ACTH modulation on corticosterone, melatonin

Comparative Biochemistry and Physiology, Part A 204 (2017) 177–184

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ACTH modulation on corticosterone, melatonin, testosterone and innate immune response in the tree frog Hypsiboas faber Adriana Maria Giorgi Barsotti ⁎, Vania Regina de Assis, Stefanny Christie Monteiro Titon, Braz Titon Junior, Zulma Felisbina da Silva Ferreira, Fernando Ribeiro Gomes Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, travessa 14, 321, 05508-900 São Paulo, SP, Brazil

a r t i c l e

i n f o

Article history: Received 15 August 2016 Received in revised form 30 November 2016 Accepted 1 December 2016 Available online 05 December 2016 Keywords: ACTH Corticosterone Immunocompetence Melatonin Testosterone Stress

a b s t r a c t The modulation exerted by glucocorticoids in physiological responses to stressors is essential for maintaining short-term homeostasis. However, highly frequent and/or prolonged activation of the hypothalamic-pituitaryadrenal/interrenal axis may inhibit processes that are important to long-term fitness and health, including reproduction and immunocompetence. The present study evaluates the response to adrenocorticotropic hormone (ACTH) injection in the adult male tree frog, Hypsiboas faber, as indicated by levels of plasma corticosterone (CORT), plasma testosterone (T), ocular melatonin (MEL), hematocrit and immune functioning (total leukocyte count and bacterial killing ability against Escherichia coli). All levels were measured 1, 3 and 6 h after treatment. ACTH increased CORT levels whilst decreasing T and MEL levels at 1 h post-treatment. 6 h after ACTH injection, hematocrit and MEL levels increased. ACTH treatment did not significantly modulate the immune measures over the time-range sampled. The hormonal changes observed in response to ACTH treatment suggest that stressors could act as inhibitors of reproductive activity, as well as differentially modulating melatonin levels at different time-points. © 2016 Elsevier Inc. All rights reserved.

1. Introduction The stress response in vertebrates comprises the activation of the hypothalamic-pituitary-adrenal/inter-renal (HPA/I) axis, culminating with the increased secretion of glucocorticoids (Romero, 2004; Norris, 2007). The activation of the HPA/I axis in vertebrates modulates several physiological functions, such as intermediate metabolism, reproduction, immune response and growth, as well as contributing to an integrative and adaptive response to short-term stressors (Wingfield et al., 1997; Sapolsky et al., 2000; Wingfield and Romero, 2001; Sapolsky, 2002). However, chronic activation of the HPA/I axis can have harmful effects, including reproductive inhibition and immunosuppression, with significant implications for an array of species (Wingfield and Romero, 2001; Sapolsky, 1992, 2002; Narayan and Hero, 2014a, 2014b; Narayan et al., 2015). Decreased androgen plasma levels, spermatogenesis and libido, due to exposure to stressors of different intensities and durations, have been observed in males of several vertebrates, including amphibians (Romero et al., 2000; Romero and Butler, 2007; Sapolsky et al., 2000; Narayan et al., 2012; Narayan et al., 2013). This stress-induced reproductive inhibition is associated with decreased activity of the hypothalamic-pituitary-gonadal (HPG) axis, via multiple steps in the ⁎ Corresponding author at: Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, Trav. 14, 101, Lab. 300, 05508-090 São Paulo, Brazil. E-mail address: [email protected] (A.M.G. Barsotti).

http://dx.doi.org/10.1016/j.cbpa.2016.12.002 1095-6433/© 2016 Elsevier Inc. All rights reserved.

activation of the HPA/I axis, including elevated plasma levels of glucocorticoids (Sapolsky et al., 2000). However, the relation between stress response and reproduction may vary across species, including stimulation of reproduction by stressors in some species (Romero, 2002; Wingfield and Kitaysky, 2002). The relation between HPA/I axis activation and immune response is temporally and functionally complex, depending on the intensity and duration of the stress response (Sapolsky et al., 2000; Fernandes et al., 2009; Martin, 2009). Dhabhar and colleagues showed that increased plasma epinephrine and glucocorticoid levels that are associated with the acute stress response, redistributes leukocytes between blood and other compartments, culminating in a reduced number of circulating leukocytes and an enhanced local immune response (Dhabhar et al., 1996). Moreover, an acute response to stressors is also associated with increased levels of antigen presentation, cell effector function, antibody production and proinflammatory cytokines (Dhabhar, 1996; Dhabhar and McEwen, 1996; Dhabhar, 1997; Dhabhar and McEwen, 1997). In contrast, chronic stress, and associated glucocorticoids, can suppress maturation, differentiation and proliferation of several immune cells (Sterberg, 2006; Martin, 2009), and trigger apoptosis of mature T cells and immature T and B cell precursors (Sapolsky et al., 2000). A complex immune response to stressors has also been observed in amphibians (Hopkins and DuRant, 2011; Graham et al., 2012; Gomes et al., 2012). In toads (Rhinella icterica), for example, the exposure to a moderate acute stressor (restraint without movement restriction for 24 h) increased plasma corticosterone (CORT) levels by 3-fold without

