Biol Reprod Lalor Postdoctoral Fellowships -- Application Deadline January 15, 2009
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Khan, I. A.
Right arrow Articles by Thomas, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Khan, I. A.
Right arrow Articles by Thomas, P.
Agricola
Right arrow Articles by Khan, I. A.
Right arrow Articles by Thomas, P.
Biology of Reproduction 64, 955-964 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Disruption of Neuroendocrine Control of Luteinizing Hormone Secretion by Aroclor 1254 Involves Inhibition of Hypothalamic Tryptophan Hydroxylase Activity1

Izhar A. Khan2,a, and Peter Thomasa

a The University of Texas at Austin, Marine Science Institute, Port Aransas, Texas 78373

ABSTRACT

Mechanisms governing the effect of polychlorinated biphenyl (PCB) toxicity on hypothalamic serotonergic function and the neuroendocrine system controlling LH secretion were investigated in Atlantic croaker (Micropogonias unulatus) exposed to the PCB mixture Aroclor 1254 (1 µg g body weight-1 day-1) in the diet for 30 days. PCB treatment caused a decrease in hypothalamic 5-hydroxytryptamine (5-HT) concentrations and significant inhibition of hypothalamic tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT synthesis, but did not alter the activity of monoamine oxidase, the catabolic enzyme. Further, PCB treatment caused significant decreases in GnRH content in the preoptic-anterior hypothalamic area. Significant decreases in pituitary GnRH receptor concentrations and the LH response to the GnRH analogue (GnRHa) were also observed in PCB-exposed fish, possibly as a consequence of a decline in GnRH release. The possible association between impaired serotonergic and neuroendocrine functions after PCB treatment was explored using serotonergic drugs. Treatment of croaker with p-chlorophenylalanine, an irreversible TPH inhibitor, mimicked the effects of PCB on the GnRH system and the LH response to GnRHa. Bypassing the TPH-dependent hydroxylation step with the administration of 5-hydroxytryptophan restored 5-HT to control levels and prevented the deleterious effects of PCB on the neuroendocrine parameters. Moreover, slow-release GnRH implants prevented the PCB-induced decline in GnRH receptors and restored the LH response to GnRHa, suggesting that GnRH therapy can reverse PCB-induced disruption of LH secretion. These results demonstrate that TPH is one of the targets of PCB neurotoxicity and indicate that a decrease in 5-HT availability in PCB-exposed croaker results in disruption of the stimulatory 5-HT/GnRH pathway controlling LH secretion.

gonadotropin-releasing hormone, luteinizing hormone, polychlorinated biphenyls, serotonin, tryptophan hydroxylase

INTRODUCTION

Considerable evidence suggests that a variety of endocrine disrupting chemicals, such as heavy metals, pesticides, and polychlorinated biphenyls (PCBs), can impair vertebrate reproduction by acting at the hypothalamic level [1–9]. Investigations of the neuroendocrine targets of these chemicals and their mechanisms of disruption are, however, complicated by the complexity and multiplicity of neuroendocrine pathways that control vertebrate reproduction and the wide variety of potential chemical interactions. For example, a variety of pesticides interfere with LH secretion and reproductive function in rats by disrupting monoamine neurotransmitter function in the hypothalamus [6–8]. PCBs can also disrupt LH secretion in both mammals and fish, and neuroendocrine pathways that control LH secretion are potential targets of PCB toxicity [9–13]. However, the precise neuroendocrine targets of PCBs are unknown, and their mechanisms of neuroendocrine disruption warrant more thorough investigation.

The studies with pesticides and PCBs suggest that certain organic compounds could potentially disrupt neuroendocrine systems by impairing the functions of neurotransmitter systems that influence GnRH and/or gonadotropin secretion. Neuroendocrine control of reproduction in fishes, as in other vertebrates, involves complex interactions of a variety of neurotransmitters and neuromodulators with GnRH to regulate the synthesis and release of gonadotropins [5, 14]. The monoamine neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) stimulates gonadotropin II (GTH II or LH) secretion in goldfish and Atlantic croaker, the two teleosts in which effects of 5-HT have been investigated in any detail, by acting on the GnRH system in the preoptic-anterior hypothalamic area (POAH) and at the pituitary gland [15–18]. In mammals, both stimulatory and inhibitory influences of 5-HT on GnRH and LH secretion have been reported, depending on the developmental stages of the animals and experimental approach [19–24]. However, most studies have shown that 5-HT and its precursor 5-hydroxytryptophan (5-HTP), regulate LH secretion in mammals, including humans, by acting centrally via the stimulation of GnRH neurons [19–22]. In addition, selective degeneration of serotonergic nerve terminals in the ventromedial region of the hypothalamus by 5,7-dihydroxytryptamine, a 5-HT neurotoxin, results in reduced LH levels in rats [25]. Therefore, lesions in the hypothalamic 5-HT system induced by environmental chemicals could potentially impair a stimulatory GnRH-LH pathway in vertebrates.

