Structural modifications of non-mammalian gonadotropin-releasing hormone (GnRH) isoforms: design of novel GnRH analogues

Three natural forms of vertebrate gonadotropin-releasing hormone (GnRH) provided the structural basis upon which to design new GnRH agonists: [His⁵,Trp⁷,Leu⁸]-GnRH, dogfish (df) GnRH; [His⁵,Asn⁸]-GnRH, catfish (cf) GnRH; and [His⁵,Trp⁷,Tyr⁸]-GnRH, chicken (c) GnRH-II. The synthetic peptides incorporated the position 6 dextro ( )-isomers -arginine ( -Arg) or -naphthylalanine ( -Nal) in combination with an ethylamide substitution of position 10. The in vitro potencies for LH and FSH release of these analogues were assessed using static cultures of rat anterior pituitary cells. Efficacious peptides were examined for their gonadotropin-II and growth hormone releasing abilities from perifused goldfish pituitary fragments. Rat LH and FSH release was measured using homologous radioimmunoassays, whereas goldfish growth hormone and gonadotropin-II release were determined using heterologous carp hormone radioimmunoassays. The receptor binding of the most potent analogues was determined in bovine pituitary membrane preparations. Substitution of -Nal⁶ into [His⁵,Asn⁸]-GnRH increased the potency over 2200-fold compared with the native ligand (cfGnRH) in cultured rat pituitary cells. This was equivalent to a 55-fold greater potency than that of the native mammal (m) GnRH peptide. Substitution of -Nal⁶ or -Arg⁶ into dfGnRH or cGnRH-II resulted in potencies that were related to the overall hydrophobicity of the analogues. The [ -Nal⁶,Pro⁹NEt]-cfGnRH bound to the bovine membrane preparation with an affinity statistically similar to that of [ -Nal⁶,Pro⁹NEt]-mGnRH (k_d = 0.40 ± 0.04 and 0.55 ± 0.10 nM, respectively) in cultured rat pituitary cells. All analogues tested released the same ratio of FSH to LH. In goldfish, the analogues did not possess superagonistic activity but instead desensitized the pituitary fragments at lower analogue doses than that of the sGnRH standard suggesting differences in receptor affinity or signal transduction.     

Differential regional distribution of gonadotropin-releasing hormones in amphibian (clawed toad, Xenopus laevis) brain

In most vertebrate species two forms of gonadotropin-releasing hormone (GnRH) are present in the brain, and their differential distribution suggests they have different functional roles. The regional distribution and relative concentrations of GnRH molecular forms in the brain of adult clawed toad (Xenopus laevis) were determined using high performance liquid chromatography and radioimmunoassay with a library of region-specific GnRH antisera. Four immunoreactive forms of GnRH were detected: mammalian, hydroxyproline mammalian, chicken II, and an unidetified form of GnRH. Mammalian GnRH was distributed throughout the brain, and hydroxyproline mammalian was present in the forebrain, midbrain (excluding hypothalamus), and hypothalamus. Chicken GnRH II also occurred throughout the brain, but was present in greater amounts in the hindbrain and midbrain (excluding hypothalamus). An unidentified form of GnRH with properties of salmon GnRH was detected in the forebrain. Considering the relative proportions of mammalian GnRH and chicken GnRH II in the major brain areas, the concentration of mammalian GnRH was high in the forebrain, midbrain (excluding hypothalamus), and in particular in the hypothalamus, and very little chicken GnRH II was present in these areas. In the hindbrain, chicken GnRH II predominated and the concentration of chicken GnRH II was highest in the medulla. These findings suggest: (1) mammalian GnRH is the prime regulator of gonadotropin release from the pituitary, and (2) chicken GnRH II has an extrapituitary role.     

