breeding species such as sifaka, elevated fGC in males reflects specific events related to reproduction rather than states or social context during the birth season.

Endocrine mechanisms of primate life history trade-offs: growth and reproductive maturation in vervet monkeys.

Life history theory predicts that the timing of maturation will result from a trade-off between growth and the age of first reproduction. This trade-off and its mechanisms of action are still poorly understood in many species and have not been well studied at the individual level. This study examined hypothesized trade-offs between growth and reproductive maturation in wild populations of vervet monkeys (Cercopithecus aethiops) from Kenya, East Africa. Individuals were sampled from four populations in widely separated sites differing in temperature, altitude, and rainfall. Biological samples and morphometric measures were collected from 50 adult males, 83 adult females, and 225 juveniles. Gonadal steroids and leptin levels were analyzed by radioimmunoassay of sera from 136 juvenile males and 90 juvenile females. Cross-sectional profiles of morphometric and endocrine data were used to assess the onset and cessation of growth in relation to sexual maturation. Gonadal steroids were used to assess sexual maturation and breeding onset. Leptin was used as an index of nutritional state. Estimates of mortality were derived from population age-structure. Across populations, higher resource productivity and nutrient status were associated with more rapid growth. Shorter growth duration was associated with earlier reproductive onset. These findings provide support for models of trade-offs between the timing of growth completion and reproductive onset, but they are contradicted by the evidence that reproduction precedes the cessation of growth in these populations. The biphasic actions of estradiol provide an alternative model and mechanism for the growth-reproduction trade-off.

Focused grooming networks and stress alleviation in wild female baboons.

We examine the relationship between glucocorticoid (GC) levels and grooming behavior in wild female baboons during a period of instability in the alpha male rank position. All females' GC levels rose significantly at the onset of the unstable period, though levels in females who were at lower risk of infanticide began to decrease sooner in the following weeks. Three factors suggest that females relied on a focused grooming network as a coping mechanism to alleviate stress. First, all females' grooming networks became less diverse in the weeks following the initial upheaval. Second, females whose grooming had already focused on a few predictable partners showed a less dramatic rise in GC levels than females whose grooming network had been more diverse. Third, females who contracted their grooming network the most experienced a greater decrease in GC levels in the following week. We conclude that close bonds with a few preferred partners allow female baboons to alleviate the stress associated with social instability.

Ecological and reproductive variance in serum leptin in wild vervet monkeys.

Leptin was originally thought to be an antiobesity hormone, but increasing evidence suggests that its ancestral role was to mobilize neuroendocrine responses to starvation. Research on wild primates is critical for interpreting the high leptin values seen in Western human populations and captive animals. This study examined natural variation in serum leptin in wild vervet monkeys (Chlorocebus aethiops), testing the hypothesis that serum leptin in vervets varies with sex, adiposity, ecology, and reproductive state. Analyses made use of a unique dataset comprised of serum and morphometric measurements obtained from vervet monkeys in four Kenyan sites differing in altitude, temperature, rainfall, and access to human foods. Leptin and gonadal steroid concentrations were analyzed in serum samples from 116 adults. Low leptin levels in males and acyclic females support the contention that levels seen in captivity are not typical for wild primates. Measures of adiposity were not correlated with serum leptin, reflecting the extremely low fat storage in wild cercopithecine primates. Associations with habitat and season, however, indicate that leptin does register ecological variation in energy balance. Leptin levels were higher in sites and seasons with higher rainfall. Moreover, leptin varied significantly with reproductive state, with higher levels in pregnant than in acyclic females. Changes in leptin with gestation stage and duration of lactation suggest that transitory and reversible elevations were an important part of its ancestral role. These data show that in this wild primate population leptin is a sensitive index of natural variation in habitat and seasonally fluctuating reproductive state.

Behavioural and hormonal responses to predation in female chacma baboons (Papio hamadryas ursinus).