Evidence has accumulated that one major class of contaminants, PCBs, impairs the function of serotonergic and other monoaminergic neurotransmitter systems [26–32]. Several laboratory studies in rats have shown that PCBs at sublethal doses influence dopamine (DA), norepinephrine, 5-HT, and their metabolite concentrations in discrete brain areas [26–29]. Moreover, PCBs consistently decrease DA concentration in nonhuman primates and mammalian cell lines [30, 31]. A decrease in DA concentrations in rats by PCBs is thought to be due to inhibition of tyrosine hydroxylase (TH), although the mechanism of this inhibition is unknown [32]. Some PCBs have also been shown to reduce 5-HT concentrations and increase the ratio of the metabolite (5-hydroxyindolacetic acid, 5-HIAA) to 5-HT in rats [28, 29]. In addition to their effects on neurotransmitter metabolism, PCBs impair LH secretion in rats [11, 12] and Atlantic croaker [10]. The disruption of LH secretion by the PCB Aroclor 1254 in croaker is accompanied by inhibition of gonadal growth [10]. Moreover, we have previously shown that exposure of croaker to this PCB in the diet during gonadal recrudescence phase results in reduced hypothalamic 5-HT and DA concentrations and increased ratios of the metabolite to the parent amine [9]. Furthermore, PCB exposure inhibits both in vivo and in vitro LH responses to stimulation by a GnRH analogue (GnRHa) in croaker [9, 13]. These results suggest that PCB-induced disruption of the hypothalamic 5-HT system is associated with the impairment of LH secretion in croaker because 5-HT exerts stimulatory influences on LH secretion in this species [17]. However, a causal relationship between PCB-induced alterations in 5-HT metabolism and the disruption of LH secretion has not been established.

The objectives of the present study were, therefore, to determine the target(s) of PCB neurotoxicity in the hypothalamic serotonergic pathway in Atlantic croaker and to investigate whether the disruption of LH secretion by PCB was related to the reduced hypothalamic 5-HT concentrations. Croaker is an excellent vertebrate model to investigate the mechanisms of neuroendocrine disruption because the neuroendocrine systems of fishes have the same basic features as those of other vertebrates, yet their relatively simple organization affords unique opportunities to study hypothalamic and pituitary function [14]. First, we investigated whether PCB can alter hypothalamic tryptophan hydroxylase (TPH), the rate-limiting enzyme for 5-HT synthesis and/or monoamine oxidase (MAO), the enzyme that converts 5-HT to its inactive metabolite, 5-HIAA. The finding of PCB-induced inhibition of hypothalamic TPH activity prompted us to try to mimic the inhibitory effect of PCB on TPH activity and 5-HT concentrations by treatment with p-chlorophenylalanine (PCPA), an irreversible TPH inhibitor [33, 34]. In addition, the 5-HT precursor, 5-HTP, was administered to bypass the TPH-dependent hydroxylation step in 5-HT biosynthesis and to restore neuronal 5-HT concentrations [34, 35] in PCB-treated fish. GnRH contents in the POAH and pituitary gland and levels of basal and GnRHa-induced LH secretion were examined under these experimental conditions. In addition, the POAH and pituitary slices were incubated in vitro to determine the functional integrity of GnRH neurons to release GnRH in response to stimulation by 5-HT. We used slow-release GnRHa implants simultaneously with PCB exposure to determine whether GnRH therapy would prevent PCB-induced decreases in the number of pituitary GnRH receptors (GnRH-R) and the LH response to GnRHa.

MATERIALS AND METHODS

Animals

Young-of-the-year Atlantic croaker were captured with shrimp trawls in Redfish Bay, Texas, during the late summer and were maintained in the laboratory in large tanks with a recirculating seawater system. Croaker undergo gonadal recrudescence in the laboratory under a simulated seasonal cycle of photoperiod/water temperature in the fall (September–November) and are routinely maintained in a reproductively active stage under fall photoperiod (11L:13D) and constant temperature (22°C ± 1°C) conditions until March. All PCB exposure experiments (15 or 30 days) were initiated in male fish during the early recrudescence phase of the gonadal cycle and were terminated when the gonads had developed fully in control fish, as evidenced by the presence of readily expressible milt. Fish weighing 45–65 g were fed a diet of chopped shrimp and commercial trout pellets (95/5, w/w) at 3% of their body weight daily in the morning throughout the experiments. They were fed an average of three pellets of prepared food per fish per day. The animal utilization protocols were approved by the Institutional Animal Care and Use Committee of the University of Texas at Austin.

Chemicals and Drugs

Aroclor 1254 was purchased from Chemservice (West Chester, PA). GnRHs were obtained from Bachem (Torrence, CA), and PCPA was from Research Biochemicals (Natick, MA). The 3H-tryptophan and tryptamine-14C-bisuccinate were purchased from New England Nuclear (Boston, MA), and the 125I-labeled mGnRH tracer was from Amersham Pharmacia Biotech (Piscataway, NJ). The scintillation fluid (CytoScint) was obtained from ICN (Costa Mesa, CA), and the charcoal (Darco G-60) was from EM Science (Gibbstown, NJ). Acetylthiocholine, BSA, catalase, 5,5'-dithio-bis-(2-nitrobenzoic acid), Dulbecco modified Eagle medium (DMEM), dithiotheritol (DTT), EDTA, hCG, Hepes, 5-HT, 5-HTP, iodogen, goat and mouse anti-rabbit IgGs, 6-methyl-5,6,7,8-tetrahydropterine (6-MPH4), and other analytical grade chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). The seabream GnRH conjugated to acetylcholinesterase (sbGnRH-AChE tracer) was purchased from Cayman Chemical Co. (Ann Arbor, MI). The sbGnRH peptide and antibody (AS-691) were gifts from Drs. K. Okuzawa and H. Kagawa [36]. The 5-HT ELISA kit (RE 591-21, IBL-Hamburg) was a gift from KMI Diagnostics (Minneapolis, MN).