Characterization of brain gonadotropin-releasing hormone (GnRH) molecular variants in brain extracts from different perciform fishes from Antarctic waters

Gonadotropin-releasing hormone (GnRH) is the hypothalamic hormone that regulates the reproductive system by stimulating release of gonadotropins from the anterior pituitary gland. The molecular variants of the reproductive neuropeptide GnRH were characterized from brain tissue of three perciform species from Antarctic waters: Pseudochaenichthys georgianus, Chaenocephalus aceratus, and Notothenia rossi. The study involved reverse phase high-performance liquid chromatography (RP-HPLC) followed by radioimmunoassay (RIA) with two antisera that recognize all GnRH variants already identified: PBL 45 and PBL 49. The results showed that brain extracts of P. georgianus, C. aceratus, and N. rossi contain, like those of other perciform fish, three forms of GnRH likely to be: sbGnRH (seabream GnRH), cGnRH-II (chicken GnRH II) and sGnRH (salmon GnRH). They also showed evidence for the presence of a fourth GnRH variant, chromatographically and immunologically different from the other known forms of the vertebrate hormone. Although final conclusions will require isolation, purification, and sequencing of these molecules, these results offer encouraging possibilities of further advances in the characterization of a multiplicity of GnRH molecular variants. Accepted: 28 August 1998     

The distribution of gonadotropin-releasing hormone (GnRH) neurons and fibers throughout the chick brain (Gallus domesticus)

Summary Nerve fibers and perikarya containing gonadotropin-releasing hormone (GnRH-like) immunoreactivity were investigated in the brain of the three-week-old chick, Gallus domesticus using the technique of immunocytochemistry. Six major groups of perikarya were found to include the olfactory bulb, olfactory tubercle/lobus parolfactorius, nucleus accumbens, septal preoptic hypothalamic region (three sub-nuclei), lateral anterior thalamic nucleus and in and about the oculomotor complex. The immunostaining was unusual in the latter group, suggesting that the neurons may contain a GnRH-II like material. Immunoreactive fibers for GnRH were found throughout the entire brain extending from the olfactory bulbs to the caudal brainstem. Two anatomical areas, not emphasized in the past literature, which had distinct GnRH-like immunoreactivity, included the lateral anterior thalamic nucleus and the preoptic recess. The former included a group of GnRH perikarya that is also known to be a retino-recipient area while the latter contained neuronal terminals some of which appeared to be contacting the cerebrospinal fluid of the preoptic recess. An attempt was made to list all anatomical structures that contained or were juxta-positioned to sites that displayed immunoreactive perikarya and fibers including circumventricular organs.Abbreviations used in figure legends Ac Nucleus accumbens - Ap Archistriatum posterior - APH Area parahippocampalis - AVT Area ventralis (Tsai) - BO Bulbus olfactorius - CA Commissura anterior (rostralis) - CDL Area corticoidea dorsolateralis - CO Chiasma opticum - CP Commissura posterior - CPi Cortex piriformis - CPP Cortex praepiriformis - CT Commissura tectalis - CTz Corpus trapezoideum - EW Nucleus of Edinger-Westphal - FV Funiculus ventralis - GCt Substantia grisea centralis - GLv Nucleus geniculatus lateralis, pars ventralis - HD Hyperstriatum dorsale - HM Nucleus habenularis medialis - Hp Hippocampus - ICo Nucleus intercollicularis - IH Nucleus inferior hypothalami - IN Nucleus infundibuli hypothalami - IP Nucleus interpeduncularis - LA Nucleus lateralis anterior (rostralis) thalami - LHy Regio lateralis hypothalami - LPO Lobus parolfactorius - LSO Organum septi lateralis (lateral septal organ) - LT Lamina terminalis - ME Eminentia mediana - INT. Z Internal zone - EXT. Z External zone - ML Nucleus mamillaris lateralis - MM Nucleus mamillaris medialis - nBOR Nucleus opticus basalis (n. of basal optic root) - nCPa Nucleus commissurae pallii - N III Nervus oculomotorius - N V Nervus trigeminus - n V M Nucleus mesencephalicus nervi trigemini - OA Nucleus olfactorius anterior (rostralis) - OMdl Nucleus nervi oculomotorii, pars dorsomedialis - OMv Nucleus nervi oculomotorii, pars ventralis - OVLT Organum vasculosum laminae terminalis - P Glandula pinealis - PA Palaeostriatum augmentatum (caudate putamen) - PHN Nucleus periventricularis hypothalami - POM Nucleus praeopticus medialis - POMn Nucleus praeopticus medianus - POP Nucleus praeopticus periventricularis - PP Palaeostriatum primitivum - PT Nucleus praetectalis - PVN Nucleus paraventricularis magnocellularis - RPaM Nucleus reticularis paramedianus - RPR Recessus praeopticus - b, RPR Basal region, RPR - F, RPR Floor, RPR - R, RPR Roof, RPR - S Nucleus tractus solitarii - SCO Organum subcommissurale - SGP Stratum griseum periventriculare - SHL Nucleus subhabenularis lateralis - SL Nucleus septalis lateralis - SM Nucleus septalis medialis - SO Stratum opticum - SSO Organum subseptale - TO Tuberculum olfactorium - TIO Tractus isthmo-opticus - TPc Nucleus tegmenti pedunculopontinus, pars compacta (substantia nigra) - TrO Tractus opticus - TSM Tractus septomesencephalicus - VeD Nucleus vestibularis descendens - VeM Nucleus vestibularis medialis - VL Ventriculus lateralis - VLT Nucleus ventrolateralis thalami - VO Ventriculus olfactorius - V III Ventriculus tertius (third ventricle)     

