The influence of age on fecal steroid hormone levels in male Budongo Forest chimpanzees (Pan troglodytes schweinfurthii).

Potential interactions between age and endocrinological functioning have been understudied in wild ape populations. Therefore, we examined the relationship between age and the secretion of androgens and glucocorticoids in 15 juvenile, subadult, and adult male chimpanzees (Pan troglodytes schweinfurthii) free ranging in the Budongo Forest of Uganda. One hundred and nine fecal samples were opportunistically collected, between 07:30 and 13:30 hr, during the wet season. Fecal samples were preserved, by oven drying, and steroid content extracted before radioimmunoassay for dehydroepiandrosterone-sulfate (DHEA-S), testosterone (TEST), cortisol (CORT), and corticosterone (CCT). Employing indexes of age as predictive factors, linear mixed-effects modeling and non-parametric statistical comparisons of fecal steroid levels were conducted. Age was observed to significantly influence the production of both glucocorticoids and androgens in male Budongo Forest chimpanzees. Basically, whereas TEST and CORT increased, DHEA-S and CCT levels slightly declined as animals matured.

Lake pigments facilitate analysis of fecal cortisol and behavior in group-housed macaques.

Fecal steroid analyses are becoming more popular among both field and laboratory scientists. The benefits associated with sampling procedures that do not require restraint, anesthesia, and blood collection include less risk to both subject and investigator, as well as the potential to obtain endocrine profiles that do not reflect the influence of stress. However, the utility of the fecal steroid method has been limited in field conditions because of problems associated with sample identification. Here, we present evidence that Lake pigments are a valuable tool for the identification of individual fecal samples from group-housed female cynomolgus macaques. Further, we present data that suggest that excreted cortisol can be assayed from such samples, leading to the finding that time of day of sample collection influences cortisol concentrations, with morning samples producing higher values (t = 2.769, P = 0.024). Finally, the collection of physiological data from group-housed animals permits the evaluation of the relationship between endocrine status and behavior. This study demonstrated that morning fecal cortisol was significantly correlated with competitive and proximity behaviors, although not with rank in two stable social groups. In conclusion, the utility and validity of fecal steroid analyses continue to expand with further investigations.

Soy isoflavone supplements antagonize reproductive behavior and estrogen receptor alpha- and beta-dependent gene expression in the brain.

Epidemiological evidence suggests that isoflavone phytoestrogens may reduce the risk of cancer, osteoporosis, and heart disease, effects at least partially mediated by estrogen receptors alpha and beta (ERalpha and ERbeta). Because isoflavone dietary supplements are becoming increasingly popular and are frequently advertised as natural alternatives to estrogen replacement therapy, we have examined the effects of one of these supplements on estrogen-dependent behavior and ERalpha- and ERbeta-dependent gene expression in the brain. In the adult female rat brain, 17beta-estradiol treatment decreased ERbeta messenger RNA signal in the paraventricular nucleus by 41%, but supplement treatment resulted in a 27% increase. The regulation of ERbeta in the paraventricular nucleus is probably via an ERbeta-dependent mechanism. Similarly, in the ventromedial nucleus of the hypothalamus, supplement treatment diminished the estrogen-dependent up-regulation of oxytocin receptor by 10.5%. The regulation of oxytocin receptor expression in the ventromedial nucleus of the hypothalamus is via an ERalpha-dependent mechanism. Supplement treatment also resulted in a significant decrease in receptive behavior in estrogen- and progesterone-primed females. The observed disruption of sexual receptivity by the isoflavone supplement is probably due to antiestrogenic effects observed in the brain. These results suggest that isoflavone phytoestrogens are antiestrogenic on both ERalpha- and ERbeta-dependent gene expression in the brain and estrogen-dependent behavior.

Cross-species and interassay comparisons of phytoestrogen action.

This paper compiles animal and human data on the biologic effects and exposure levels of phytoestrogens in order to identify areas of research in which direct species comparisons can be made. In vitro and in vivo assays of phytoestrogen action and potency are reviewed and compared to actions, dose-response relationships, and estimates of exposure in human subjects. Binding studies show that the isoflavonoid phytoestrogens are high-affinity ligands for estrogen receptors (ERs), especially ER beta, but have lower potency in whole-cell assays, perhaps because of interactions with binding proteins. Many other enzymatic actions require concentrations higher than those normally seen in plasma. In vivo data show that phytoestrogens have a wide range of biologic effects at doses and plasma concentrations seen with normal human diets. Significant in vivoresponses have been observed in animal and human tests for bone, breast, ovary, pituitary, vasculature, prostate, and serum lipids. The doses reported to be biologically active in humans (0.4--10 mg/kg body weight/day) are lower than the doses generally reported to be active in rodents (10--100 mg/kg body weight/day), although some studies have reported rodent responses at lower doses. However, available estimates of bioavailability and peak plasma levels in rodents and humans are more similar. Steroidogenesis and the hypothalamic-pituitary-gonadal axis appear to be important loci of phytoestrogen actions, but these inferences must be tentative because good dose-response data are not available for many end points. The similarity of reported proliferative and antiproliferative doses illustrates the need for fuller examination of dose-response relationships and multiple end points in assessing phytoestrogen actions.