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A.M.G. Barsotti et al. / Comparative Biochemistry and Physiology, Part A 204 (2017) 177–184

effects on immune parameters. Exposure to a more intense acute stressor (restraint with movement restriction for 24 h) increased CORT 9-fold, whilst also increasing neutrophil/lymphocyte ratio (N:L) and decreasing plasma bacterial killing ability (BKA) (Assis et al., 2015). Furthermore, keeping R. icterica in captivity for three months led to a further BKA decrease along with a 3-fold increase in CORT (Assis et al., 2015). In addition to inter-renal hormones, melatonin also has immunomodulatory effects in birds and mammals (Ortega et al., 1996; Rodriguez et al., 1999; Barriga et al., 2002). In healthy animals, pineal melatonin acts as an anti-inflammatory mediator, controlling the bone marrow cell flow to blood and tissues (Cavalcanti et al., 2007; Fernandes et al., 2009) and partially inhibiting leukocyte rolling and adhesion on endothelium (Lotufo et al., 2001, 2006; Fernandes et al., 2009). In rats, corticosterone shows a dual modulation of the sympathetic-induced melatonin synthesis accordingly to different phases of the inflammatory response (Markus et al., 2007). During a proinflammatory phase, glucocorticoids inhibit pineal melatonin secretion favoring leukocyte migration to the injured site (Lotufo et al., 2001; Fernandes et al., 2009). However, during the resolution phase, the lower sympathetic output allows glucocorticoids to act synergistically with noradrenaline, recovering nocturnal melatonin pineal secretion and thus contributing to inhibition of leukocytes transmigration, as well as decreasing inflammatory process (Markus et al., 2007). In addition, activated leukocytes at inflammatory sites produce melatonin, contributing to the recovery phase and dampening the inflammatory response (Markus and Ferreira, 2011). In ectothermic vertebrates such as amphibians, the retina is the main source of melatonin (Pang and Allen, 1986; Delgado and Vivien-Roels, 1989; Skene et al., 1991; Valenciano et al., 1997). Studies conducted in birds have suggested that melatonin produced in the retina does not have an immunomodulatory role (Moore et al., 2002). Although levels of ocular melatonin (MEL) contribute to plasma melatonin in amphibians (Delgado and Vivien-Roels, 1989), there are no studies on its relationship with the immune system in these animals. Increased hematocrit is another stress-induced physiological response in tetrapods (Hart, 2006; Johnstone et al., 2015). Stress-driven increased hematocrit has been causally associated with different physiological mechanisms, including: increased blood pressure; catecholamines-induced splenic contraction (Hart, 2006); stimulated release of immature erythrocytes from bone marrow; and increased erythropoiesis (Fisher and Crook, 1962; Schall et al., 1982; O’Brien et al., 2001; Teague et al., 2007; Hart, 2006; Johnstone et al., 2015). Amphibian populations show severe decline, with many species driven to extinction (Phillips, 1990; Vial and Saylor, 1993; Carey et al., 1999; Wake and Vredenburg, 2008). Several stressors have contributed to this, including increased ultraviolet radiation, climate change, habitat loss, pollution and pathogens, as well as combinations thereof (Daszak et al., 1999). Given the relative lack of knowledge on the physiological effects of the stress response in amphibians, the objective of the present study was to evaluate ACTH injection effects on CORT and plasma levels of testosterone (T), MEL and hematocrit as well as indicants of immunocompetence (total leukocytes count and plasma BKA) in Hypsiboas faber males. Measures were taken at 1, 3 and 6 h after ACTH injection, to monitor any temporal alterations of these measures. The following hypotheses were tested: ACTH injection will promote: 1) increase CORT in a period of 30 to 60 min; 2) reduce T and MEL; 3) decrease total leukocytes count and BKA; and 4) increase hematocrit. 2. Materials and methods 2.1. Ethical procedures All procedures for the collection and use of biological material were performed under the approval of the Comissão de Ética no Uso de Animais (CEUA, process number 145/2011), of Instituto de Biociências da Universidade de São Paulo. The animals were collected under a