Development of gonadotropin-releasing hormone (GnRH) neuron regulation in the female rat

Summary 1. After reaching its final destination the GnRH neuronal network develops under the influence of both excitatory and inhibitory inputs.2. In the first 2 weeks of life, the immaturity of the GnRH neuronal system is reflected in sporadic unsynchronized bursts of the decapeptide, which determine the pattern of serum gonadotropin levels observed in female rats: high FSH levels and transient bursts of LH. The main inhibitory neuronal systems that operate in this period are the opioid and dopaminergic systems. A decrease in their inhibitory effectiveness may not be sufficient correctly to activate and synchronize the GnRH neuronal system.3. There is a concomitant increase in excitatory inputs, mainly noradrenaline, excitatory amino acids, and NPY, which increase the synthesis and release of GnRH at the beginning of the juvenile period and participate in the coupling of GnRH neural activity to the ongoing rhythmic activity of a hypothalamic circadian oscillator.4. The morphological changes of GnRH neurons which take place during the third and fourth weeks of life, and which are probably related to increasing estradiol levels, reflects the increasing complexity of the GnRH neuronal network, which establishes synaptic contacts to enable the expression of pulsatility and of the positive feedback of estradiol, both necessary components for the occurrence of puberty.     

Thyrotropin releasing hormone (TRH) binding sites in the adult human brain: localization and characterization

In the current study, we found evidence for the existence of binding sites for TRH in synaptic membrane preparations of several regions of the postmortem adult human brain. High levels of specific binding (fmol [3H]Me-TRH/mg protein/2 hr) were found in limbic structures: amygdala (7.1 +/- 0.6, Mean +/- SE), hippocampus (2.8 +/- 0.3), and temporal cortex (2.4 +/- 0.8). Intermediate levels of binding were found in the hypothalamus and nucleus accumbens whereas binding was low to undetectable in frontal and occipital cortex, cerebellum, pons, medulla and corpus striatum. Binding of the radioligand was linear over protein concentrations of 0.05-1.5 mg, and greater than 6 hr of incubation was required to achieve maximal binding. In the amygdala, binding was inhibited in the presence of TRH and Me-TRH but not in the presence of up to 1 microM concentrations of cyclo (His-Pro), TRH-OH, pGlu-His or peptides unrelated to TRH. Pretreatment of amygdala synaptic membranes with detergents, proteases or phospholipases disrupted [3H]Me-TRH binding; pretreatment with DNase or collagenase had no effect on binding. Saturation and association/dissociation analyses of the binding of [3H]Me-TRH to purified amygdala synaptic membranes revealed the presence of a high affinity (KD = 2.0 nM), low capacity (Bmax = 180 +/- 16 fmoles/mg protein) binding site. These results demonstrate that a highly specific membrane associated receptor for TRH is present in the adult human brain. The specific role that this receptor plays in brain function remains to be elucidated.     