Regulation of estrogen receptor beta mRNA in the brain: opposite effects of 17beta-estradiol and the phytoestrogen, coumestrol.

Estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) are differentially distributed in the brain and likely mediate different estrogen-dependent processes. ERbeta is abundant in the bed nucleus of the stria terminalis, medial preoptic nucleus, paraventricular nucleus of the hypothalamus and the amygdala of the rat. In the paraventricular nucleus, which is devoid of ERalpha, ERbeta is colocalized with the neuropeptides, oxytocin and vasopressin, suggesting a potential functional role for ERbeta in the regulation of these peptides. We examined the regulation of ERbeta mRNA expression in the rat brain by 17beta-estradiol and the phytoestrogen, coumestrol. 17beta-Estradiol treatment decreased ERbeta mRNA in situ hybridization signal by 44.5% in the paraventricular nucleus of the hypothalamus (PVN), but had no effect in the bed nucleus of the stria terminalis (BnST) or the medial preoptic nucleus (MPA). In contrast, dietary exposure to coumestrol increased ERbeta mRNA signal by 47.5% in the PVN but had no effect in the BnST or the MPA. These data demonstrate that like ERalpha, ERbeta is down regulated by estrogen in a region specific manner in the rat brain. Furthermore, exposure to coumestrol may modulate ERbeta-dependent processes by acting as an anti-estrogen at ERbeta. This data contradicts results from cell transfection assays which suggest an estrogenic activity of coumestrol on ERbeta, indicating that the mode of action may be tissue specific, or that metabolism of dietary coumestrol may alter its effects. Because the highest concentrations of phytoestrogens are found in legumes, vegetables and grains, they are most prevalent in vegetarian and traditional Asian diets. Understanding the neuroendocrine effects of phytoestrogens is particularly important now that they are being marketed as a natural alternative to estrogen replacement therapy and sold in highly concentrated pills and powders.

Reproduction in free-ranging male Propithecus verreauxi: the hormonal correlates of mating and aggression.

Endocrine studies of captive strepsirrhine primates suggest that physical environment and social factors mediate inter-individual variations in testicular function and serum testosterone (sT) in males. While these studies have made major contributions to our understanding of the individual proximate mechanisms influencing androgen activity in male strepsirrhines, none have investigated how these mechanisms work coincidentally in free-ranging populations. In this study we used fecal steroid analysis to examine androgen-behavior interactions associated with reproduction in free-ranging male Propithecus verreauxi. Behavioral and hormone data were collected from two social groups during the 1990-91 and 1991-92 breeding seasons at Beza Mahafaly, Madagascar. Solid phase and radioimmunoassay techniques were used to quantify testosterone (T) in 105 desiccated fecal samples collected weekly from seven males. Results suggest that 1) solid phase extraction and radioimmunoassay techniques were reliable and accurate methods for quantifying T in sifaka feces; 2) fecal T (fT) elevations spanned a minimum of 4 months, peak levels occurring 1 month prior to the January onset of the breeding season; 3) fecal T concentrations were influenced by developmental factors and, among mature males, social factors associated with rank, intergroup aggression, and group instability.

Response of fecal cortisol to stress in captive chimpanzees (Pan troglodytes).

This study examined whether fecal cortisol could be used as an index of stress responses. The stress responsiveness of fecal cortisol was tested with a stressor known to stimulate adrenal activity, the stress of anesthesia. Daily fecal and urine samples were collected from four captive chimpanzees (Pan troglodytes) before and after anesthetizations with Telazol/Ketasat. Tests of assay validity indicated that cortisol was measurable in chimpanzee fecal extracts. Fecal cortisol concentrations were significantly elevated 2 days after anesthetization, with elevations in seven of the eight treatments. The posttreatment peak was significantly greater than baseline values in three of the four subjects. Both fecal concentrations and proportionate increases in responses to stress were significantly correlated with the corresponding values in urinary cortisol, confirming the stressfulness of these procedures and the stress responsiveness of fecal cortisol. These findings provide evidence for the application of fecal cortisol as a noninvasive index of physiologic stress in nonhuman primates.