Experimental Protocols

Experiments were conducted in 1500-L recirculating seawater tanks (24–30 fish/tank). The hormones and neuropharmacological agents were injected i.p. in a final volume of 100-µl saline/fish. The pharmacological treatments and fish sampling were carried out between 0900 and 1100 h to avoid diurnal differences. All the experimental fish were anesthetized with quinaldine sulfate (20 mg/L) prior to handling. However, hypothalamic 5-HT concentrations and TPH and MAO activities were measured in fish killed by decapitation within 15 sec of capture without the use of the anesthetic. Brains and pituitaries either were stored at -80°C until analyzed for various parameters or were processed immediately according to the requirements of particular assays. The hypothalamic tissue for TPH and MAO activity contained POAH and medial and posterior hypothalamus, which included nucleus recessus lateralis (NRL) and nucleus recessus posterioris (NRP), the homologues of mammalian paraventricular organ [18]. The NRL and NRP contain intrinsic 5-HT neurons in fish [18]. Plasma samples collected after 20-min centrifugation were stored at -20°C until assayed for LH levels. The gonadosomatic index (GSI; gonad weight expressed as a percentage of body weight) was determined in all the experiments.

Effects of PCB on Hypothalamic TPH and MAO Activities

Forty-eight fish were separated into two equal groups during the early recrudescence phase of their gonadal cycle and were maintained under ambient photoperiod and constant temperature (22°C ± 1°C) conditions. One group of fish was exposed to the PCB mixture Aroclor 1254 (1 µg g body weight [BW]-1 day-1) for 30 days in the diet, whereas the other group was given a control diet. Twelve fish from each group were rapidly killed by decapitation, and their brains were collected and frozen in liquid nitrogen for the measurement of hypothalamic TPH activity. The remaining 12 fish from each group were used for the determination of hypothalamic MAO activity.

Dose-Response and Time-Course Effects of PCPA on Hypothalamic TPH Activity

The effects of different concentrations of PCPA (1, 10, 100, and 1000 µg/g BW) on hypothalamic TPH activity at 1 h postinjection were investigated in croaker during the period of gonadal recrudescence to determine which treatment caused a reduction in hypothalamic TPH activity comparable to that due to the PCB exposure. The 1-h sampling time was based on a previous study in fish where significant reduction in hypothalamic 5-HT levels was achieved within 1 h of PCPA administration [37]. A time course (0–96 h) experiment was conducted to determine the duration and frequency of PCPA injections.

Effects of PCB, PCPA, and PCB + 5-HTP on the 5-HT and GnRH Systems and on LH Secretion

To minimize handling stress due to multiple injections of the pharmacological agents and to achieve a similar PCB body burden, the duration of PCB exposure was reduced from 30 to 15 days and the dose was increased from 1 to 2 µg g BW-1 day-1 in this experiment. Two hundred forty fish were separated into four equal treatment groups (30 fish/tank, two tanks/treatment). Fish in two groups (A and B) were fed uncontaminated diet, and fish in the other two groups (C and D) were exposed to PCB in the diet. The fish in group A were given saline, and those in group B received PCPA (10 µg/g BW; five injections at 3-day intervals). The fish in groups C and D were injected with either saline or 5-HTP (20 µg/g BW; seven injections every other day). This dose of 5-HTP previously reversed the inhibitory effect of PCPA on hypothalamic 5-HT concentrations and restored LH secretion in catfish [35]. The experiments were terminated 24 h after the last injection, and hypothalamic tissues were processed for determination of TPH activity and 5-HT concentrations (n = 8 for each measurement). GnRH content (n = 8 fish/group) was determined in POAH and pituitaries, and the GnRH response to 5-HT was determined in the POAH and pituitary slices incubated in vitro (n = 16/group: 8 unstimulated, 8 stimulated with 5-HT). In addition, 20 fish from each group were further separated into two subgroups at the end of the exposure period and were injected with either saline or GnRHa (10 ng/g BW). Blood samples were collected 1 h after the injections for the measurement of plasma LH levels.