Identification of Phe313 of the gonadotropin-releasing hormone (GnRH) receptor as a site critical for the binding of nonpeptide GnRH antagonists

The dog GnRH receptor was cloned to facilitate the identification and characterization of selective nonpeptide GnRH antagonists. The dog receptor is 92% identical to the human GnRH receptor. Despite such high conservation, the quinolone-based nonpeptide GnRH antagonists were clearly differentiated by each receptor species. By contrast, peptide antagonist binding and functional activity were not differentiated by the two receptors. The basis of the differences was investigated by preparing chimeric receptors followed by site-directed mutagenesis. Remarkably, a single substitution of Phe313 to Leu313 in the dog receptor explained the major differences in binding affinities and functional activities. The single amino acid replacement of Phe313 of the human receptor with Leu313 resulted in a 160-fold decrease of binding affinity of the nonpeptide antagonist compound 1. Conversely, the replacement of Leu313 of the dog receptor with Phe313 resulted in a 360-fold increase of affinity for this compound. These results show that Phe313 of the GnRH receptor is critical for the binding of this structural class of GnRH antagonists and that the dog receptor can be "humanized" by substituting Leu for Phe. This study provides the first identification of a critical residue in the binding pocket occupied by nonpeptide GnRH antagonists and reinforces cautious extrapolation of ligand activity across highly conserved receptors.     

Calcium regulation in wild populations of a freshwater cartilaginous fish, the lake sturgeon Acipenser fulvescens

Lake sturgeon, Acipenser fulvescens, are one of a few species of cartilaginous fishes that complete their life cycle entirely in freshwater. Sturgeons maintain very low concentrations of circulating calcium (Ca²⁺) compared with other vertebrates, and therefore, face unique challenges in regard to Ca²⁺ regulation, which are likely to be magnified during vitellogenic stages of the reproductive cycle. In the present study, Ca²⁺ concentrations and associated hormones of female and male lake sturgeon were examined in two wild populations, and were related to reproductive stage. In both populations, free, bound and total Ca²⁺ were low, peaking in mid-late vitellogenic females. Internal Ca²⁺ and phosphate (PO₄³⁻) concentrations were inversely related to environmental concentrations, suggesting that these ions are preferentially retained and that mechanisms for mobilization are up-regulated under diminished environmental concentrations. Plasma 17β-estradiol, 11-ketotestosterone and testosterone, peaked in mid-late vitellogenic females, while the androgens peaked in spawning males. Urine Ca²⁺ was more tightly regulated than other divalent ions and decreased in spawning fish. Therefore, the increases in free plasma Ca²⁺, the very low circulating concentrations of free and total Ca²⁺, and the increase in PO₄³⁻ and bound Ca²⁺ in low Ca²⁺ environments indicate unique adaptations to Ca²⁺ regulation in the lake sturgeon.     

Pattern of luteinizing hormone (LH) and follicle stimulating hormone (FSH) in bovine blood plasma after injection of a synthetic gonadotropin-releasing hormone (Gn-RH)

A synthetic gonadotropin-releasing hormone (Gn-RH) was administered to female and male adult bovines in order to study the release of luteinizing and follicle-stimulating hormones into blood by the pituitary gland. Plasma LH and FSH were determined by means of a radioimmunological method. In females as well as in males, increasing doses of Gn-RH (range 50 to 1500 μg) administered i.v. or i.m. caused a linear increase in plasma LH. The release of FSH evidently was curvilinear over the same dosage range.After 2 or 3 injections of Gn-RH every 3 hours, or every 24 hours or more, smaller amounts of LH were released; repeated treatment did not result in reduction of FSH. Thus pituitary depletion of LH occurred more readily than FSH. The effect of Gn-RH on plasma levels of LH and FSH at various stages of the estrous cycle shows a tendency for an increasing release of both gonadotropins on Days 17 – 18 in comparison to Days 4 – 5 or Days 11 – 12.The results suggest that, within the limits allowed by the heterogenous FSH assay and the method of administration used in these experiments, synthetic Gn-RH does not evoke completely normal physiological responses. Therefore, further work is needed to determine its role in improving reproductive function.     