Recent advances in noninvasive techniques to monitor hormone-behavior interactions.

This paper reviews recent advances in field endocrinology, a focus as well as a method in primatology and behavioral ecology that permits the examination of social behavior and life history through hormonal investigations in natural settings. Endocrine data complements the traditional behavioral data collected by field scientists by providing quantitative measures for the examination of adaptive tradeoffs, costs of social strategies, and reproductive and social significance of mating events. Further, investigations of the physiological mechanisms of reproductive constraint provide tests of the adaptive significance of reproductive skew in cooperative and competitive breeders. Hormone data also can provide insights into the costs of competition and aggression and the role of temperament in individual reproductive success and the evolution of social systems. New, noninvasive methods for the collection of this information have augmented and expanded field endocrinology through the use of techniques that do not require potentially confounding physical or physiological manipulations. Specifically, urine and fecal samples can be collected from free-ranging animals and contain gonadal and adrenal hormones that parallel profiles of serum hormones. Sampling, preservation, extraction, and assay methods for the analysis of excreted steroids are reviewed along with the species and questions to which these methods have been applied.

Reproductive actions of phytoestrogens.

This chapter reviews the reproductive actions of phytoestrogens, comparing mechanisms of action, dose-response relationships, and human exposures. Although a wide range of biochemical actions have been reported for phytoestrogens, in vitro tests suggest that phytoestrogens may be more likely to act through receptor-mediated mechanisms than through enzyme inhibition. Epithelial cell proliferation in the reproductive tract and anestrus are well-documented actions of isoflavonoids in experimental studies of animals. However, thus far, soy-based diets have generally failed to produce epithelial proliferation in ovariectomized rats and monkeys or menopausal women, and clinical studies have produced mixed evidence for effects of soy isoflavones on the human menstrual cycle or post-menopausal gonadotropin secretion. There has been considerable interest in the use of phytoestrogens as oestrogen replacement therapy in menopausal women. Reported results of initial clinical trials have been mixed, and it is unclear whether isoflavones in presently advised doses can substantially reduce menopausal symptoms. Some recent trials with oral isoflavone supplements report reductions in hot flushes, vaginal dryness, and breast pain. There is also limited clinical evidence for protective actions of isoflavones in mammary cancer. Like other oestrogenic substances, the isoflavonoids are effective differentiating agents in rodent models of development. The consequences of these actions for humans is of interest due to the high concentrations of isoflavonoids in some infant formulae. Thus, it is likely that some humans may experience greater exposure to phytoestrogens in infancy than in any other lifestage. At the time of writing, no ill effects of such exposure have been reported.

Sex determination of prehistoric human paleofeces.

Analysis of 12 prehistoric human paleofecal specimens from the Mammoth Cave System, Kentucky has produced the first estimate of biological sex using fecal material from ancient humans. Accelerator mass spectrometry (AMS) radiocarbon dating indicates that the specimens range in age from ca. 2700 B.P. to 2300 B.P. Dietary contents and steroids were extracted and analyzed. Chromatography and radioimmunoassay were used to measure levels of testosterone and estradiol in both modern fecal reference samples and in ancient feces. Results indicate that all 12 paleofeces were probably deposited by males whose diet included a variety of native crops and wild plants. These preliminary analyses have the potential to revolutionize the investigation of gender difference in diet, health, and nutrition.

Reproduction in free-ranging Propithecus verreauxi: estrus and the relationship between multiple partner matings and fertilization.

Female sifaka mate selectively with one or more resident and/or non-resident males during the breeding season. Various adaptive explanations have been advanced to explain why female primates mate with multiple males including that 1) females seek to confuse paternity and thereby forestall male infanticide and/or ensure male infant care or 2) females seek to ensure fertilization. Assessing the power of fertilization insurance to explain mating patterns in females requires information on the temporal relationship between mating and ovarian hormones. The hormonal correlates of reproduction and mating in free-ranging Propithecus verreauxi were investigated using excreted steroids as indices of reproductive state. Solid-phase extraction and radioimmunoassay techniques were used to measure unconjugated estradiol (E(2)) and progesterone (P(4)) in 485 desiccated fecal samples collected from five female sifaka before and during the breeding season at Beza Mahafaly, Madagascar. Results suggest that behavioral estrus was characterized by 10 to 15-day elevations in E(2); hormonal activity was observed to be similar to pseudo-estrus reported for other lemur species; apparent conception was associated with sustained P4 elevations beginning 1 to 3 days post-estrus with gestational phase elevations of E2 beginning 42 to 45 days post-conception; and mating with multiple partners appeared not to be a prerequisite to fertilization, as conception resulted from both monoandrous and polyandrous matings. These preliminary data suggest that fertilization insurance is not adequate to explain polyandrous mating in sifaka at Beza Mahafaly.

