Role of γ melanocyte-stimulating hormone-renal melanocortin 3 receptor system in blood pressure regulation in salt-resistant and salt-sensitive rats

Melanocortin 3 receptor (MC3-R) has high affinity and specificity to γ melanocyte-stimulating hormone (γMSH), a natriuretic peptide involved in regulation of blood pressure (BP) and sodium excretion. Recent studies showing increased MC3-R expression and elevated plasma γMSH in normal rats fed a high-salt diet support the role of this system in sodium homeostasis. We hypothesized that dysregulation of MC3-R response to dietary salt may contribute to salt retention and BP elevation in salt-sensitive hypertension. We examined renal MC3-R expression, plasma γMSH concentration, and response to MC3-R agonist and antagonist in Dahl salt-sensitive (DSS) and Dahl salt-resistant (DSR) rats fed high-salt (8%) or low-salt (0.07%) diets for 3 weeks. Consumption of high-salt diet significantly increased BP in the DSS but not the DSR group. High-salt diet led to a 5-fold increase in plasma γMSH and a 2-fold increase in renal MC3-R in DSR rats. Plasma γMSH and renal MC3-R abundance in DSS rats were maximally elevated on low-salt diet and remained unchanged on high-salt diet. Administration of MC3-R agonist melanotan II significantly lowered BP and raised fractional Na excretion in the DSR but not the DSS rats consuming high-salt diet. In contrast, MC3-R antagonist SHU9119 significantly raised BP and lowered fractional Na excretion in both groups. Thus, the data suggest that γMSH-renal MC3-R pathway is activated and appears to be biologically functional in the DSS rats.     

Expression profiles of LHβ, FSHβ and their gonadal receptor mRNAs during sexual differentiation of Xenopus laevis tadpoles

The gonadotropins, luteinising hormone (LH) and follicle stimulating hormone (FSH), are important hormones regulating reproductive biology in vertebrates, especially the processes of steroidogenesis and gamete maturation. Despite the role of gonadotropins during the reproductive cycle in amphibians is well established, much less is known about the functional maturation of the hypothalamus-pituitary-gonad axis during larval development. Therefore, the present study aimed to analyze the expression profiles of hypophyseal LHβ and FSHβ mRNA and of their corresponding gonadal receptors (LH-R, FSH-R) in Xenopus laevis tadpoles during their ontogeny and sexual differentiation. The first significant elevation of LHβ and FSHβ mRNA was observed at late premetamorphosis. A clear raise of LHβ mRNA was present during prometamorphic stages especially in males, while the LH-R only slowly increased during ontogeny with highest levels during metamorphic climax. In contrast, FSHβ mRNA expression only slightly increased during ontogeny, however in both sexes the FSH-R mRNA was considerably elevated at prometamorphosis and further at metamorphic climax. Our results suggest that LHβ and LH-R mRNA expression might be involved in initial maturation events of gametes, at least in males, while the gradually increase of FSH-R mRNA coincided with the advancing process of gamete maturation in both sexes. The present study provides for the first time evidence based on expression of gonadotropins and their corresponding gonadal receptors that the hypothalamus-pituitary-gonad axis evolves already at early stages of ontogeny and sexual differentiation in amphibians.     

Characterization of testicular expression of P450 17α-hydroxylase, 17,20-lyase in zebrafish and its perturbation by the pharmaceutical fungicide clotrimazole

The aim of the present study was to characterize P450 17α-hydroxylase/17,20-lyase (cyp17a1) expression in zebrafish and to assess the effect of the pharmaceutical clotrimazole, a known inhibitor of various cytochrome P450 enzyme activities, on testicular gene and protein expression of this enzyme as well as on the testicular release of 11-ketotestosterone (11-KT), a potent androgen in fish. We first showed that cyp17a1 is predominantly expressed in gonads of zebrafish, notably in male. In vivo, clotrimazole induced a concentration-dependent increase of cyp17a1 gene expression and Cyp17-I protein synthesis in zebrafish testis. Using zebrafish testicular explants, we further showed that clotrimazole did not directly affect cyp17a1 expression but that it did inhibit 11-KT release. These novel data deserve further studies on the effect of azole fungicides on gonadal steroidogenesis.     