Effects of GnRH Implants on GnRH-R Concentrations and LH Secretion in PCB-Exposed Fish

One hundred twenty fish were separated into four equal treatment groups (30 fish/tank). Fish in the first group were fed uncontaminated diet, and fish in the other three groups were exposed to PCB in the diet (2 µg g BW-1 day-1 for 2 wk). A day before the start of the feeding regimen, fish in two PCB-exposure groups were implanted with slow-release cholesterol pellets (5 mm length x 1.5 mm diameter; 95% cholesterol, 5% cellulose) of GnRHa (5 and 50 ng/g BW). Similar slow-release cholesterol implants have been shown to release GnRHa in striped bass for up to 2 wk [38]. Fish in the other two groups received the pellets without GnRHa. At the end of 2 wk, 20 fish were injected with either saline or GnRHa (10 ng/g BW), and blood samples were collected 1 h later for the determination of plasma LH levels. The pituitaries of the 10 saline-injected and remaining 10 uninjected fish were pooled from each group to determine GnRH-R concentrations.

TPH and MAO Activities

TPH activity was measured using a radioenzymatic assay [39] validated for croaker hypothalamic tissues. Tissue homogenate (25 µl) was added to a reaction mixture containing 0.05 mM tryptophan, 50 mM Hepes (pH 7.6), 5 mM DTT, 0.01 mM Fe(NH4)2(SO4)2, 0.5 mM 6-MPH4, 0.1 mg/ml catalase, and 3H-tryptophan (1 µCi/reaction). The enzymatic reaction was allowed to proceed at 25°C for 20 min or as specified for the time-course experiment. Unreacted tryptophan and the product 5-HTP were adsorbed with 500 µl of 7.5% charcoal in 1 M HCl at the end of the incubation. The samples were vortexed thoroughly and centrifuged at 14,000 x g for 2 min. The supernatant (350 µl) was centrifuged, and a 200-µl aliquot of the final supernatant was added to 3 ml of scintillation fluid (CytoScint) and the radioactivity was measured by a liquid scintillation counter (Beckman LS 6000SC). Blank values were obtained by performing the reaction in the absence of tissue homogenate and in the presence of boiled homogenate. The counts per minute were converted to pmoles of 5-HTP formed per milligram of tissue per hour using the formula described by Vrana et al. [39].

MAO activity was determined by a radioisotopic method [40]. Hypothalamic tissues were homogenized and incubated in the presence of tryptamine-14C-bisuccinate, and the enzyme activity was determined by measuring radioactivity of 14C-indole acetic acid formed during 20-min incubation at 25°C and expressed as pmoles of amine deaminated per milligram of tissue per hour.

5-Hydroxytryptamine ELISA

Hypothalamic tissue was homogenized in 20 volumes of 1 N formic acid/acetone (15/85, v/v) according to the method of Smith et al. [41]. After centrifugation and reextraction of the pellet with formic acid/acetone, the combined supernatants were washed by shaking for 10 min with 3 ml of heptane/chloroform (8/1, v/v) for every 1 ml of formic acid/acetone. The organic phase and lipid interface were aspirated, and the samples were dried under vacuum. The samples were reconstituted in the enzyme immunoassay (EIA) buffer (phosphate buffer with BSA and sodium azide) and analyzed following the protocol provided with the ELISA kit. The 5-HT concentrations determined with the ELISA were comparable to those reported previously in croaker using an HPLC method [9].

GnRH Release from POAH and Pituitary Slices Incubated In Vitro

The POAH and pituitary tissues were transferred to DMEM in 48-well culture plates kept on ice. The tissues were divided into four slices (thickness: POAH, ~0.5 mm; pituitary, ~0.25 mm), preincubated for 1 h, and then incubated with 5-HT (20 µg/ml) or medium alone for 1 h at 24°C under oxygen as previously described [42].

Gonadotropin-Releasing Hormone ELISA

The POAH and pituitary tissues were homogenized and extracted following the procedure described by Holland et al. [43]. A specific sbGnRH antibody (AS-691) and sbGnRH-AChE as tracer were used in the ELISA. The sbGnRH-AChE conjugate was prepared by the Cayman Chemical Co., according to the method of Pradelles et al. [44]. Assays were performed in 96-well microtiter plates precoated with mouse anti-rabbit IgG. The plates were washed three times with 0.01 M potassium phosphate buffer (pH 7.4) containing 0.05% Tween-20 prior to each assay. The incubation mixture consisted of 50 µl of antiserum (1:50000), 50 µl of GnRH-AChE tracer (1:1000), and 50 µl of standard or sample. Samples were assayed in triplicate. Two wells on each plate were used for tracer activity (TA), and three were used for total binding (B0). The plates were incubated for 3 days at 4°C and then washed four times with the washing buffer. Freshly prepared Ellman reagent (20 mg acetylthiocholine and 21.5 mg 5,5'-dithio-bis-(2-nitrobenzoic acid)/100 ml; 200 µl/well) was added to each well, and 5 µl of tracer was added only to the TA wells. Optical density was read with a microplate reader (Uvmax; Molecular Devices, Sunnyvale, CA) at 405 nm after 3 h of incubation in complete darkness and under constant horizontal agitation. The GnRH concentrations were calculated using a Beckman EIA program. The interassay variation was 11.4%, and the intraassay variation ranged from 7.2% to 10.9%. The minimum and maximum limits of detection were 4 and 2000 pg/sample, respectively.