Influence of exogenous gonadotropin-releasing hormone on seasonal reproductive behavior of the coyote (Canis latrans)

Wild Canis species such as the coyote (C. latrans) express a suite of reproductive traits unusual among mammals, including perennial pair-bonds and paternal care of the young. Coyotes also are monestrous, and both sexes are fertile only in winter; thus, they depend upon social and physiologic synchrony for successful reproduction. To investigate the mutability of seasonal reproduction in coyotes, we attempted to evoke an out-of-season estrus in October using one of two short-acting gonadotropin-releasing hormone (GnRH) agents: (1) a GnRH analogue, deslorelin (6-d-tryptophan-9-(N-ethyl-l-prolinamide)-10-deglycinamide), 2.1 mg pellet sc; or (2) gonadorelin, a GnRH (5-oxoPro-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH₂) porcine hypothalamic extract, 2.0 μg/kg im once daily for 3 consecutive days. A transient increase in serum concentrations of estradiol and progesterone (1 and 2 wk, respectively) was detected after treatment with deslorelin but not gonadorelin. Also, socio-sexual behaviors reminiscent of winter mating (including courtship, mate-guarding, precoital mounts, and copulatory ties) were observed among the deslorelin group. During the subsequent breeding season (January and February), however, preovulatory courtship behavior and olfactory sampling appeared suppressed; emergence of mounts and copulations were delayed in both deslorelin and gonadorelin treatment groups. Furthermore, whereas 8 of 12 females treated in October ovulated and produced healthy litters in the spring, 4 naïve coyotes failed to copulate or become pregnant. Thus, perturbation of hormones prior to ovulation in species with complex mating behaviors may disrupt critical intrapair relationships, even if fertility is not impaired physiologically.     

Origin of the prolactin-releasing hormone (PRLH) receptors: evidence of coevolution between PRLH and a redundant neuropeptide Y receptor during vertebrate evolution

We present seven new vertebrate homologs of the prolactin-releasing hormone receptor (PRLHR) and show that these are found as two separate subtypes, PRLHR1 and PRLHR2. Analysis of a number of vertebrate sequences using phylogeny, pharmacology, and paralogon analysis indicates that the PRLHRs are likely to share a common ancestry with the neuropeptide Y (NPY) receptors. Moreover, a micromolar level of NPY was able to bind and inhibit completely the PRLH-evoked response in PRLHR1-expressing cells. We suggest that an ancestral PRLH peptide started coevolving with a redundant NPY binding receptor, which then became PRLHR, approximately 500 million years ago. The PRLHR1 subtype was shown to have a relatively high evolutionary rate compared to receptors with fixed peptide preference, which could indicate a drastic change in binding preference, thus supporting this hypothesis. This report suggests how gene duplication events can lead to novel peptide ligand/receptor interactions and hence spur the evolution of new physiological functions.     

Anatomy and reproduction of viviparous Pisidium (Parapisidium) reticulatum Kuiper, 1966: Implications for the phylogeny of Sphaeriidae (Mollusca: Bivalvia: Heterodonta)