In humans, bereavement is associated with an increase in glucocorticoid (GC) levels, though this increase can be mitigated by social support. We examined faecal GC levels and grooming behaviour of free-ranging female baboons to determine whether similar effects were also evident in a non-human species. Females who lost a close relative experienced a significant increase in GC levels in the weeks following their relative's death compared with the weeks before, whereas control females showed no such increase. Despite the fact that females concentrate much of their grooming on close kin, females who lost a close female relative did not experience a decrease in grooming rate and number of grooming partners; instead, both grooming rate and number of grooming partners increased after a relative's death. While the death of a close relative was clearly stressful over the short term, females appeared to compensate for this loss by broadening and strengthening their grooming networks. Perhaps as a result, females' GC levels soon returned to baseline. Even in the presence of familiar troop-mates and other relatives, females experienced a stress response when they lost specific companions, and they apparently sought to alleviate it by broadening and strengthening their social relationships.

Sensitive high-performance liquid chromatographic method using coulometric electrode array detection for measurement of phytoestrogens in dried blood spots.

As the epidemiological and physiological investigation of isoflavones and lignans expands, the need for sensitive methods for analyzing large numbers of samples intensifies. We have developed a method using high-performance liquid chromatography (HPLC) equipped with a coulometric electrode array detector for separation and sensitive detection of daidzein (Da), equol (Eq), genistein (Ge) and enterolactone (Enl) in dried blood spots (DBS). Detection limits ranged from 4.5 pg or 0.09 ng/mL (Eq) to 19 pg or 0.38 ng/mL (Ge) on column. Signal linearities ranged from detection limits to 200 ng/mL (Eq, Enl) and 600 ng/mL (Da, Ge) sample concentration. Correlations between DBS and serum concentrations were 0.66 (Enl), 0.88 (Eq), 0.98 (Ge) and 0.99 (Da). Intra-assay coefficients of variation (CVs) were less than 8% and inter-assay CVs ranged from 2.4 to 20.2% for Da, Eq and Ge for three levels of controls. Enl intra-assay CV was 13.6% for the low pooled control. Analytic recovery ranged from 87% (inter-assay Ge) to 98% (inter-assay Enl). DBS concentrations of Da, Ge and Eq were stable for at least 8 weeks at 4 and 25 degrees C, and at 37 degrees C for at least 5 weeks, with Enl showing greater variability at all temperatures but relative stability for 7 weeks. Measurement of samples from 135 perimenopausal Japanese women consuming habitual diets in Kyoto and Fukushima prefectures showed the former to have the expected lower concentrations of Da and Eq (416 and 87 nM) as well as Enl (49 nM) compared to the latter locale (566, 145 and 72 nM, respectively). This method could be useful in large epidemiological research or detailed physiological studies.

Genistein affects ER beta- but not ER alpha-dependent gene expression in the hypothalamus.

Isoflavone phytoestrogens are growing increasingly popular because of their reported cardiovascular and anticarcinogenic properties, but the effects of these compounds in the brain are largely unknown. In a previous study, we found that an isoflavone supplement, containing a mixture of soy phytoestrogens, inhibited estrogen-dependent female sexual behavior and was antiestrogenic for both ER alpha- and ER beta-dependent gene expression in the hypothalamus. Here we examined the impact of the soy isoflavone genistein, a major component of the supplement, on estrogen-dependent female sexual behavior and ER alpha- and ER beta-dependent gene expression in the rat brain. Genistein, at a dietary concentration of 100 or 500 ppm had no effect on lordosis behavior in rats. However, at 500 ppm genistein had differential activity through ER alpha and ER beta in the hypothalamus. Genistein had no effect, in either the presence or absence of 17 beta-E2, on oxytocin receptor density in the ventromedial nucleus of the hypothalamus, an estrogen-dependent action thought to be regulated via ER alpha. However, genistein increased ER beta mRNA expression in the paraventricular nucleus of the hypothalamus by 24%, whereas 17 beta-E2 decreased ER beta mRNA expression by 26%, a process likely mediated by ER beta itself. These results suggest that at this dose, genistein has antiestrogenic action through ER beta in the paraventricular nucleus but negligible activity through ER alpha in the brain.

Modifications of a field method for fecal steroid analysis in baboons.