license for capture from Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA, process number 29896-1). 2.2. Collection site, species studied and maintenance in captivity Hypsiboas faber (sensu Faivovich et al., 2005) is a large Hylidae, with wide geographical distribution, being found near permanent rivers and lakes in Atlantic rain-forests from Argentina to Northeastern Brazil (Martins and Haddad, 1988). Thirty one adult males were collected in October 2012, at the Jardim Botânico de São Paulo (23° 38′‘ 08″ S, 46° 36′‘ 48″ W) – São Paulo/Brazil. These individuals were transported to the laboratory and kept individually in plastic boxes [20 L - 43.0 (L) × 28.5 (W) × 26.5 (H) cm] with free access to water. Each box contained a PVC pipe cut (10 × 12 cm) to be used as shelter for the animals. The lids of the boxes had holes to allow air circulation. Animals were kept within a climatic chamber in the Physiology Department, Institute of Biosciences, University of São Paulo (23° 33′‘ 45″ S, 46° 43′‘ 40″ W) for two months at a light;dark (LD) 13:11 h, with 13 h of light (light turned on 06:30 h) and 11 h of dark (light turned off 19:30 h) at 22 ± 2 °C. The animals were fed with crickets (Gryllus sp.) and cockroaches (Pcynoscelus sp.) offered once a week. Additionally, 9 males were collected in November 2013 at the city of Luiz Antônio, São Paulo/Brazil (21° 22′ 18″ S, 47° 42′ 14″ W), and 6 males in January 2014 at the Jardim Botânico de São Paulo. These animals were kept for two weeks under the same conditions described above, and used for an additional experiment designed to corroborate the influence of light on ocular melatonin levels (item 2.5). 2.3. Exogenous treatment with ACTH (adrenocorticotropic hormone) After two months held in captivity, the animals collected in October 2012 were divided into two groups. Animals at the experimental group (N = 15) were subjected to an intraperitoneal injection of ACTH (Sigma-Aldrich, A6303 - 0.446 μg g−1, as in Narayan, 2011), diluted in saline (0.9% NaCl). Individuals in the control group (N = 15) were injected with an equivalent volume of saline (0.9% NaCl). The injections were performed at 19:30 h with the assistance of red lights, immediately after the light were turned off in the climatic chamber. The animals were returned to their individual containers immediately after injections until further processing. 2.4. Blood samples and eye collection Blood samples from five individuals of each group (injected with ACTH or saline) were collected at: 20:30 h (1 h after injection), at 23:30 h (3 h after injection) and 01:30 h (6 h after injection). Animals were manually removed from their individual containers and a blood sample was collected (200 μL) via cardiac puncture by using previously heparinized 1 mL syringes and 26 G × 1/2″ needles. Blood samples were only considered for analyses if collected within 3 min, given that CORT can be influenced by the stress of capture and handling after 3 min (Romero and Reed, 2005). All blood samples were identified and kept on ice until further processing on the same night. Blood samples were transferred to microcentrifuge tubes, and an aliquot was separated for total leukocyte count (TLC) and leukocyte profile, with hematocrit analyses using microhematocrit tubes. The second aliquot was centrifuged (4 min at 218g), and plasma samples were aliquoted into separate microcentrifuge tubes, being kept in a − 80 °C freezer. These plasma samples were used later for BKA assays, and determination of CORT and T. After blood collection, the snout-vent length (0.01 mm) and body mass (0.01 g) of the animals were measured, and they were euthanized with intraperitoneal injection of sodium thiopental (Thiopenthax®) solution (25 mg/mL), at a dose of 75 mg/kg (Close et al., 1996; Close et al., 1997). The collection of the eyes was carried out according to Wright et al. (1999). The eyes were dissected and frozen in a −80 °C freezer, for