Investigating the key membrane protein changes during in vitro erythropoiesis of protein 4.2 (?) cells (mutations Chartres 1 and 2)

Background

Protein 4.2 deficiency caused by mutations in the EPB42 gene results in hereditary spherocytosis with characteristic alterations of CD47, CD44 and RhAG. We decided to investigate at which stage of erythropoiesis these hallmarks of protein 4.2 deficiency arise in a novel protein 4.2 patient and whether they cause disruption to the band 3 macrocomplex.

Design and Methods

We used immunoprecipitations and detergent extractability to assess the strength of protein associations within the band 3 macrocomplex and with the cytoskeleton in erythrocytes. Patient erythroblasts were cultured from peripheral blood mononuclear cells to study the effects of protein 4.2 deficiency during erythropoiesis.

Results

We report a patient with two novel mutations in EPB42 resulting in complete protein 4.2 deficiency. Immunoprecipitations revealed a weakened ankyrin-1-band 3 interaction in erythrocytes resulting in increased band 3 detergent extractability. CD44 abundance and its association with the cytoskeleton were increased. Erythroblast differentiation revealed that protein 4.2 and band 3 appear simultaneously and associate early in differentiation. Protein 4.2 deficiency results in lower CD47, higher CD44 expression and increased RhAG glycosylation starting from the basophilic stage. The normal downregulation of CD44 expression was not seen during protein 4.2(−) erythroblast differentiation. Knockdown of CD47 did not increase CD44 expression, arguing against a direct reciprocal relationship.

Conclusions

We have established that the characteristic changes caused by protein 4.2 deficiency occur early during erythropoiesis. We postulate that weakening of the ankyrin-1-band 3 association during protein 4.2 deficiency is compensated, in part, by increased CD44-cytoskeleton binding.     

Involvement of soluble receptor activator of nuclear factor-κB ligand (sRANKL) in collagenase-induced murine osteoarthritis and human osteoarthritis

Joint destruction and excessive bone formation are associated with high expression of soluble receptor activator of nuclear factor-κB ligand (sRANKL). This study was undertaken to investigate the role of sRANKL in collagenase-induced osteoarthritis (CIOA) in mice and in patients with osteoarthritis (OA). The initial phase of CIOA was associated with severe proteoglycan depletion, decreased collagen density, and up-regulation of bone morphogenetic protein (BMP)-2. At the late stage of CIOA, bone remodeling was related with increased BMP2 and RANKL expression in the joints, high sRANKL, and decreased number of activated neutrophils in synovium. CIOA mice showed elevated plasma level of sRANKL but low RANKL expression on blood neutrophils. The percentage of RANKL-positive blood neutrophils was higher in patients with OA than in healthy individuals. Our data indicate that increased local and systemic levels of soluble RANKL might be indicative for OA disorders in mouse and human.     

Differential effects of 17β-estradiol and 11-ketotestosterone on the endocrine stress response in zebrafish (Danio rerio)

Sexually dimorphic stress responses are present in species across all vertebrate taxa and it has been suggested that these effects are mediated by circulating sex steroids. While a few species of fish have been identified as having a sexually dimorphic stress response, there is conflicting evidence as to the effects of sex steroids on the stress axis. In this study, we tested whether zebrafish exhibit a sexually dimorphic cortisol stress response and whether 17β-estradiol (E2) or 11-ketotestosterone (11KT) modulate the activity of the hypothalamic-pituitary-interrenal (HPI) axis. To accomplish this, we quantified the whole body cortisol response to a physical stressor, cortisol release in vitro, and the expression of key HPI axis regulating genes of control and E2- or 11KT-exposed zebrafish. Under control conditions no dimorphisms in the HPI axis were apparent at rest or in response to a standardized stressor. In contrast, E2-exposure blunted the cortisol response of male fish in vivo and in vitro and as well as corticotropin-releasing factor (crf) expression in the pre-optic area (POA) of the brain. While the expression of some interrenal genes was suppressed by E2-exposure, these changes occurred in both male and female zebrafish. 11KT-exposure increased whole-body cortisol of males at rest and vortex-exposed females, but had no impact on the rate of cortisol synthesis in vitro or on POA crf expression. Therefore, while we found no evidence that zebrafish exhibit a sexually dimorphic cortisol stress response, both E2 and 11KT can modulate the activity of the HPI axis in this species and do so via different mechanisms.     