The pea clams Sphaeriidae represent a major molluscan freshwater radiation with cosmopolitan distribution in all kinds of lotic and lentic habitats. Their phylogenetic relationships are still controversial, with comprehensive taxonomic sampling and examination of morphological characters still challenging. Here, based on rare and rediscovered original material, we study in detail the anatomy and aspects of brood protection of the African Pisidium reticulatum Kuiper, 1966. Representing the monotypic subgenus Parapisidium Kuiper, 1966, this species is characterized by its peculiar combination of shell and anatomical features of potentially high phylogenetic relevance. While similar to other congeners in several anatomical characters (e.g. reduction of inhalant siphon and descending lamella of outer demibranch, simplified structure of intestine coil and nephridium), P. reticulatum differs from other Pisidium species in retaining both pairs of retractor muscles of the inhalant siphon, and particularly in its peculiar mode of brooding. The yolky eggs are relatively large (160–170 μm in diameter) and are incubated in the gill, albeit in the absence of the formation of brood pouches. During later stages of incubation the larvae are surrounded by large cells similar to nourishing cells in other sphaeriids and probably with similar function. This unique combination of reproductive features is hypothesized to represent an intermediate stage between the typical ovoviviparity of Euperidae and euviviparity (i.e. nourishment by the parent animal) as found exclusively in Sphaeriidae, the latter being characterized by the possession of closed brood pouches. Phylogenetic analyses based on a comprehensive set of morphological characters reveal Parapisidium as the most basal lineage within a clade Pisidium. Evaluating the phylogenetic reconstructions based also on available molecular data for Sphaeriidae, we discuss alternative scenaria of (parallel) evolution of brood pouches and viviparity in this group.     

Effects of the gonadotropin-releasing hormone associated peptides (GAP) on the release of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) in vivo

Six peptide sequences residing between basic amino acid residues in GAP were tested for effects on the release of FSH, LH and PRL in vivo in ovariectomized, estrogen-progesterone-primed (OEP) rats. Synthetic GAP peptides (1–13, 1–23, 15–23, 25–36, 38–53 and 41–53) were injected intravenously (IV) into conscious OEP rats and plasma levels of FSH, LH and PRL were measured by RIA. The activity of GAP peptides in the control of PRL was further examined in ether-stressed male rats which were injected IV with GAP peptides just prior to a 1-min etherization. GAP(1–13) significantly stimulated FSH release at doses of 1, 10 and 100 μg, whereas it stimulated LH release only at the highest dose of 100 μg. GAP(1–23) elevated plasma levels of FSH and LH only at a dose of 100 μg. The other 4 peptides had no effect on the release of gonadotropins. Of these 6 peptides, only GAP(1–13) partially lowered the plasma levels of PRL at the high dose of 100 μg in OEP rats, but it had no effect on the ether-induced PRL surge at doses of 10 and 100 μg. In conclusion, both GAP(1–13) and GAP(1–23) stimulate FSH and LH release in vivo; these 2 peptides are much less potent in stimulating gonadotropin release than is LHRH. GAP(1–13) exerts a preferential FSH-releasing activity, but its PRL-inhibiting activity is minimal.     

Localization and identification of Neuropeptide Y (NPY)-like immunoreactivity in the frog brain

The distribution of neuropeptide Y (NPY) in the central nervous system of the frog Rana ridibunda was determined by immunofluorescence using a highly specific antiserum. NPY-like containing perikarya were localized in the infundibulum, mainly in the ventral and dorsal nuclei of the infundibulum, in the preoptic nucleus, in the posterocentral nucleus of the thalamus, in the anteroventral nucleus of the mesencephalic tegmentum, in the part posterior to the torus semicircularis, and in the mesencephalic cerebellar nucleus. Numerous perikarya were also distributed in all cerebral cortex. Important tracts of immunoreactive fibers were found in the infundibulum, in the preoptic area, in the lateral amygdala, in the habenular region, and in the tectum. The cerebral cortex was also densely innervated by NPY-like immunoreactive fibers. A rich network of fibers was observed in the median eminence coursing towards the pituitary stalk. Scattered fibers were found in all other parts of the brain except in the cerebellum, the nucleus isthmi and the torus semicircularis, where no immunoreactivity could be detected. NPY-immunoreactive fibers were observed at all levels of the spinal cord, with particularly distinct plexus around the ependymal canal and in the distal region of the dorsal horn. At the electron microscope level, NPY containing perikarya and fibers were visualized in the ventral nuclei of the infundibulum, using the peroxidase-antiperoxidase and the immunogold techniques. NPY-like material was stored in dense core vesicles of 100 nm in diameter. A sensitive and specific radioimmunoassay was developed. The detection limit of the assay was 20 fmole/tube. The standard curves of synthetic NPY and the dilution curves for acetic acid extracts of cerebral cortex, infundibulum, preoptic region, and mesencephalon plus thalamus were strictly parallel. The NPY concentrations measured in these regions were (pmole/mg proteins) 163±8, 233±16, 151±12 and 60±13, respectively. NPY was not detectable in cerebellar extracts. After Sephadex G-50 gel filtration of acetic acid extracts from whole frog brain, NPY-like immunoreactivity eluted in a single peak. Reverse phase high performance liquid chromatography (HPLC) and radioimmunoassay were used to characterize NPY-like peptides in the frog brain. HPLC analysis revealed that infundibulum, preoptic area and telencephalon extracts contained a major peptide bearing NPY-like immunoreactivity. The retention times of frog NPY and synthetic porcine NPY were markedly different. HPLC analysis revealed also the existence, in brain extracts, of several other minor components cross-reacting with NPY antibodies. These results provide the first evidence for the presence of NPY in the brain of a non-mammalian chordate and indicate that the structure of NPY is preserved among the vertebrate phylum. The abundance of NPY producing neurons in the hypothalamus and telencephalon suggests that this peptide may play both neuroendocrine and neurotransmitter functions in amphibians.     

