The adrenocortical response of greater sage grouse (Centrocercus urophasianus) to capture, ACTH injection, and confinement, as measured in fecal samples.

Investigators of wildlife populations often utilize demographic indicators to understand the relationship between habitat characteristics and population viability. Assessments of corticosterone may enable earlier detection of populations at risk of decline because physiological adjustments to habitat disturbance occur before reproductive diminutions. Noninvasive methods to accomplish these assessments are important in species of concern, such as the greater sage grouse (GRSG). Therefore, we validated a radioimmunoassay that measures immunoreactive corticosterone metabolites (ICM) in fecal samples and used it to characterize the adrenocortical response of 15 GRSG exposed to capture, intravenous injection of 50 IU/kg adrenocorticotrophic hormone (ACTH) or saline, and 22 h of confinement. Those animals injected with ACTH exhibited a more sustained (P = 0.0139) and less variable (P = 0.0012) response than those injected with saline, indicating different levels of adrenocortical activity. We also found that potential field-collection protocols of fecal samples did not alter ICM concentrations: samples held at 4 degrees C for up to 16 h contained similar levels of ICM as those frozen (-20 degrees C) immediately. This study demonstrates a multiphasic adrenocortical response that varied with the level of stimulation and indicates that the assay used to measure this phenomenon is applicable for studies of wild GRSG.

Social suppression of cortisol in female marmoset monkeys: role of circulating ACTH levels and glucocorticoid negative feedback.

Behaviorally subordinate female common marmoset monkeys (Callithrix jacchus) exhibit pronounced, chronic reductions of circulating cortisol levels. Cortisol suppression in these animals is mediated in part by adrenocortical hyporesponsiveness to adrenocorticotropic hormone (ACTH). In addition, we hypothesized that social subordination may activate a central, neurally mediated mechanism to further inhibit hypothalamo-pituitary-adrenal function. In this study, therefore, we evaluated basal plasma cortisol and ACTH concentrations, as well as cortisol and ACTH responses to dexamethasone (DEX), in dominant and subordinate females to initially characterize such a mechanism. Morning plasma cortisol and ACTH levels were determined before, and 1, 2, and 3 days following administration of DEX (0.5, 1.0, or 5.0 mg/kg, IM) or saline. Baseline cortisol concentrations prior to DEX treatment were significantly lower in subordinate females than in dominants, as previously reported. However, ACTH concentrations in the same blood samples did not differ between the two groups. Furthermore, dominant and subordinate females showed similar cortisol and ACTH responses to DEX. These results indicate that reduced circulating cortisol levels in subordinate females are not associated with either altered circulating ACTH concentrations or enhanced responsiveness to glucocorticoid negative feedback. However, the finding that basal ACTH levels are not elevated in subordinate females as compared to dominants, in spite of low circulating cortisol concentrations, suggests that ACTH secretion in subordinate females is restrained by a steroid-independent inhibitory mechanism operating at the level of the brain or pituitary.

Lipophorin of lower density is formed during immune responses in the lepidopteran insect Galleria mellonella.

Injection of heat-killed bacteria into larvae of the greater wax moth Galleria mellonella is followed by changes in lipoprotein composition in the hemolymph. Density gradient centrifugation experiments revealed that within the first four hours after injection, a part of larval lipoprotein, high-density lipophorin (HDLp), was converted into a lipoprotein of lower density. SDS-polyacrylamide gel electrophoresis analysis of the gradient fractions and sequencing of protein fragments, established that the exchangeable apolipoprotein apolipophorin III (apoLp-III), a potent immune-activator, was associated with this newly formed lipophorin. To investigate further the influence of lipophorin-associated apoLp-III on immune-related reactions, we performed in vitro studies with isolated hemocytes from G. mellonella and lipophorins from the sphinx moth Manduca sexta, as a natural source of high amounts of low-density lipophorin (LDLp) and HDLp. The hemocytes were activated to form superoxide radicals upon incubation with LDLp, but not with HDLp. Fluorescence-labeled LDLp was specifically taken up by granular cells. This process was inhibited by adding an excess of unlabeled LDLp, but not by HDLp. We hypothesize that larval lipophorin formed in vivo is an endogenous signal for immune activation, specifically mediated by the binding of lipid-associated apoLp-III to hemocyte membrane receptors.

Presence of IL-1- and TNF-like molecules in Galleria mellonella (Lepidoptera) haemocytes and in an insect cell line Fromestigmene acraea (Lepidoptera).