GPR30 activation opposes estrogen-dependent uterine growth via inhibition of stromal ERK1/2 and estrogen receptor alpha (ERα) phosphorylation signals

Although estradiol-17β (E2)-regulated early and late phase uterine responses have been well defined, the molecular mechanisms linking the phases remain poorly understood. We have previously shown that E2-regulated early signals mediate cross talk with estrogen receptor (ER)-α to elicit uterine late growth responses. G protein-coupled receptor (GPR30) has been implicated in early nongenomic signaling mediated by E2, although its role in E2-dependent uterine biology is unclear. Using selective activation of GPR30 by G-1, we show here a new function of GPR30 in regulating early signaling events, including the inhibition of ERK1/2 and ERα (Ser118) phosphorylation signals and perturbation of growth regulation under the direction of E2 in the mouse uterus. We observed that GPR30 primarily localizes in the uterine epithelial cells, and its activation alters gene expression and mediates inhibition of ERK1/2 and ERα (Ser118) phosphorylation signals in the stromal compartment, suggesting a paracrine signaling is involved. Importantly, viral-driven manipulation of GPR30 or pharmacological inhibition of ERK1/2 activation effectively alters E2-dependent uterine growth responses. Overall, GPR30 is a negative regulator of ERα-dependent uterine growth in response to E2. Our work has uncovered a novel GPR30-regulated inhibitory event, which may be physiologically relevant in both normal and pathological situations to negatively balance ERα-dependent uterine growth regulatory functions induced by E2.     

Expressions of parathyroid hormone‐related protein (PTHrP) and PTH/PTHrP‐receptor (PTH/PTHrP‐R) in gastrointestinal stromal tumours (GISTs), leiomyomas and schwannomas

Background: Gastrointestinal stromal tumours (GISTs) are rare, c‐kit and CD34 positive, and different from other mesenchymal tumours of the gastrointestinal tract (e.g. leiomyomas and schwannomas). The purpose of this study was to investigate the roles of parathyroid hormone‐related protein (PTHrP) and parathyroid hormone/parathyroid hormone‐related protein‐receptor (PTH/PTHrP‐R) in the growth and differentiation of GISTs. Methods: Nineteen GISTs, six leiomyomas and five schwannomas were examined in this study. Results: All of the GISTs and leiomyomas, and four of the schwannomas (80.0%) were positive for PTHrP. Immunohistochemical staining revealed that all of the leiomyomas, 90% of the GISTs and 80% of the schwannomas expressed PTH/PTHrP‐R. Furthermore, both PTHrP and PTH/PTHrP‐R were expressed in the cytoplasm of identical cells in all of these tumours. Conclusion: Our results suggest that both PTHrP and PTH/PTHrP‐R play an important role in the growth and differentiation of GISTs, leiomyomas and schwannomas.     

Serum levels of IL-6 and its soluble receptor (sIL-6R) in Waldenström's macroglobulinemia

Abstract: Background: Interleukin‐6 (IL‐6) is a multifunctional cytokine that plays roles in the immune response, inflammation and hematopoiesis. Serum IL‐6 levels have reported to reflect disease severity and high tumor burden in multiple myeloma (MM) patients and to correlate with several other laboratory parameters. Serum‐soluble IL‐6 receptor (sIL‐6R) plays an agonist role in IL‐6 signaling, enhancing its biological activity tenfold. Purpose–Methods: We measured IL‐6 and sIL‐6R levels in 11 patients (7 male, 4 female, mean age 66.9 yr) with Waldenström's macrobulinemia (WM) using a commercially available enzyme‐linked immunoassay in order to investigate their biological role and to find any possible relationship with disease severity, tumor burden or response to treatment. Results: Serum IL‐6 and sIL‐6R concentrations at diagnosis were significantly higher than in healthy controls (Mann–Whitney U‐test, p<0.001 and p<0.01, respectively). Patients who were effectively treated had a significant reduction in IL‐6 levels (p=0.017). With regard to sIL‐6R levels, no specific tendency was observed. In some of the responsive patients the levels increased whereas in others they decreased. No correlation was found between IL‐6 and sIL‐6R levels at diagnosis (p=0.9, r=0.036) or after treatment (p=0.083, r=0.3). Conclusions: Our results suggest that IL‐6 may be a marker reflecting tumor burden, disease severity and response to treatment in WM. With regard to sIL‐6R, we believe that it does not seem to be of much value, and its role remains to be clarified. However, future studies are needed to confirm and further extend the present results.     