Human disturbance alters endocrine and immune responses in the Galapagos marine iguana (Amblyrhynchus cristatus)

Anthropogenic disturbance is a relevant and widespread facilitator of environmental change and there is clear evidence that it impacts natural populations. While population-level responses to major anthropogenic changes have been well studied, individual physiological responses to mild disturbance can be equally critical to the long-term survival of a species, yet they remain largely unexamined. The current study investigated the impact of seemingly low-level anthropogenic disturbance (ecotourism) on stress responsiveness and specific fitness-related immune measures in different breeding stages of the marine iguana (Amblyrhynchus cristatus). Specifically, we found stress-induced elevations in plasma corticosterone among tourist-exposed populations relative to undisturbed populations. We also found changes in multiple immunological responses associated with stress-related effects of human disturbance, including bacterial killing ability, cutaneous wound healing, and hemolytic complement activity, and the responses varied according to reproductive state. By identifying health-related consequences of human disturbance, this study provides critical insight into the conservation of a well-known species that has a very distinct ecology. The study also broadens the foundation of knowledge needed to understand the global significance of various levels of human disturbance.     

Characterization of gonadotropin-releasing hormone (GnRH) binding sites in the pituitary and testis of the frog, Rana esculenta

Frog, Rana esculenta, pituitary and testis gonadotropin-releasing hormone (GnRH) receptors were characterized by using 125I-chicken IIGnRH (cIIGnRH) as radiolabeled ligand. At 4 C equilibrium binding of 125I-cIIGnRH to pituitary homogenates was achieved after 90 min of incubation; binding of 125I-cIIGnRH to testis membrane fractions reached its maximum at 60 min of incubation. Binding of the radioligand was a function of tissue concentration, with a positive correlation over the range 0.5-2 tissue equivalents per tube. One pituitary and one testis per tube were used as standard experimental condition. Incubation of the pituitary homogenate with increasing concentrations of 125I-cIIGnRH indicated saturable binding at radioligand concentrations of 1 nM and above while for the testis membrane preparation saturation was achieved using 5 nM 125I-cIIGnRH. The binding of 125I-cIIGnRH was found to be reversible after addition of the cold analog and the displacement curves could be resolved into one linear component for both tissues. Scatchard analysis suggested the presence of one class of binding sites for both pituitary and testis (Pituitary: Kd = 1.25 +/- 0.14 nM and Bmax = 8.55 +/- 2.72 fmol/mg protein; testis: Kd = 2.23 +/- 0.89 nM and Bmax = 26.48 +/- 7.39 fmol/mg protein). Buserelin displaced the labeled 125I-cIIGnRH with a lower IC50 as compared with cIIGnRH cold standard, while Arg-vasopressin (AVP) was completely ineffective, confirming the specificity of binding.