GnRH-R Assay

GnRH-R concentrations were measured in croaker following the methods previously described for seabream [45] with minor modifications. Seabream GnRH, the major releasing form present in the preoptic area and pituitary of all the perciform fish investigated to date, differs from the mammalian GnRH decapeptide (mGnRH) by only one amino acid [36]. Therefore, we used commercially available 125I-labeled mGnRH tracer for the binding assays. Pituitaries were homogenized in ice-cold 0.25 M sucrose in 10 mM Tris-HCl, pH 7.4 (25 µl/pituitary) with a glass homogenizer. The homogenate was brought to a volume of 3 ml and centrifuged at 500 x g for 5 min at 4°C. The supernatant was centrifuged at 12 000 x g for 30 min. The crude membrane fraction (pellet) was resuspended in 10 mM Tris-HCl (pH 7.4) to 1 pituitary equivalent/50 µl and used immediately for receptor binding studies.

The microfuge tubes used for binding assays were precoated overnight with 1% BSA in 10 mM Tris-HCl (pH 7.4). The membrane fraction (50 µl) was added to 125I-labeled mGnRH (7.5 x 10-10 M in all but the saturation experiment) with or without excess unlabeled mGnRH (10-6 M) in a final volume of 250 µl assay buffer containing 0.1% BSA. Bound and free hormone were separated after 40 min of incubation by centrifugation for 5 min at 14 000 x g through 100 µl of 10% sucrose in 10 mM Tris-HCl. Supernatant was aspirated, and the last 2–3 mm of the tips of the tubes were cut off and transferred to polypropylene tubes for counting on a gamma counter. Specific binding was determined by subtracting nonspecific binding from total binding.

Pilot studies included GnRH-R assays to determine specific binding as a function of pituitary membrane concentrations (0.125–4 pituitary equivalents/tube), time course of incubation (0, 10, 20, 30, 40, 60, 120, and 180 min), saturation of the binding with increasing concentrations of 125I-labeled mGnRH (9 x 10-11 to 12 x 10-9 M), and displacement of 125I-labeled mGnRH binding with a range of concentrations (10-10–10-6 M) of mGnRH, sbGnRH, chicken GnRH II (cGnRH-II), and salmon GnRH (sGnRH) and 10-7–10-6 M hCG (used as a negative control).

Luteinizing Hormone RIA

Plasma LH concentrations were measured using a homologous RIA [42]. The assay has a sensitivity of 5 pg/tube equivalent to 50 pg/ml in plasma samples. Interassay variance was <10%, and the recovery of LH added to croaker plasma ranged from 85% to 112%.

Statistical Analyses

Data were analyzed by Student t-test assuming unequal variances for comparison between two groups, or by analyses of variance followed by Tukey test for comparison of multiple group means. P values of <0.05 were considered significant. A computerized nonlinear least-square curve-fitting program (LIGAND) was used for Scatchard analysis of GnRH-R binding data.

RESULTS

Effects of PCB on Hypothalamic TPH and MAO Activities

A pilot study to determine the appropriate incubation time for TPH activity measurement showed that enzyme activity increased linearly up to 20 min, remained stable for 40 min, and declined thereafter (data not included). Therefore, a 20-min incubation period was selected for subsequent assays. PCB exposure (1 µg g BW-1 day-1 for 30 days) resulted in a significant reduction (38.1%, P < 0.02) in hypothalamic TPH activity (from 29.1 ± 2.5 to 18.0 ± 2.1 pmol mg tissue-1 h-1), whereas MAO activity was not significantly altered (Fig. 1).



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 1. Effects of PCB on hypothalamic TPH (A) and MAO (B) activities. Each bar represents mean ± SEM of 12 observations (a, significantly different from the control)

Dose-Response and Time-Course Effects of PCPA on Hypothalamic TPH Activity

PCPA at doses of 10 µg/g BW or higher significantly inhibited hypothalamic TPH activity (P < 0.05) within 1 h after the injection (Fig. 2A). Therefore, the 10 µg/g BW dose was used for the time-course and other experiments. The time-course study revealed that enzyme activity remained low for 72 h postinjection (Fig. 2B). Based on these results, an injection protocol was developed where PCPA was administered in acidified saline at 3-day intervals in an attempt to mimic the effect of PCB on hypothalamic TPH activity and 5-HT concentrations.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 2. Dose-response (A) and time-course (B; 10 µg/g BW) studies of the effects of PCPA on hypothalamic TPH activity. Each point represents mean ± SEM of 8–10 observations. Different letters denote significant differences

Effects of PCB, PCPA, and PCB + 5-HTP on the 5-HT and GnRH Systems and on LH Secretion

A similar decrease in hypothalamic TPH activity (36.6% versus 38.1%, P < 0.03) was observed in this experiment when the dose of PCB was increased from 1 to 2 µg per g of BW-1 day-1 and the duration of exposure was reduced from 30 to 15 days (Fig. 3A). Moreover, PCPA treatment caused a decrease in TPH activity comparable to that induced by PCB (45.8% versus 36.6%, P < 0.01). Cotreatment with 5-HTP did not influence enzyme activity as compared with that observed with the PCB treatment alone (42.5% versus 36.6% decrease). Similar decreases in TPH activity were accompanied by comparable decreases in hypothalamic 5-HT concentrations after PCB (P < 0.03) and PCPA (P < 0.01) treatments, whereas treatment of the PCB-exposed group with 5-HTP restored the neurotransmitter levels to those observed in control fish (Fig. 3B).