Identification of the resistance of a novel molecule heat shock protein 90α (HSP90α) in Microtus fortis to Schistosoma japonicum infection

Microtus fortis is a naturally resistant vertebrate host of Schistosoma japonicum by preventing completion of parasite's life cycle. Sera of M. fortis were found to have anti-schistosome effect in vitro and in vivo. In order to identify genes associated with the anti-schistosome effect of M. fortis, we screened a M. fortis marrow cDNA expression library by expression cloning and identified a 331-bp clone gC14.75. It was the homologue of heat shock protein 90α (HSP90α). Full-length of M. fortis HSP90α gene, Mf-HSP90α, was amplified according to gC14.75 and Cricetulus griseus HSP90α. To test the potential anti-schistosome function of Mf-HSP90α, we prepared conditioned medium of Mf-HSP90α and added it to schistosomula cultured in vitro. It caused 27.0% schistosomula death rate in 96 h, which was considerably higher than that of negative control. We transferred Mf-HSP90α by retroviral expression vector pLXSN into mice to investigate its anti-schistosome effect in vivo. Compared with those of DMEM injection control, mice injected with Mf-HSP90α recombinant retrovirus had 40.8% worm burden reduction and 57.9% reduction in liver eggs per gram (LEPG) indicating its anti-schistosome effect in vivo. Taken together, our results suggested Mf-HSP90α as a novel anti-schistosome molecule in vitro and in vivo.     

Gender-related expression of TRα and TRβ in the protandrous black porgy, Acanthopagrus schlegeli, during sex change processes

We cloned the thyroid hormone receptor α (TRα) and β (TRβ) cDNAs from the ovaries of the protandrous black porgy and compared the expression levels of TRα and TRβ mRNA during the sex change in black porgy. We observed that the TRα mRNA by quantitative real-time PCR and protein levels by Western blot were highest in the mature ovaries. Additionally, TRβ mRNA levels were only expressed highly in the mature ovaries when compared to any other gonadal stages. Then, we injected gonadotropin-releasing hormone analogue (GnRHa) to know the effects on TRs mRNA in immature black porgy. Injection with GnRHa resulted in a significant increase in TRα level while significantly reducing TRβ level after 12 h. We concluded that TRα was related in testicular development as well as ovarian development and TRβ was only affect to ovarian development in black porgy. These results will provide a framework for better understanding of the role of TRs during sex change processes in this fish.     

Convergence of IRBIT,phosphatidylinositol (4,5) bisphosphate,and WNK/SPAK kinases in regulation of the Na+-HCO3? cotransporters family