Potential adverse effects of phytoestrogens.

Evaluation of the potential benefits and risks offered by naturally occurring plant estrogens requires investigation of their potency and sites of action when consumed at natural dietary concentrations. Our investigations have examined the effects of a range of natural dietary concentrations of the most potent plant isoflavonoid, coumestrol, using a rat model and a variety of estrogen-dependent tissues and endpoints. Treatments of immature females demonstrated agonistic action in the reproductive tract, brain, and pituitary at natural dietary concentrations. Experiments designed to test for estrogen antagonism demonstrated that coumestrol did not conform to the picture of a classic antiestrogen. However, coumestrol did suppress estrous cycles in adult females. Developmental actions were examined by neonatal exposure of pups through milk of rat dams fed a coumestrol, control, or commercial soy-based diet during the critical period of the first 10 postnatal days or throughout the 21 days of lactation. The 10-day treatment did not significantly alter adult estrous cyclicity, but the 21-day treatment produced in a persistent estrus state in coumestrol-treated females by 132 days of age. In contrast, the 10-day coumestrol treatments produced significant deficits in the sexual behavior of male offspring. These findings illustrate the broad range of actions of these natural estrogens and the variability in potency across endpoints. This variability argues for the importance of fully characterizing each phytoestrogen in terms of its sites of action, balance of agonistic and antagonistic properties, natural potency, and short-term and long-term effects.

Phytoestrogen influences on the development of behavior and gonadotropin function.

The effect of phytoestrogens on the sexual differentiation of gonadotropin function was examined by neonatal exposure of pups through milk of rat dams fed a coumestrol (100 micrograms/g), control, or chow diet during the "critical period" of the first 10 postnatal days or throughout the 21 days of lactation. In females, exposure to coumestrol throughout the period of lactation produced growth suppression and an acyclic condition in early adulthood resembling the premature anovulatory syndrome. When the period of treatment was restricted to the first 10 postnatal days, however, no effects on vaginal cyclicity were seen. The 10-day exposure period produced more marked effects in males, resulting in transitory reductions in body weight in weanling males and reductions in mount and ejaculation frequency and a prolongation of the latencies to mount and ejaculate. Testicular weights and plasma testosterone levels did not differ among treatment groups, suggesting that the deficits in male sexual behavior were not due to deficits in adult gonadal function. Few effects of chow treatment were observed. However, significant differences from controls were apparent for weight at vaginal opening in females, and mount rate for chow-treated males was intermediate between that of controls and that of the coumestrol-treated group. These data provide evidence that lactational exposure to phytoestrogen diets can alter neuroendocrine development in both female and male rats.

Influence of phytoestrogen diets on estradiol action in the rat uterus.

The influence of coumestrol on the action of estradiol was examined in oral and parenteral tests. Coumestrol did not antagonize the uterotrophic action of estradiol when administered either prior to, or jointly with, E2 treatment, or when administered orally or parenterally. Additive effects on estradiol stimulation of uterine weight and reduction of cytosolic estrogen receptor binding were observed following oral, but not parenteral, administration of coumestrol. On the other hand, coumestrol pretreatment did appear to dampen estradiol's induction of progestin receptors, uterine protein, and nuclear estrogen receptor binding. However, even at those endpoints where coumestrol pretreatment did dampen estradiol action, coumestrol itself produced an estrogenic response. These findings contradict the assumption that all phytoestrogens are necessarily antiproliferative agents and argue for specific identification of the actions of each chemical.

A phytoestrogen diet induces the premature anovulatory syndrome in lactationally exposed female rats.

The effects of a phytoestrogen diet on sexual differentiation were examined in lactationally exposed rat pups. Rat dams were provided a semipurified diet containing the isoflavonoid coumestrol at a concentration (0.01%) previously found to be uterotrophic. Coumestrol treatment did not significantly alter the time of vaginal opening, although vaginal opening did occur at a lighter body weight. By 132 days of age, 83% of coumestrol-treated females exhibited the cornified smears of a persistent estrous state. By contrast, 91% of control animals were cycling regularly at 132 days of age. Estradiol stimulation failed to elicit an LH elevation in the coumestrol-treated animals, suggesting the possibility of neuroendocrine impairments. These findings indicate that the female offspring of mothers fed a low-level phytoestrogen diet during lactation manifest early and nearly universal disruption of cyclicity of the persistent-estrus type.