posterior measurements of melatonin levels. Blood collection, euthanasia and eyes dissection were performed in the dark, with the assistance of red lights. 2.5. Influence of light on ocular melatonin levels Animals collected in 2013 and 2014 (Section 2.2) were mixed and randomly divided in two groups. One group had their eyes collected 1 h before lights off (18:30 h), and the other group had their eyes collected 1 h after lights off (20:30 h). The animals were euthanized under the same protocol (see Section 2.4), and the eyes were subsequently processed for melatonin according to the procedures described (see Section 2.8). The effect of light on MEL was clearly indicated, with animals sampled 1 h after lights off showing a two-fold increase versus 1 h before lights off (t = −2.541, df = 10, P = 0.029; Fig. 3). MEL from individuals sampled 1 h after lights off did not differ from those injected with saline or those receiving no injection (t = 0.274, df = 3, P = 0.802). 2.6. Blood parameters 2.6.1. Total leukocytes count (TLC) On the same night as the blood collection, 5 μL of blood was diluted on 120 μL of toluidine blue saline solution (0.01%). 10 μL of this dilution was placed in a hemocytometer, and the TLC was performed in an optical microscope (40× lens, Nikon E200-104c). The number of leukocytes counted was multiplied by the dilution factor (25×). 2.6.2. Leukocyte profile A drop of blood was pipetted on a microscope slide and a smear was performed with a second slide. The smear was dried for 30 min and then fixed for 2 min with methanol. The slides were made in duplicate for each individual, and one of them was stained with Giemsa solution (10%) for 15 min and observed through optical microscopy at 1000 × magnification (Nikon E200, 104c), using immersion oil. One hundred white blood cells were counted and identified based on cellular morphology in amphibians, according to Campbell (2006). The N:L was calculated as the number of neutrophils divided by the number of lymphocytes counted on the slides. 2.6.3. Hematocrit (HEM) The HEM was calculated as the proportion of red blood cells in relation to the total volume of blood after centrifuging the blood contained in the microhematocrit tube (4 min at 218g).

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2.8. Hormonal assays For the determination of CORT and T, steroids were initially extracted with ether according to Mendonça et al. (1996). 3 mL of ethyl ether was added in 10 μL of each plasma sample, the mixture was vortexed for 30 s, and then centrifuged at 4 °C for 9 min at a g-force of 218. Subsequently, the samples were frozen at −80 °C for 7 min, then decanted. The liquid phase was then transferred to new test tubes, which were kept in a laminar flow hood at room temperature (20 ± 2 °C) for ether evaporation. CORT and T were measured via ELISA kits (Cayman Chemical, cat no. 500655 and cat no. 582701, respectively). Before the test, the samples were resuspended in assay buffer. The assays were carried out according to the manufacturer's instructions. Intra-assay variation for CORT and T were, respectively, 8.63% and 7.80%. The inter-assay variation, estimated using the average of four intermediate values from the standard curve (as recommended by the kit instructions), for CORT and T were, respectively, 8.59% and 7.35%. Sensitivity of the assays (mean ± SD), calculated as 80% B/B0 of curve value, was 38.48 pg/mL for CORT and 5.84 pg/mL for T. Validation of the use of the corticosterone assay kit from Cayman Chemicals (number 500655) for tree frogs, was conducted with a parallelism test, including plasma samples of H. faber under baseline condition. Pooled plasma samples (250 μL) were initially extracted with 5 mL of ethyl ether and followed the same procedures mentioned above (according to Mendonça et al., 1996). At the end, these pooled samples were resuspended and diluted in EIA buffer. The top standard of the corticosterone kit and the pooled plasma samples were used for a serial dilution (neat, 1:2, 1:4; 1:8, 1:16, 1:32, 1:64 and 1:128) and assayed on the same plate. The standard and sample curves were plotted on the same XY axes, and the 50% binding point was considered indicative of the best dilution factor to run the samples. The standard and sample curves were parallel, not crossing each other (Fig. 1), corroborating the functionality of the assay for tree frogs. The best dilution factor for baseline pooled plasma samples from H. faber corresponds to 1:32 (Fig. 1). The eyes were partially defrosted and homogenized in 700 μL of PBS. After homogenization, the mixture was centrifuged at 218g for 20 min at room temperature, and the supernatant was maintained at −20 °C for subsequent measure of melatonin. For MEL determination, the samples were initially extracted through silica columns (Waters Sep-Pak® Vac) and were measure with ELISA kits (IBL-RE54021, Hamburg, Germany), according to manufacturer's instructions. The analytical sensitivity of the melatonin assay was 1.6 pg/mL. The intra- and inter-assay coefficients of variation (CV %) were 0.2–3.7% and 7.0–14.9% respectively.

2.7. Plasma bacterial killing ability (BKA) The plasma BKA of H. faber against Escherichia coli (non-pathogenic Microbiologics, # 24311-ATCC 8739), in vitro, was evaluated according to Assis et al. (2013). Briefly, plasma samples diluted (1: 20) in Ringer's solution (10 μL plasma: 190 μL Ringer) were mixed with 10 μL of E. coli working solution (~104 microorganisms). Positive controls consisted of 10 μL of E. coli working solution in 200 μL of Ringer's solution, and negative control contained 210 μL of Ringer's solution. All samples and controls were incubated for 60 min at 37 °C. After the incubation time, 500 μL of TSB was added to each sample. The bacterial suspensions were thoroughly mixed and 300 μL of each sample was transferred (in duplicate) to a 96 wells microplate. The microplate was incubated at 37 °C for 2 h, and thereafter the optical density of the samples was measured hourly in a plate spectrophotometer (wavelength 600 nm), totaling 4 readings. The BKA was calculated according to the formula: 1 − (optical density of sample / optical density of positive control), which represents the proportion of killed microorganisms in the samples compared to the positive control. The BKA was evaluated at the beginning of the bacterial exponential growth phase.