Fluid and HCO₃⁻ secretion is a vital function of secretory epithelia, involving basolateral HCO₃⁻ entry through the Na⁺-HCO₃⁻ cotransporter (NBC) NBCe1-B, and luminal HCO₃⁻ exit mediated by cystic fibrosis transmembrane conductance regulator (CFTR) and solute carrier family 26 (SLC26) Cl⁻/HCO₃⁻ exchangers. HCO₃⁻ secretion is highly regulated, with the WNK/SPAK kinase pathway setting the resting state and the IRBIT/PP1 pathway setting the stimulated state. However, we know little about the relationships between the WNK/SPAK and IRBIT/PP1 sites in the regulation of the transporters. The first 85 N-terminal amino acids of NBCe1-B function as an autoinhibitory domain. Here we have identified a positively charged module within NBCe1-B(37-65) that is conserved in NBCn1-A and all 20 members of the NBC superfamily except NBCe1-A. This module is required for the interaction and activation of NBCe1-B and NBCn1-A by IRBIT and their regulation by phosphatidylinositol 4,5-bisphosphate (PIP₂). Activation of the transporters by IRBIT and PIP₂ is nonadditive but complementary. Phosphorylation of Ser65 mediates regulation of NBCe1-B by SPAK, and phosphorylation of Thr49 is required for regulation by IRBIT and SPAK. Sequence searches using the NBCe1-B regulatory module as a template identified a homologous sequence in the CFTR R domain and Slc26a6 sulfat transporter and antisigma factor antagonist (STAS) domain. Accordingly, the R and STAS domains bind IRBIT, and the R domain is required for activation of CFTR by IRBIT. These findings reveal convergence of regulatory modalities in a conserved domain of the NBC that may be present in other HCO₃⁻ transporters and thus in the regulation of epithelial fluid and HCO₃⁻ secretion.Fluid and HCO₃⁻ secretion is a vital function of secretory epithelia that involves basolateral HCO₃⁻ entry through the Na⁺-HCO₃⁻ cotransporter (NBC) NBCe1-B and luminal HCO₃⁻ exit mediated by the concerted activity of cystic fibrosis transmembrane conductance regulator (CFTR) and members of the solute carrier family 26 (SLC26) transporter family (1). HCO₃⁻ secretion is osmotically active owing to an influx of Na⁺-2HCO₃⁻ (2, 3) and the exchange of Cl⁻/2HCO₃⁻ by Slc26a6 (4, 5), resulting in net osmolyte secretion in the form of HCO₃⁻. HCO₃⁻ secretion is a highly regulated activity, with several signaling pathways converging to regulate key transporters activity to tune the secretion (1); however, very little is known about the molecular mechanisms that regulate fluid and HCO₃⁻ secretion, particularly the regulation of NBCe1-B.NBCe1-B was originally designated pancreatic NBC 1 (6) and was later renamed NBCe1-B as a member of the electrogenic NBCe1 subfamily of the Na⁺-coupled bicarbonate transporter (NCBT) superfamily (2, 3). NBCe1-B is is expressed in the basolateral membrane of most secretory epithelia, including the pancreas, salivary glands, airway, and intestines (1). The NCBTs encompass 13 transmembrane domains with varying cytoplasmic N and C termini among the isoforms (3). Most members of the NCBT superfamily have a unique N terminus (the first 85 residues in NBCe1-B) (7). This domain has been shown to function as an autoinhibitory domain (AID) in NBCe1-B (8–10). Very little is known about the regulation of NBCe1-B or other members of the superfamily beyond that NBCe1-B may be modestly activated by cAMP (11, 12), although inhibition of NBCe1-B by cAMP was subsequently reported by the same group (13). NBCe1-B appears to be constitutively phosphorylated by protein kinase A in Thr49 (11); however, its role in activation of the transporter is not clear, given that both the T49A and T49D mutations were found to prevent activation by cAMP (11). Activation of NBCe1-B and NBCe1-C by intracellular Ca²⁺ through an unknown mechanism was reported recently (5). NBCe1-A is activated by phosphatidylinositol 4,5-bisphosphate (PIP₂) (14), but direct activation of NBCe1-B and NBCe1-C by PIP₂ has not been examined. The site of interaction of PIP₂ in regulating the activity of any NCBT family member remains to be determined.NBCe1-B (9, 10, 15, 16) and NBCe1-C (17) are potently activated by the inositol 1,4,5-triphosphate (IP₃) receptors binding protein released with IP₃ (IRBIT) and are inhibited by the with no lysine kinase (WNK) and Ste20-related proline alanine rich kinase (SPAK) (15). IRBIT also regulates CFTR (15, 16) and sodium-hydrogen exchanger 3 (NHE3) (18). IRBIT activates NBCe1-B by recruiting protein phosphatase 1 (PP1), reversing inhibition by the WNK/SPAK pathway through dephosphorylation of NBCe1-B (15) and relief of inhibition by the AID (9, 10, 15, 16). The WNKs function as scaffolds to recruit SPAK to NBCe1-B, which in turn phosphorylates the transporter at unknown sites. Similarly, the site of IRBIT–AID interaction is unknown. Deletion of the first 16 residues of NBCe1-B prevents activation by IRBIT (9), although the effect of this truncation on IRBIT binding is unclear. On the other hand, an in vitro binding assay revealed binding of IRBIT to NBCe1-B(1-62), but not to NBCe1-B(1-37) (10). This finding suggests that the IRBIT binding site may be located within NBCe1-B(37-62); however, the possible binding of IRBIT to NBCe1-B(37-62) or a site within has not yet been examined.The extent to which these pathways regulate other members of the NBC family is unknown, although IRBIT may activate an unspecified member of the NBCn1 subfamily (2). The NBCn1 subfamily was established with the discovery of NBC3, later renamed NBCn1-A. NBCn1-A is a widely expressed electroneutral NBC (3, 7) that mediates HCO₃⁻ salvage in secretory epithelia (19, 20). Given that sequence analysis has shown significant conservation of the N termini of NBCe1-B and NBCn1-A, we deemed it useful to compare the regulation of these NBCs by IRBIT/PP1, PIP₂, and SPAK to evaluate the generality of this regulation. In the present studies, we also investigated whether these multiple regulatory pathways converge on the same domain to regulate the transporters.We have identified a positively charged domain within NBCe1-B(37-65) that is conserved in NBCn1-A and most members of the NCBT superfamily and is required for interaction and activation of the transporters by IRBIT. The same domain mediates regulation of NBCe1-B and NBCn1-A by PIP₂. Importantly, activation of the transporters by IRBIT and PIP₂ is nonadditive but complementary. Phosphorylation of Ser65 within this domain mediates regulation of NBCe1-B by SPAK, and phosphorylation of Thr49 within NBCe1-B(37-65) is required for regulation by the activator IRBIT and the inhibitor SPAK. Moreover, a sequence search using the conserved module identified a similar module in Slc26a6 and CFTR that is required for regulation of CFTR by IRBIT. These findings reveal convergence of regulatory modalities in the AID of the NBCs and thus in the regulation of epithelial fluid and HCO₃⁻ secretion.     