View larger version (36K):
[in this window]
[in a new window]
  		FIG. 3. Effects of PCB, PCPA, and PCB + 5-HTP on the hypothalamic TPH activity (A) and 5-HT concentrations (B). Each bar represents mean ± SEM of eight observations (a, significantly different from the respective control)

The same treatments that decreased hypothalamic TPH activity and 5-HT concentrations also caused a decrease in reproductive neuroendocrine function. Both PCB and PCPA treatments elicited significant decreases in GnRH contents in the POAH (PCB: 86.5%, P < 0.001; PCPA: 72.6%, P < 0.003; Fig. 4A). The 5-HTP treatments prevented the PCB-induced decline in the concentrations of the decapeptide in the POAH. However, pituitary GnRH content was not significantly altered by these treatments (Fig. 4B).



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 4. Effects of PCB, PCPA, and PCB + 5-HTP on the GnRH content in the POAH (A) and pituitary (B). Each bar represents mean ± SEM of eight observations (a, significantly different from the control group)

5-HT significantly stimulated GnRH release in vitro from the POAH and pituitary slices in all four treatment groups (control, PCB, PCPA, and PCB + 5-HTP; Table 1). PCB and PCPA treatments significantly reduced (P < 0.05) both spontaneous and 5-HT-induced GnRH release from the POAH, and the combined treatments of PCB + 5-HTP prevented the PCB-induced declines in GnRH release. However, none of these treatments significantly altered spontaneous or 5-HT-induced GnRH release from pituitary slices.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Effects of PCB, PCPA, and PCB + 5-HTP treatments on the basal and 5-HT-induced GnRH release from the POAH and pituitary slices incubated in vitro

Administration of GnRHa (10 ng/g BW) 1 h prior to blood sampling induced a significant increase in plasma LH levels (P < 0.02; Fig. 5). PCB and PCPA treatments significantly decreased GnRHa-induced LH secretion (PCB: 62%, P < 0.01; PCPA: 75.1%, P < 0.005). In addition, coadministration with 5-HTP prevented the PCB-induced decline in the LH response to GnRHa.



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 5. Effects of PCB, PCPA, and PCB + 5-HTP on the basal (saline) and GnRHa-induced LH secretion. Each bar represents mean ± SEM of 10 observations (a, significantly different from the saline-injected control; b, significantly different from the GnRHa-injected control)

The GSIs of immature croaker at the beginning of exposure were <0.5%, whereas those of fully mature males at the end of the experiment ranged from 4% to 12%. Both PCB and PCPA treatments significantly inhibited gonadal growth (P < 0.01 and 0.007, respectively), and the PCB-induced inhibition was partially reversed by 5-HTP cotreatments (GSIs: control, 9.82 ± 1.673; PCB, 3.05 ± 0.57; PCPA, 2.26 ± 0.48; PCB + 5-HTTP, 4.98 ± 0.87).

Effects of GnRH Implants on GnRH Receptors and LH Secretion in PCB-Exposed Fish

Results of the pilot studies showed that the specific binding of the radioligand was tissue concentration dependent (linear increase over the range of 0.125–2 pituitary equivalents/tube), was maximum within 20 min and declined after 60 min, and was displaced by mGnRH, sbGnRH, cGnRH-II, and sGnRH but not by hCG (data not included). Of the four GnRHs tested, sGnRH was the least potent form, whereas sbGnRH and cGnRH-II were slightly more potent than mGnRH in displacing the binding. Scatchard analysis of the saturation data suggested the presence of a single class of high affinity binding sites (Ka = 1.223 x 109 M-1; Bmax = 0.89 pmol/mg protein; Fig. 6).



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 6. Specific binding of pituitary membrane fractions (1 pituitary equivalent/tube) to the increasing concentrations of the 125I-labeled m;chGnRH tracer (9 x 10-11 to 12 x 10-9 M). Duplicate aliquots were incubated for 40 min at 4°C in the presence (nonspecific binding) or absence (specific binding) of 10-6 M mGnRH. Inset: Scatchard plot

PCB exposure significantly reduced GnRH-R content in the pituitary (52.1%, P < 0.01; Fig. 7A). The low-dose GnRHa implants (5 ng/g BW) in combination with PCB exposure restored GnRH-R content almost to the level observed in control fish. The high-dose GnRHa implants (50 ng/g BW) reduced the receptor content by 40% (nonsignificant, P = 0.11) as compared with PCB treatment alone. In addition, PCB significantly decreased LH secretion (72%, P < 0.02) and the LH response to GnRHa (54.8%, P < 0.04; Fig. 7B). The low-dose GnRHa implants elicited a significant increase in plasma LH levels (P < 0.01) and restored the LH response to GnRHa administration in PCB-exposed fish, whereas the high-dose GnRHa implants completely blocked the LH response to GnRHa.