In this study the authors give immunocytochemical evidence for the presence of interleukin (IL)-1alpha- and tumour necrosis factor (TNF) alpha-like molecules in the haemocytes of last instar larvae from the greater wax moth Galleria mellonella. Similar results are demonstrated in a continuous haemocyte line (BTI-EA-1174-A) from the salt marsh caterpillar Estigmene acraea. In Galleria mellonella larvae granular cells show a strong positive reaction with both primary antibodies, whereas plasmatocytes are stained to a lesser extent. Cell line haemocytes also react positively with both antibodies. After activating the cells with lipopolysaccharide (LPS) staining of Estigmene acraea cells is decreased, whereas Galleria mellonella haemocytes show no visible reaction in comparison to non-activated cells.

Seasonal and social correlates of fecal testosterone and cortisol levels in wild male muriquis (Brachyteles arachnoides).

Fecal testosterone and cortisol levels were analyzed from six wild male muriquis (Brachyteles arachnoides) over a 19-month period at the Estação Biológica de Caratinga in Minas Gerais, Brazil, to investigate the hormonal correlates of seasonal sexual behavior and environmental conditions. Group mean testosterone levels based on weekly samples from the six males did not differ between copulatory and noncopulatory periods or between rainy and dry seasons. Cortisol levels did change with copulatory periods, and were significantly higher during the second dry season, when mating continued following an exceptionally heavy rainy season, than during the first dry season, when mating ceased. Males exhibited individual variation in the timing of their hormone shifts relative to their sexual activity, but neither hormone levels nor sexual activity were related to male age. Despite individual differences in the timing of testosterone fluctuations around the onset and offset of the copulatory season, all males exhibited elevated cortisol concentrations following a slight increase in testosterone at the beginning of the copulatory season. Both the lack of significant changes in testosterone levels with the onset of the rainy and copulatory season and the lack of prebreeding increases in cortisol may be related to the low levels of overt aggression displayed by male muriquis over access to mates.

Suppression of cortisol levels in subordinate female marmosets: reproductive and social contributions.

Socially subordinate female common marmosets (Callithrix jacchus) have markedly lower plasma cortisol levels than dominant females. Subordinate females also undergo hypoestrogenemic anovulation, and estrogen can elevate glucocorticoid levels. Therefore, we previously hypothesized that this cortisol difference is mediated by rank-related differences in reproductive hormones, probably estradiol. To test this possibility, we characterized the effects of the ovarian cycle and ovariectomy on plasma cortisol concentrations. Beginning in the early follicular phase, basal blood samples were collected from seven cycling female marmosets daily for 16 days and at 2- to 3-day intervals for another 16 days. Samples were collected identically from seven anovulatory subordinate females and seven long-term ovariectomized females. Cortisol levels changed reliably across the ovarian cycle, with levels in the mid- to late follicular, peri-ovulatory, and early luteal phases higher than those in the remainder of the cycle. Cortisol levels of cycling females were significantly higher than those of subordinates at all parts of the cycle, but were significantly higher than those of ovariectomized females only during the midcycle elevation. Unexpectedly, subordinates had significantly lower cortisol levels than ovariectomized females, as well as higher estradiol and estrone levels and lower progesterone and luteinizing hormone (LH) levels. These results confirm that circulating cortisol concentrations are modulated by reproductive function in female marmosets but also indicate that low cortisol levels in subordinate females cannot be attributed simply to hypoestrogenemia. Instead, other factors, such as direct effects of social subordination or suppression of LH levels, contribute to suppression of cortisol in subordinates.

Insect cell stimulation by LPS requires the activity of cell-released proteases.

The hemocyte line BTI-EA-1174-A from the lepidopteran insect Estigmene acraea responds to bacterial lipopolysaccharide (LPS) by an enhanced phagocytic reaction and a dose-dependent increase of lysozyme release [Wittwer et al., Dev Comp Immunol 21:323 (1997)]. This paper provides evidence for a strong proteolytic activity in cell culture supernatants occurring after addition of LPS (1 mg/ml). The proteolysis is caused by cell-released proteases and seems to be necessary for cell activation. Its inhibition by alpha 2-macroglobulin results in a dose-dependent reduction in cellular response strength. Phagocytic reactions, as well as lysozyme release, are lowered to about half in the presence of 0.0001 mg/ml alpha 2-macroglobulin. A nearly complete abolishment of activation was achieved with final concentrations of 1.0 mg/ml alpha 2-macroglobulin. The data presented allow us to conclude that the LPS-triggered proteolytic activity is an important part of the activation process; it occurs outside of the cells and delivers immune response activating factors.