Effects of a normal, human-concentration, phytoestrogen diet on rat uterine growth.

The estrogenic action of the prototype natural phytoestrogen coumestrol was examined in rats in in vitro and in vivo tests. To establish the binding specificity of coumestrol and its relation to biological activities, saturation analyses and uterine weight assays were performed. These assays indicated that coumestrol competitively inhibited binding to the estrogen receptor and induced increases in uterine weight in keeping with its estrogen receptor affinity constant. Most importantly, coumestrol was uterotrophic when incorporated in a semipurified diet at natural dietary concentrations. Significant increases occurred in both uterine wet weight and dry weight, indicating that coumestrol produces true uterine growth. Effects appeared to be cumulative, raising questions of time-related interactions with other estrogen-sensitive mechanisms and clearance of isoflavonoids. Coumestrol induced uterine growth over a 90-hour period at dietary concentrations of 0.01 to 0.1%. Lower doses not active over this period were active when provided over a longer period of time: a 0.005% concentration was not active over a 90-hour period, but was active when provided over a 180-hour period. Coumestrol-induced uterine growth was accompanied by the induction of cytosolic progestin receptors and increases in nuclear estrogen binding. Scatchard analyses verified that these changes were due to changes in receptor number. These studies show that the naturally occurring phytoestrogens have dramatic estrogenic effects at natural dietary levels. These actions may be expressed via traditional receptor-mediated actions and therefore may have the same implications for development, health, and disease as do the steroidal estrogens produced by the body. Because rats have no sex hormone-binding globulin, further studies must be conducted in humans. However, these findings suggest that the natural dietary phytoestrogen coumestrol is a potent estrogen that must be considered in calculating the total estrogenic load to which humans are exposed during normal life.

Effects of a phytoestrogen diet on estrogen-dependent reproductive processes in immature female rats.

The study reported here examined the effects of a phytoestrogen diet on progestin receptor induction, vaginal opening, and the onset and maintenance of vaginal cycles in developing female rats. A natural dietary concentration (0.01%) of the isoflavonoid coumestrol was incorporated into the AIN semipurified diet and fed from 21 to 24 days (acute treatment) or from 22 to 60 days (chronic treatment). Progestin receptor induction was observed in the uterus, pituitary, and hypothalamus-preoptic area following acute treatment. Responses were more marked in the uterus and pituitary than in the hypothalamus-preoptic area. Vaginal opening was accelerated by 4 days during chronic coumestrol treatment and occurred at a lighter body weight. Vaginal cycles began on vaginal opening and did not differ in regularity from those of control animals. However, irregular cycles were observed in coumestrol-treated animals at 116 to 131 days, suggesting that chronic coumestrol treatment may have induced some permanent changes in reproductive function. These findings demonstrate that plant estrogens, at natural dietary levels, produce significant, agonistic actions in several estrogen-dependent tissues and processes.

Preliminary observations on the role of Acacia gum chemistry in Acacia utilization by Galago senegalensis in Kenya.

A census and preliminary observations on the ranging and feeding behavior of Galago senegalensis were made in a savannah-woodland site in Kenya. Population densities in three slightly different habitats were all approximately 1.5 animals/ha. Ranging patterns suggested the existence of territorial behavior. The diet of the galagos was exclusively insects and gums of two species of Acacia. The gum of A. drepanolobium appeared to be preferred to that of A. xanthophloea. The chemistry of the gums suggests that this preference is not due simply to the total level of phenolics or to avoidance of condensed tannins but may relate to the presence of compounds (e. g., flavonoids) having nutritional or hormonelike action.

Pregnancy outcome in free-ranging vervet monkeys (Cercopithecus aethiops).

A preliminary assessment of individual female differences in conception rate and fetal wastage has been determined for a population of wild vervet monkeys (Cercopithecus aethiops). One of three troops of vervet monkeys, the subjects of a long-term behavioral study, was trapped and blood was obtained for electrophoretic analysis. Pregnant females exhibited a distinctive serum aminopeptidase phenotype allowing a conclusive determination of pregnancy. Of the seven females diagnosed as pregnant, three later gave birth. Of the females that aborted, two were nulliparous and one was very old. Studies of captive animals have indicated that age and rank may affect a female's ability to carry a fetus to term. These factors, rather than the trapping procedure, may have been responsible for most of the fetal loss in the trapped troop. A comparison of all three troops for a 3-year period indicated that there were fluctuations in yearly birth success of individual females, as well as a relatively high miscarriage rate. The results of this study indicate the advantages of obtaining joint behavioral and biological data.