By extracting steroid metabolites from feces, researchers can track endocrine activity noninvasively in free-ranging animals. Sample preservation is a critical component of such methods because steroid metabolites rapidly decompose. Here, we describe a method for preservation, field extraction, and radioimmunoassay of steroid metabolites (estradiol, progesterone, glucocorticoids, and testosterone) from the feces of wild female baboons (Papio spp.). This method is a modification of that developed by Stavisky [Socioendocrinology: noninvasive techniques for monitoring reproductive function in captive and free-ranging primates. PhD, Emory University, 1994.], which employs reversed-phase octadecylsilane cartridges to extract steroids from feces. In addition to providing physiological validation for this method, we examine variation in steroid concentration across different (1) collection times (morning vs. afternoon), (2) methanol extraction treatments (homogenized vs. hand-mixed), and (3) solid-phase extraction times (2 vs. 10 h after collection). We then examine the stability of sample storage at ambient and subzero temperatures to determine whether storage time significantly alters steroid concentrations. Our results show that hormone concentrations do not differ between morning and afternoon samples, homogenization yields significantly higher fecal steroid concentrations, and fecal steroids are stable in a methanol/acetone solution for up to 10 h. When stored at ambient temperatures, only glucocorticoid metabolites had some degradation over a period of up to 40 days. However, when stored at -10 degrees C, no significant steroid changes were observed for up to 400 days. This method is particularly suited for behavioral research because it permits delays between sample collection and sample processing, thus allowing behavioral observations to continue.

Neurobehavioral actions of coumestrol and related isoflavonoids in rodents.

Isoflavonoids are plant estrogens that are increasingly advocated as a natural alternative to estrogen replacement therapy (ERT) and are available as dietary supplements. As weak estrogen agonists/antagonists with a range of other enzymatic activities, the isoflavonoids provide a useful model for the actions of endocrine disruptors. This paper reviews the responses of rodents to diets containing coumestrol or an isoflavone supplement in comparison to animals fed the phytoestrogen-free AIN76A diet. Neural mechanisms were investigated by examining isoflavonoid effects on ER(alpha)-dependent (regulation of oxytocin receptor [OTR] binding in the ventromedial nucleus of the hypothalamus [VMN]) and ERbeta-dependent (regulation of ERbeta mRNA in the paraventricular nucleus [PVN]) endpoints. Activational as well as organizational effects on sexual behavior and gonadotropin secretion were observed for coumestrol. Treatment of rat dams with a 100-ppm coumestrol diet from birth to postnatal day (PND) 21 induced premature anovulation in female offspring, and treatment from birth to PND 10 suppressed sexual behavior in male offspring. One-week treatment of ovariectomized (OVX) female rats with the same coumestrol diet increased ERbeta mRNA expression in the PVN, an effect opposite to that of estradiol. Ten days of treatment with a 200-ppm coumestrol diet increased LH secretion in OVX wild-type mice, an effect opposite to the normal negative feedback effects of estradiol. No effects were observed in ER(alpha) knockout (ER(alpha)KO)-OVX females, indicating that coumestrol's action on LH was mediated through ER(alpha). Similar activational effects were observed for the isoflavone diet. The lordotic response to estrogen was significantly reduced by 2 days of treatment of OVX adult females with an isoflavone diet providing 13 ppm genistein and 33 ppm daidzein. One week of treatment with the same isoflavone diet produced an effect opposite to that of estradiol in the PVN, increasing ERbeta mRNA expression above control levels. These investigations show that, in spite of their preferential affinity for ERbeta, isoflavonoids act through both ER(alpha) and ERbeta. Moreover, their neurobehavioral actions were antiestrogenic, either antagonizing or producing an action in opposition to that of estradiol. This work demonstrates that even small, physiologically relevant exposure levels can alter estrogen-dependent gene expression in the brain and complex behavior.

Diet and dominance among female vervet monkeys (Cercopithecus aethiops).

Behavioral observations of free-ranging female vervet monkeys (Cercopithecus aethiops) suggest that priority of access to food enhances individual fitness. Seventeen adult females living in two social groups were observed for 26 months in the Samburu/Isiolo Game Reserves, northern Kenya. Priority of access to feeding and sitting sites was associated with significant differences in diet and reproduction. Food species distribution altered rank-related differences in diet. Rank-related differences in diet occurred when food items were clumped in distribution, but not when they were randomly distributed. The relation of individual food items to reproduction influenced rank-related differences in reproduction. Rank was significantly correlated with birth rate when a preferred food species was clumped in distribution.

Information content of sexual swellings and fecal steroids in sooty mangabeys (Cercocebus torquatus atys).