View larger version (36K):
[in this window]
[in a new window]
  		FIG. 7. Effects of PCB alone and in combination with the GnRHa implants on the GnRH-R content (A) and on the basal (saline) and GnRHa-induced LH secretion (B). The number of GnRH-R was determined by single point assays (n = 5; 20 pituitaries/group) using membrane fractions of 1 pituitary equivalent and 7.5 x 10-10 M 125I-labeled mGnRH/tube in duplicate in the presence or absence of 10-6 M mGnRH. The bars for plasma LH levels represent mean ± SEM of 10 observations (a, significantly lower than the control group in A; b, significantly lower than the saline-injected control; c, significantly higher than the saline-injected control; d, significantly lower than the GnRHa-injected control; e, significantly lower than the other GnRHa-injected groups)

The PCB-induced inhibition of gonadal growth (P < 0.04) was partially reversed by the low-dose GnRHa implants but not by the high-dose implants (GSIs: control, 4.93 ± 0.67; PCB, 1.92 ± 0.34; PCB + low-dose GnRHa, 3.01 ± 0.42; PCB + high-dose GnRHa, 1.65 ± 0.28).

DISCUSSION

The results of the present study demonstrate for the first time in any vertebrate species that impairment of the hypothalamic serotonergic system by PCB involves inhibition of TPH activity, the rate-limiting step in 5-HT synthesis. In addition, they provide clear evidence that reduced 5-HT synthesis in the hypothalamus of PCB-exposed croaker results in disruption of the stimulatory 5-HT-GnRH pathway that controls LH secretion. The finding that inhibition of TPH activity by pharmacological treatment with the TPH inhibitor PCPA mimicked the inhibitory effects of Aroclor 1254 on GnRH function and LH secretion indicates involvement of the hypothalamic 5-HT system in the neuroendocrine toxicity induced by PCBs. The ability of 5-HTP treatments to prevent PCB-induced impairment of the GnRH system and LH secretion is further evidence that inhibition of this enzyme is an important mechanism of PCB neuroendocrine toxicity. Moreover, the efficacy of slow-release GnRHa implants in preventing PCB-induced decreases in GnRH-R concentrations and the LH response to GnRHa suggests that GnRH therapy can ameliorate disruption of LH secretion after PCB exposure.

The complexity of the neuroendocrine system controlling LH secretion complicates investigations of the sites and mechanisms of chemical interference and their overall effects on neuroendocrine function. Therefore, several components of the GnRH system were investigated in Atlantic croaker, a well-characterized model with a relatively simple neuroendocrine organization, to obtain a more comprehensive understanding of the neuroendocrine effects of altered serotonergic function after PCB exposure. The results show that PCB-induced inhibition of TPH activity in croaker is accompanied by alterations of many components of the GnRH system. Such extensive alterations of the GnRH system have not been reported in any other vertebrate model after exposure to PCBs. A consistent finding was that inhibition of TPH activity by PCB and PCPA resulted in a decrease in GnRH content in the POAH. One interpretation of these results is that disruption of the stimulatory 5-HT input leads to impairment of GnRH synthesis. The observation that coadministration of 5-HTP prevented the PCB-induced decline in GnRH content in the POAH is consistent with this possibility. 5-HT has been shown to regulate GnRH gene expression in the rat [24], although similar evidence is currently lacking in teleosts. PCB also may act directly on GnRH neurons. In a preliminary study, PCB directly influenced GnRH neurons (GT1–7 cells) in vitro by causing retraction of neuronal processes and reduction of GnRH mRNA levels ([46], personal communication). However, the ability of PCPA to mimic the effects of PCB on the GnRH system and the ability of 5-HTP treatments to reverse these effects indicate that inhibition of hypothalamic TPH activity is at least partially responsible for the PCB-induced impairment of the GnRH system and LH secretion.

Pituitary GnRH-R concentrations vary during the reproductive cycle in both fish and mammals [47–50] and gradually increase during gonadal recrudescence in goldfish [47, 48] and croaker (data not included). Moreover, there is evidence that GnRH regulates GnRH-R concentrations in these two species ([47, 48], present study). Therefore, one of the reasons for the lower pituitary GnRH-R concentrations observed in PCB-exposed croaker may be insufficient GnRH release from the pituitary nerve terminals, resulting in reduced upregulation of the GnRH-R, although a direct effect of PCB on GnRH-R cannot be ruled out. The finding that the low-dose GnRHa implants upregulated GnRH-R, increased basal LH secretion, and fully restored the LH response to GnRHa in PCB-exposed croaker is consistent with a mechanism of PCB toxicity involving decreased GnRH secretion. However, the complete loss of the LH response to further stimulation by GnRHa in fish with high-dose GnRHa implants was likely due to downregulation of GnRH-R and/or depletion of pituitary LH stores.