LPS (lipopolysaccharide)-activated immune responses in a hemocyte cell line from Estigmene acraea (Lepidoptera).

The suitability of the hemocyte cell line BTI-EA-1174-A from Estigmene acraea (Lepidoptera) to serve as a tool for studying insect immune reactions in vitro was investigated. Addition of bacterial lipopolysaccharides to the cultures caused enhanced phagocytosis of silica beads, as well as increased lysozyme activity in the cell culture supernatants. Addition of fungal beta 1,3-glucans did not result in any activation. The LPS-influenced (1 mg/mL) increase of phagocytic reactions against the silica beads was at its highest within 24 h after LPS-addition. Activated cells exhibited drastic changes in their morphology in connection with reduced cell numbers in the cultures but without increased mortality rates. LPS-dosages higher than 10 micrograms/mL LPS provoked significantly enhanced lysozyme activities. A maximal induction took place with 1 mg/mL LPS. The lysozyme activity started to rise 2 days after LPS-addition, further increase was observed up to the seventh day. The responsible protein was isolated from cell culture supernatants and N-terminally sequenced. The exact molecular mass was determined by mass spectrometry as 14.080 kDa. The amino acid sequence of the analysed portion revealed high sequence-similarity to the lysozymes of other lepidopteran insects as well as to hen egg lysozyme. Further results presented in this paper give indications for the existence of soluble molecules which are released by the cells and which enhance the LPS-triggered activation.

Metabolism of reproductive steroids during the ovarian cycle in two species of callitrichids, Saguinus oedipus and Callithrix jacchus, and estimation of the ovulatory period from fecal steroids.

Gonadal steroids were measured in daily fecal samples providing comparative data on steroid metabolism in two genera of New World primates. Circulating bioactive LH and progesterone concentrations and fecal progesterone, pregnanediol, estradiol, and estrone concentrations were measured by collecting blood and daily fecal samples from four captive common marmoset females and four cotton-top tamarin females for 30 days. High recoveries (> 80%) of labeled steroids that were added directly to the feces before extraction were recovered from feces of both species. Because of the presence of complex steroid conjugates, only one fifth the amount of estradiol was measured without solvolysis as compared to the amount measured with solvolysis. In tamarins, steroids were metabolized rapidly, with all postovulatory increases occurring within two days after the circulating LH peak (an increase of 2 SD higher than mean follicular levels). In marmosets, steroid excretion was slower; increased steroid levels occurred 2-4 days after the LH peak except in the case of estrone, which did not consistently increase after the LH peak. Circulating estrone and estradiol both contributed to the high excretion of estradiol in the feces from both species. The timing in the delay in excretion of fecal steroids was used to accurately determine the ovulatory period to within a 2-day window. This degree of accuracy is possible when the duration of the delay to the LH peak is known for a given species. Additionally, steroid concentrations were highly correlated between frozen and lyophilized fecal samples (0.81 +/- 0.07 SEM), indicating that fluid removal from the feces did not effectively alter steroid profiles.

Long-term effects of prenatal stress on HPA axis activity in juvenile rhesus monkeys.

The effect of stress to the pregnant mother on hormonal responses of the offspring to stressful events was investigated in juvenile rhesus monkeys. Six pregnant monkeys were repeatedly removed from their home cages and exposed to unpredictable noise during mid- to late gestation (Days 90-145 postconception), while six undisturbed pregnant mothers served as controls. Blood samples were collected from the juvenile offspring under anesthesia on four occasions and assayed for ACTH and cortisol. In a second experiment, blood samples were collected from the awake offspring under a baseline and four progressively stressful conditions. Offspring of stressed mothers showed higher ACTH and cortisol levels than control offspring at all four anesthesia samples and at a nonanesthesized home cage baseline. Prenatally stressed offspring also showed higher ACTH values in all four stress conditions. Cortisol values were similar for the two groups under the stress conditions. The disparity between the two groups in the relationship between ACTH and cortisol was greatest in the most stressful condition, suggesting regulatory differences between the two groups. These results indicate that offspring of primate mothers stressed during pregnancy show enhanced HPA axis responsivity to stressors later in life, and concur with rodent findings indicating that prenatal stress may have long-term effects on HPA axis regulation.