The accuracy and precision of sexual swellings and fecal steroids as measures of ovarian function and the periovulatory period were compared in 4 sexually mature, individually housed, sooty mangabey females. Fecal samples were collected daily over a 10-week period during the normal breeding season. Serum was collected 3×/week, daily during peak swelling, and sex skin was rated 5×/week on a 0-5 relative scale. Both fecal estradiol (fE2) and progesterone (fP4) were significantly correlated with serum values in composite E2-aligned profiles and within the cycles of individual females with average correlations of rs = 0.6. Follicular phase means for fE2 and luteal phase means for fP4 were significantly correlated with the serum means across cycles, suggesting that fecal concentrations could be used to accurately evaluate cycle phases within and across females. In contrast, the timing of peak swelling relative to the periovulatory period varied considerably across the cycles of individual females. Although maximum tumescence appears to bracket the periovulatory period, individual differences in the duration of peak swelling and the timing of its onset and end tend to obscure the exact time of ovulation in relation to maximal tumescence. These data illustrate the utility of fecal steroid analysis as a tool for further evaluation of the signal value of sexual skin and its role in mating interactions. © 1996 Wiley-Liss, Inc.

Application of fecal steroid techniques to the reproductive endocrinology of female verreaux's Sifaka (Propithecus verreauxi).

Solid phase extraction, high performance liquid chromatography, and radioimmunoassay were used to test the validity of fecal steroid analysis for assessing ovarian function in sifaka (Propithecus verreauxi). Daily fecal samples were collected over a 4 month period from two cycling female sifaka, and single samples were collected from females during normal gestation and males while housed at the Duke University Primate Center. Tests of radioimmunoassay validity indicated that solid phase extraction and microradioimmunoassay techniques were reliable and accurate methods for quantifying ovarian steroids in sifaka feces. The progesterone (P4 ) antibody specifically quantitated only P4 , while several estrogen metabolites made small contributions to immunoreactive measures of estradiol (E2 ). A 1:10 dilution reduced these contributions to 3-15% of the estimated E2 concentration. Although the spectral data suggested that E2 was not the major metabolite present, it accounted for the majority of the immunoreactivity at normal assay dilutions. Fecal profiles of immunoreactive E2 and P4 in the conceptive female resembled serum profiles of other strepsirhines. E2 and P4 were elevated at the end of the conceptive cycle and were more markedly increased in late pregnancy in the two pregnant females. Mating behavior and indices of sexual interest were observed in conjunction with E2 peaks, although not all peaks were accompanied by observations of sexual behavior. © 1995 Wiley-Liss, Inc.

Fecal steroid analysis of ovarian cycles in free-ranging baboons.

This paper reports field and laboratory tests of serial sampling, solid phase extraction, and microradioimmunoassay methods for the collection, preservation, and analysis of fecal steroids. The field study was conducted in a troop of 87 yellow baboons (Papio cynocephalus) in the Tana River Primate Reserve, Kenya. Serial samples of four focal females and opportunistic sampling of 18 additional females over 22 days of sampling yielded a total of 62 samples, X = 3.1 ± 0.4/day, demonstrating the feasibility of regular field collection and extraction. Estradiol and progesterone concentrations in the field-extracted samples exhibited high recovery and statistically significant correlations (P < 0.05) with concentrations in the lab-extracted samples, suggesting that solid phase extraction could provide a useful alternative to freezing in sites where electricity or liquid nitrogen is not available. Tests of microradioimmunoassays demonstrated that these assays were sensitive, accurate, and precise when applied to the assay of fecal extracts, providing estimates of ovarian steroids that varied significantly with reproductive state. The demonstration that testosterone could be accurately and reliably assayed in fecal extracts suggests that these techniques also could be applied to the study of male reproductive function. Parallels between fecal profiles of cycling and pregnant baboons with patterns reported for serum steroids in baboons suggest that fecal steroids might be useful in distinguishing amenorrhea from early pregnancy in free-ranging baboons as well as in species lacking external indices of reproductive state. © 1995 Wiley-Liss, Inc.

Non-invasive methods for estradiol recovery from infant fecal samples.