Fig. 1. Parallelism curve. Binding displacement curves of serially diluted Hypsiboas faber pooled plasma at baseline conditions, against the corticosterone standard used in the corticosterone enzyme-immunoassay. The y-axis shows the % Hormone Bound/Total Binding measured at 412 nm. The 50% binding point is denoted using a dashed line, which determined dilution factors for the extracted plasma samples.

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2.9. Statistical analysis All data were initially submitted to descriptive statistics and the Shapiro-Wilk normality test. The following variables were transformed to log10 to conform to the prerequisites of parametric tests: neutrophil counts, basophil counts, monocyte counts, N:L, CORT and T. Two tailed t-tests for independent samples were used to compare ACTH and saline groups, at each time sampled, in order to verify the effects of the ACTH treatment on all variables tested, except T. A t-test was also used to compare the individuals sampled 1 h after lights off, with and without saline injection. One-tailed t-tests for independent samples were used to assess if T is lower after the injection of ACTH when compared to the injection of saline on each time sampled. One-way ANOVA were used to evaluate the temporal response of all dependent variables to the treatments with ACTH and saline. All ANOVAs were followed by tests for mean multiple comparisons, using the Bonferroni adjustment. All statistical analysis was performed in SPSS 13.0 for windows. 3. Results

Parameter

N

Group

Minimum

Maximum

Mean ± SD

HEM (%)

5 6 5 6 5 6 5 6 5 6 5 6 4 5 5 6 5 6 5 6 5 6 5 6 5 6

Saline ACTH Saline ACTH Saline ACTH Saline ACTH Saline ACTH Saline ACTH Saline ACTH Saline ACTH Saline ACTH Saline ACTH Saline ACTH Saline ACTH Saline ACTH

11.0 18.0 75.0 625.0 0.44 0.3 0.0 0.0 6.0 2.2 0.3 0.4 0.0 0.0 26.8 21.5 22.0 19.0 0.0 1.0 0.0 2.0 13.0 16.0 1.0 3.0

43.0 36.0 1100.0 1175.0 5.3 4.4 68.0 58.0 47.4 86.9 14.3 4.7 0.2 0.2 52.9 43.7 69.0 75.0 8.0 8.0 17.0 15.0 50.0 63.0 21.0 12.0

27.4 ± 12.1 24.6 ± 7.1 650.0 ± 448.2 937.5 ± 184.2 2.1 ± 1.9 2.0 ± 1.9 17.0 ± 29.4 22.1 ± 25.6 25.0 ± 16.1 39.7 ± 32.1 7.3 ± 6.1 1.5 ± 1.6 0.1 ± 0.0 0.1 ± 0.0 39.3 ± 10.1 36.6 ± 8.0 48.8 ± 18.1 45.8 ± 23.4 4.2 ± 3.0 6.5 ± 2.7 5.4 ± 6.8 5.6 ± 4.8 32.4 ± 14.2 36.5 ± 20.5 9.2 ± 7.3 5.5 ± 3.2

TLC (cells/μL) N:L BKA (%) CORT (ng/mL) TEST (ng/mL) MEL (ng/mL) Mass (g) Leukocyte profile (%)

The descriptive statistics of the experiments investigating intraperitoneal ACTH injection and the influence of light on MEL are shown in Tables 1, 2, 3 and 4. Animals treated with ACTH showed 6.7 times higher CORT versus the saline group, at 1 h after injection (t = −8.138, df = 7, P ≤ 0.0001; Fig. 2A). ACTH treatment resulted in a 7-fold decrease in T (t = 2.316, df = 3, P = 0.050 [1-tailed]; Fig. 2B) and 2.5-fold decrease in MEL (t = 2.689, df = 5, P = 0.041; Fig. 3) versus the saline group, at 1 h after injection. There were no significant differences in the ACTH and saline groups in any variables 3 h after injection (P ≥ 0.150). Hematocrit (t = −2.488, df = 9, P = 0.035; Fig. 2C) and MEL (t = − 2.909, df = 5, P = 0.033; Fig. 3) were 1.5 times higher after ACTH versus saline injection, 6 h after treatment. ACTH treatment did not affect any of the immune aspects studied within the time range sampled (P ≥ 0.183). There was a significant temporal difference