Distribution of estrogen receptors (ERα and ERβ) and androgen receptor in the testis of big fruit-eating bat Artibeus lituratus is cell- and stage-specific and increases during gonadal regression

The testis is a classical target for androgens, especially testosterone, acting via androgen receptor (AR). Alternatively, androgens can be aromatized to produce estrogens which act via specific receptors ERα and ERβ. Although estrogen action is essential for maintenance of male fertility, studies regarding the expression of ERα and ERβ in testis are restricted to a few species of rodent and domestic animals, but rarely in wild species. To our knowledge, there are no studies in Chiroptera species. Chiroptera represent one of the largest and most diversified orders of mammals, which possess several interesting reproductive features, including higher affinity of SHBG for estrogens than androgens. Therefore, we thought that bats would constitute a good model for investigation of the role of estrogens in the male. In this study, the distribution of ERα, ERβ and AR were evaluated in the testis of the big fruit-eating bat Artibeus lituratus and their levels were compared during reproductive and regressive periods. The results showed that ERα and AR were restricted to the somatic cells of the testis, whereas ERβ was widely distributed in both somatic and spermatogenic cells in a cellular and stage-specific fashion. We demonstrated for the first time by immunohistochemistry, and confirmed by Western blotting, that ERβ and AR increased during regression. The localization of ERα, ERβ and AR in a seasonal, cell and stage-specific fashion in the testis of A. lituratus suggests that these receptors may play important roles in testis function during reproductive and non-reproductive periods.     

Monitoring reproduction in the critically endangered marsupial, Gilbert’s potoroo (Potorous gilbertii): Preliminary analysis of faecal oestradiol-17β, cortisol and progestagens

Gilbert’s potoroo (Potorous gilbertii) was rediscovered in 1994 after having been presumed extinct for 120 years. Estimates indicate fewer than 40 individuals remain at Two Peoples Bay Nature Reserve on the south coast of Western Australia although a translocated population of approximately 20 animals has recently been established on nearby Bald Island. A captive breeding facility has been established adjacent to the mainland population but few young have been produced (8 since 1995). Faecal levels of oestradiol-17β (E₂) were monitored over a 2-year period in an effort to determine cyclic reproductive activity, and faecal cortisol levels were also monitored to gauge whether chronic stress may be a factor limiting breeding in captivity.Faecal steroids were monitored in six captive females, and four captive male potoroos, and four wild females. The only captive births recorded after 1998 were one in August 1999 and one in February 2001, both to the same female. Peaks in E₂ concentration, up to 10 ng g⁻¹ of dried faecal mass were measured and results to date suggest the main breeding period to be November-December based on elevated E₂ levels at this time. Clear patterns of reproductive activity in the captive females, however, were not evident. Analysis of epithelial cell counts from urinogenital swabs and faecal E₂ and progestagen (PM) levels from a single female kept at the Perth Zoo, suggest that Gilbert’s potoroo has an oestrous cycle of approximately 39 days. Faecal cortisol levels in captive females were significantly lower than those in wild-caught individuals and thus there is no indication that elevated cortisol levels per se inhibited reproduction in captive females.