While the activation of the infant hypothalamic-pituitary-gonadal axis and the existence of a postnatal gonadotropin surge were first documented in the early 1970s, study of the longitudinal development of gonadal hormones in infancy, and the potential physiological and behavioral correlates of this development, have been hampered by reliance on infrequent serum sampling. The present study reports the validation of a non-invasive method for repeated assessment of steroid hormones in infant fecal samples. Fecal samples were collected in and excised from cotton diaper liners and extracted using methanol. Extracts were analyzed for estradiol using a diluted assay modification. Method validity was supported by a steroid recovery rate of at least 80%, a sensitivity of 0.35 pg/ml, and inter- and intra-assay coefficients of variations of less than 10 and 20%, respectively. Variation in estradiol concentration was assessed across (1) sample type (scraped vs. cut from diaper liner), (2) time of day (morning vs. afternoon/evening samples), (3) time interval between samples, and (4) time-to-assay (1 day vs. 489 days after collection). Of these characteristics, only the time interval between samples within an individual was significantly associated with estradiol concentration. This is the first report of human infant fecal estradiol levels. The results support fecal recovery as a novel and powerful non-invasive tool for longitudinal studies of human infants, expanding research opportunities for investigating development of sex-specific behaviors in infancy, and the potential effects of endocrine disruptors on development.

Social stressors and coping mechanisms in wild female baboons (Papio hamadryas ursinus).

We examined the social correlates of fecal glucocorticoid (GC) levels in wild female baboons during a period of social and demographic stability. Females' GC levels were not affected by individual attributes such as number of kin or dominance rank, nor could we detect any significant seasonal effects. Instead, GC levels were influenced by behavioral attributes that varied between individuals and within individuals across time. Pregnant and cycling females who received high rates of aggression had higher GC levels than others. In contrast, pregnant and cycling females who received grunts - vocal signals of benign intent - at high frequencies from dominant females had lower GC levels than females who received grunts at lower frequencies. Lactating females showed the opposite trend, apparently as a consequence of the high rate of grunting and intense, unsolicited attention that their infants received from others. All females experienced lower GC levels in months when they concentrated their grooming among a small number of partners than when their grooming was more evenly distributed among many partners. Although GC levels in female baboons are most strongly influenced by events that directly affect their reproductive success, subtle social factors associated with the loss of predictability and control also seem to exert an effect. Loss of control may be mitigated if a female is able to predict others' intentions - for example, if others grunt to her to signal their intentions - and if she is able to express some preference over the timing and identities of her grooming partners.

Female testosterone, dominance rank, and aggression in an Ethiopian population of hybrid baboons.

Studies on the relationship between female testosterone (T) measures and behavior, particularly in free-ranging primate populations, remain scant. In this study we used fecal steroid analysis to examine the effects of seasonal, reproductive, and social factors on female T in a group of free-ranging hybrid baboons (Papio sp.) in the Awash National Park of Ethiopia. We collected behavioral and hormonal data from 25 adult females across an 11-month period. Solid phase extraction and radioimmunoassay (RIA) techniques were used to quantify T in 776 fecal samples collected weekly from each female. The results indicate that 1) the females had elevated T during pregnancy and during the wet season relative to other periods, 2) female dominance rank was positively related to T measures, and 3) female T and aggression were positively related within subjects but not across subjects. Higher T concentrations during pregnancy are consistent with other published profiles of pregnancy in primates. In combination with data on foraging, wet season increases in T may indicate contest competition for females. The rank-T relationship may be mediated by supplants or aggression. Finally, we discuss the different interpretations of the hormone-behavior relationship based on within- and across-subject analyses.

Dominance rank and fecal testosterone levels in adult male chimpanzees (Pan troglodytes schweinfurthii) at Ngogo, Kibale National Park, Uganda.

In an attempt to describe hormone-behavior interactions in a sample of wild male chimpanzees, we quantified testosterone in 67 fecal samples obtained from 22 adult male chimpanzees at Ngogo, Kibale National Park, Uganda. A mixed-model methodology that controlled for age-class identified a significant positive association between testosterone levels and dominance rank. The results are consistent with those reported from a separate, smaller chimpanzee community in the same population in a study that analyzed testosterone levels in urine [Muller & Wrangham, 2004]. As in that earlier study, our results held during a period of social stability, which is not consistent with predictions of the "challenge hypothesis." We concur with Muller and Wrangham [2004] that the challenge hypothesis requires modification to explain the chimpanzee data, because fission-fusion sociality in chimpanzees makes challenges unpredictable. We also discuss the utility of fecal samples and a mixed-model statistical method for behavioral endocrinology studies.