Table 1 Descriptive statistics of blood parameters, bacterial killing ability, corticosterona and testosterone plasma levels, melatonin ocular levels and body mass for individuals of Hypsiboas faber 1 h after experiment. Parameter

N

Group

Minimum

Maximum

Mean ± SD

HEM (%)

5 4 5 4 5 4 5 5 5 4 2 3 4 4 5 5 5 4 5 4 5 4 5 4 5 4


11.0 18.0 225.0 475.0 0.07 0.14 0.0 0.0 11,3 74,2 2,2 0.35 0.0 0.0 22.5 29.2 6.0 11.0 0.0 0.0 1.0 2.0 40.0 55.0 2.0 2.0

27.0 33.0 1100.0 1250.0 1 0,5 84.0 78.0 21.2 177.2 10.6 1.2 0.2 0.1 41.3 42.7 43.0 31.0 3.0 5.0 12.0 4.0 82.0 79.0 13.0 10.0

19.8 ± 6.0 25.7 ± 6.1 655.0 ± 345.6 900.0 ± 363.4 0.4 ± 0.4 0.3 ± 0.1 16.8 ± 37.5 15.6 ± 34.8 15.7 ± 4.2 105.1 ± 48.7 6.4 ± 5.9 0.9 ± 0.4 0.1 ± 0.0 0.0 ± 0.0 33.5 ± 7.3 36.7 ± 5.2 22.4 ± 14.3 20.7 ± 8.6 1.4 ± 1.3 2.2 ± 2.2 6.2 ± 5.4 2.7 ± 0.9 63.6 ± 16.7 68.2 ± 12.0 6.4 ± 4.8 6.0 ± 4.0


Table 2 Descriptive statistics of blood parameters, bacterial killing ability, corticosterona and testosterone plasma levels, melatonin ocular levels and body mass for individuals of Hypsiboas faber 3 h after experiment.

Neutrophil Eosinophil Basophil Lymphocyte Monocyte

HEM: Hematocrit; TLC: Total Leukocytes Count; N:L: Neutrophil/Lymphocyte Ratio; BKA: Bacterial Killing Ability; CORT: Corticosterone Plasma Levels; T: Testosterone Plasma Levels; MEL: Melatonin Ocular Levels; ACTH: Adrenocorticotropic Hormone; SD: standard deviation.



following ACTH injection in regard to CORT (F2,13 = 4.226, P = 0.039; Fig. 2A) and MEL (F2,9 = 5.996, P = 0.022; Fig. 3). The difference was observed between 1 h and 6 h after ACTH injection, for both CORT (P = 0.041) and MEL (P = 0.025). 4. Discussion In Hypsiboas faber males ACTH injection increased CORT and decreased T and MEL, 1 h after treatment. Moreover, ACTH injection increased MEL and hematocrit 6 h after treatment. ACTH treatment therefore leads to physiological changes that are variable over time, which is consistent with the stress response observed in several groups of vertebrates (Hunt et al., 2004; Cockrem, 2007; Narayan et al., 2010). Immune measures, as investigated in this study, were not affected in the temporal range sampled. The increase in CORT in response to ACTH injection was expected, given that CORT is the main glucocorticoid in amphibians, where its secretion is stimulated by ACTH (Norris, 2007). A peak in CORT was detected in H. faber 1 h after ACTH injection, returning to values close to those found in the group injected with saline, 3 h after treatment. Concomitant to the CORT elevation, T decreased 1 h after ACTH administration. Previous studies have indicated a negative correlation of plasma CORT with androgen levels and manifestation of vocal behavior in anurans when CORT is high, either as a result of natural causes or experimental induction (Burmeister et al., 2001; Leary et al., 2006; Marler and Ryan, 1996; Orchinik et al., 1988). However, the current investigation found no evidence of such a negative correlation between CORT and T in male H. faber. Although, by different mechanisms, glucocorticoids can inhibit the mammalian HPG axis (Sapolsky et al., 2000). Glucocorticoids decrease gonadotropin releasing hormones secretion; reduce gonadol luteinizing hormone receptor levels (Johnson et al., 1982; Sapolsky, 1985; Hayashi and Moberg, 1990; Sapolsky et al., 2000); and inhibit gonadal steroid synthesis (Norris and Lopez, 2011). Furthermore, CORT can compete with T for the binding sites of


181

Table 3 Descriptive statistics of blood parameters, bacterial killing ability, corticosterona and testosterone plasma levels, melatonin ocular levels and body mass for individuals of Hypsiboas faber 6 h after experiment. Parameter

N

Group

Minimum

Maximum

Mean ± SD

HEM (%)

5 5 5 5 5 5 5 5 5 5 5 5 4 2 5 5 5 5 5 5 5 5 5 5 5 5


11.0 25.0 525.0 125.0 0.1 0.1 0.0 0.0 1.8 3.2 0.2 0.3 0.0 0.1 29.7 26.4 9.0 11.0 0.0 0.0 3.0 3.0 37.0 36.0 2.0 7.0

28.0 33.0 1125.0 900.0 0.7 1.2 100.0 36.0 38.3 26.3 2.6 6.0 0.1 0.2 45.8 45.4 38.0 45.0 25.0 4.0 7.0 11.0 80.0 76.0 10.0 19.0

19.6 ± 7.6 29.0 ± 3.7 675.0 ± 253.1 675.0 ± 318.6 0.4 ± 0.2 0.5 ± 0.4 28.6 ± 44.0 13.8 ± 18.9 24.4 ± 16.2 17.5 ± 9.3 0.9 ± 0.9 2.2 ± 2.3 0.1 ± 0.0 0.1 ± 0.0 35.8 ± 6.6 36.8 ± 7.7 24.0 ± 10.9 27.0 ± 15.1 7.0 ± 10.2 1.0 ± 1.7 4.4 ± 1.6 6.8 ± 3.0 59.4 ± 17.1 54.8 ± 16.1 5.2 ± 3.2 10.4 ± 5.0




corticosterone binding globulins (Deviche et al., 2001; Narayan et al., 2011), leading to an acute increase in free T, which can negatively feedback on the HPG axis, thereby reducing T secretion during the stress response (Narayan et al., 2011). A typical night-time MEL increase was observed in H. faber, a result consistent with studies conducted in many species with a day or nighttime active phase (Jessop et al., 2002, 2014). Moreover, this nocturnal pattern of increased MEL was temporally differentially affected by ACTH treatment, with MEL decreasing at 1 h after lights off versus saline injection, although MEL was increased at 6 h following ACTH injection. ACTH transiently increases noradrenaline production by the superior cervical ganglia in rats (Sabban et al., 2004; Serova et al., 2008). According to Markus and Ferreira (2011), the noradrenaline elevation during an acute stress response may stimulate pineal α1 and β adrenoceptors, with this noradrenaline increase, in association with raised glucocorticoid levels, reducing melatonin production (Markus and Ferreira, 2011). Accordingly, night-time physical activity in toads, turtles, birds and humans reduces pineal melatonin production, indicating that increased HPA/I axis activity triggers a transient reduction of pineal activity in different tetrapods (Montelone et al., 1990; Van Reeth et al., 1994; Gwinner, 1996; Buxton et al., 1997; Jessop et al., 2002, 2014). Interestingly, 6 h after lights off, the ACTH treatment increased MEL and CORT versus saline injection. These results are consistent with those obtained by experiments carried out in the cultured rat pineal gland, where moderate concentrations of corticosterone stimulate melatonin

Table 4 Descriptive statistics of melatonin ocular levels for individuals of Hypsiboas faber in the light and dark controls. Parameter

N

Group

Minimum

Maximum

Mean ± SD

MEL (ng/mL)

6 6

Light Dark

0.020 0.070

0.090 0.190

0.05 ± 0.02 0.11 ± 0.05

MEL: Melatonin Ocular Levels; SD: standard deviation.

Fig. 2. A. Corticosterone plasma levels post ACTH treatment. Corticosterone plasma levels post ACTH treatment of adult males of Hypsiboas faber after saline and ACTH injection in three separate times (1 h, 3 h and 6 h after the light turned off). The bars represent mean ± standard error; ⁎represents significant differences between the groups injected with saline and ACTH after 1 h (P ≤ 0.0001); #represents the temporal response of corticosterona plasma levels after ACTH injection (P ≤ 0.039). B. Testosterone plasma levels post ACTH treatment. Testosterone plasma levels post ACTH treatment of adult males of Hypsiboas faber after saline and ACTH injection in three separate times (1 h, 3 h and 6 h after the light turned off). The bars represent mean ± standard error; ⁎represents significant differences between the groups injected with saline and ACTH after 1 h (P ≤ 0.05 [1-tailed]). C. Hematocrit variation post ACTH treatment. Hematocrit of adult males of Hypsiboas faber after saline injection and ACTH in three separate times (1 h, 3 h and 6 h after the light turned off). The bars represent mean ± standard error; ⁎represents significant differences between the groups injected with saline and ACTH after 6 h (P ≤ 0.035).

Fig. 3. Melatonin ocular levels. Melatonin ocular levels of adult males of Hypsiboas faber on light control (1 h before the light turned off) and dark control (1 h after the light turned off) and after saline injection and ACTH in three separate times (1 h, 3 h and 6 h after the light turned off). The bars represent mean ± standard error; ⁎represents significant differences between the controls groups and groups injected with saline and ACTH after 1 and 6 h (⁎P ≤ 0.029; ⁎⁎P ≤ 0.041; ⁎⁎⁎P ≤ 0.033); #represents the temporal response of melatonin ocular levels after ACTH injection (P ≤ 0.022).

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synthesis induced by noradrenaline (Ferreira et al., 2005; Fernandes et al., 2006; Fernandes et al., 2009; Markus and Ferreira, 2011). According to Fernandes (2009), corticosterone enhances the production of noradrenaline-induced melatonin at the range of noradrenaline concentrations known to activate only β adrenoceptor in the pineal gland (Fernandes, 2009). In this way, these results are consistent with the differential temporal effect of the HPA/I axis stimulation on the immune-pineal axis during the acute stress associated with an inflammatory event (Markus and Ferreira, 2011), suggesting that this is an evolutionarily conserved physiological mechanism. Although there are no studies on the effects of glucocorticoids on retinal melatonin production, it is plausible that it is similar to its role on pineal melatonin production. Additionally, the role of this modulation on the control of leukocyte trafficking and associated inflammatory processes in amphibians remains to be investigated. The hematocrit level of the control group peaked 3 h after saline injection, thereafter decreasing, whilst the hematocrit level following ACTH treatment remained high at the three time-points. Hutchison and Hazard (1984) showed that the hematocrit of Rana berlandieri shows a circadian rhythm, with one peak at 12:00 midnight. This peak coincides with the increased hematocrit values of H. faber 3 h after saline injection (11:30 pm). Although we sampled the hematocrit for only a short period, it is possible that the temporal changes observed in the control group reflect its circadian rhythm. Otherwise, the maintenance of high hematocrit values for the group injected with ACTH resembles the increased hematocrit values following the exposure of different tetrapods to stressors. Increased hematocrit following shortterm stress has been causally associated to increased arterial pressure (Patterson et al., 1995; Johnstone et al., 2015) or to splenic contraction induced mainly by catecholamines (Baković et al., 2003, 2005, Abdo, 2013, Hart, 2006), which are synthesized and secreted in response to ACTH in fish, reptiles and amphibians (Laforgia and Varano, 1978; Perry et al., 1993; Bernier and Perry, 1996; Reid et al., 1996; Laforgia and Muoio, 1997; Capaldo et al., 2004). Several authors have also associated the increased hematocrit following frequent or prolonged exposure to stressors, as well as ACTH injection, with stimulated release of immature erythrocytes from the bone marrow and/or increased erythropoiesis (Fisher and Crook, 1962; Schall et al., 1982; O’Brien et al., 2001; Teague et al., 2007; Johnstone et al., 2015). Given the short-time period we monitored physiological changes following ACTH injection in H. faber, it is more plausible that the increased hematocrit observed might be due to increased blood pressure and/or splenic contraction, but further studies are necessary to elucidate these relations. BKA showed no changes in response to ACTH treatment in H. faber. Changes in immune parameters in response to stressors, such as in leukocyte counts, can take hours to days for a full response in several vertebrates, including amphibians (Bennett and Harbottle, 1968; Bennett et al., 1972; Davis et al., 2008). Submission to restraint stress involving movement restriction resulted in a 10% decrease in BKA after 12 h in Rhinella marina (Graham et al., 2012) and after 24 h in R. icterica (Assis et al., 2015). Maintenance in captivity for three months also decreased BKA of R. icterica toads by 41% (Assis et al., 2015). It is therefore likely that the time of blood sampling after ACTH treatment was insufficient to observe changes in BKA resulting from ACTH injection in H. faber. As previously mentioned, the changes in CORT and MEL observed in response to ACTH treatment in H. faber resemble those that have been temporally associated to profound changes in inflammatory response in rats (Couto-Moraes et al., 2009). It is therefore possible that other aspects of the innate immune response, perhaps especially cell-mediated immune responses, may have a temporal influence associated to the hormonal changes observed in this study. The results of this study indicate that ACTH injection increases CORT and hematocrit and reduces T, whilst having differential temporal effects on MEL in Hypsiboas faber. The hormonal changes observed in response to ACTH treatment suggest that stressors could act as inhibitors of reproductive activity, as well as modulating MEL, which also has gonadal regulatory effects. Additional studies are needed to

extend and clarify the influence of stress intensity and duration on the variables tested, as well as to which aspects of the immune response may be influenced by the CORT modulation of MEL.

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