5-HT stimulates GnRH release in both mammals and fish [16, 19, 20, 22], including Atlantic croaker as shown in the present study. The decreases in GnRH content observed in the POAH of croaker after the PCB and PCPA treatments were reflected in both spontaneous and 5-HT-induced GnRH release from POAH slices in vitro. The observation that 5-HT induced two- to threefold increases in GnRH release from the POAH and pituitary slices of fish treated with PCB and PCPA indicates that the ability of GnRH neurons to release GnRH in vitro in response to 5-HT is retained after these treatments, although the response was attenuated in the POAH. The POAH and pituitary slice preparations in fishes ([16], present study) and median eminence of rats [19] contain GnRH neurons that are not intact. Therefore, the precise physiological significance of the in vitro GnRH release data in the present study is unclear. GnRH content did not decline in the pituitaries of PCB- and PCPA-exposed fish despite the drastic reduction of GnRH in the POAH. One possible explanation for the lack of a decline is that these treatments disrupt GnRH release from the nerve terminals in the pituitary. However, in vivo GnRH release was not measured because of its complicated nature in teleosts due to the absence of a hypothalamohypophysial portal system and the release of GnRH from nerve terminals directly in the anterior pituitary [14]. Thus, although alterations of GnRH content, GnRH release, and GnRH receptors together point to the impairment of pituitary GnRH release as an additional site of neuroendocrine disruption by PCB, further studies will be required to confirm this possibility.

PCB also may interfere with other neuromodulators that influence LH secretion. A depletion of 5-HT in the pineal gland could impair the production of melatonin, a hormone that has been shown to stimulate LH secretion in croaker by acting at the POAH and pituitary [51]. PCB exposure also results in lower hypothalamic DA concentrations in croaker [9], a finding consistent with those of several studies in mammals [26, 30, 31]. The decline in hypothalamic DA concentrations in croaker may also partially contribute to the PCB-induced disruption of LH secretion, because apomorphine (a DA agonist) potentiates and pimozide and domperidone (DA antagonists) inhibit GnRHa-induced LH secretion in this species [52]. The reduction in DA concentrations in croaker after PCB exposure is likely due to inhibition of TH (the rate-limiting enzyme in DA synthesis); this enzyme is inhibited in rats exposed to PCBs [32], and no alteration of MAO, the enzyme that metabolizes monoamines, including DA, was observed in the present study.

PCBs could inhibit the activities of TPH and TH by similar mechanisms. TPH and TH are aromatic amino acid hydroxylases that share many physical, structural, and catalytic properties [53], and activation of the two enzymes involves coupling with similar second messenger pathways [54]. Alterations of DA metabolism in rats after PCB exposure are also associated with interference of Ca2+ channels and protein kinase pathways [55]. Similar studies have not been conducted to determine the mechanisms of PCB action on 5-HT metabolism. Our results suggest that the decreases in 5-HT concentrations in rat brains after exposure to PCB mixtures, including Aroclor 1254 [28, 29], are due to inhibition of TPH activity. Clearly, the mechanisms of the effects of PCB toxicity on 5-HT metabolism and TPH activity warrant further investigation.

Despite a ban on the manufacture of PCBs in the United States and Europe in the late 1970s, these compounds continue to pose a threat to wildlife and certain human populations because of their persistence, bioaccumulation, biomagnification, and toxicity [56–60]. The PCB exposure regime used in the present study resulted in tissue burdens in croaker brains, livers, and testes of 13, 25, and 3 ppm, respectively [9]. These concentrations are comparable to those reported in tissues of Atlantic tomcod collected from the Hudson River estuary [61] but are somewhat higher than those typically detected in environmental samples. Thus, the broad environmental relevance and public health implications of our present findings of inhibition of TPH activity and impaired neuroendocrine function remain uncertain. However, there is evidence of neuroendocrine and reproductive dysfunction in women exposed to PCBs occupationally or in the diet [57, 58] and of neurodevelopmental and behavioral deficits in their children [59, 60]. The possibility that these adverse effects of PCBs on neural functions in humans are partly attributable to inhibition of TPH activity needs to be explored.

ACKNOWLEDGMENTS

The gifts of sbGnRH peptide and antibody from Drs. K. Okuzawa and H. Kagawa, National Research Institute of Aquaculture, Japan, and the 5-HT ELISA kit from KMI Diagnostics are gratefully acknowledged. We also acknowledge the helpful suggestions from Drs. Hamid Habibi and Debananda Pati for the development of the GnRH-R assay and the assistance of Ms. Sonya Mathews with the GnRH ELISA.

FOOTNOTES

First decision: 22 September 2000.

1 This research was supported by PHS grant ESO7672. An abstract of the preliminary results was published in the Proceedings of the 4th International Symposium on Fish Endocrinology, August 2000, Seattle, WA. Back

2 Correspondence: Izhar A. Khan, The University of Texas at Austin, Marine Science Institute, 750 Channelview Drive, Port Aransas, TX 78373. FAX: 361 749 6777; ikhan{at}utmsi.utexas.edu Back

Accepted: October 26, 2000.

Received: August 17, 2000.

REFERENCES

  1. Klein D, Wan YY, Kamyab S, Okuda H, Sokol RZ. Effects of toxic levels of lead on gene regulation in the male axis: increase in messenger ribonucleic acid and intracellular stores of gonadotrophs within the central nervous system. Biol Reprod 1994; 50:802–811[Abstract]
  2. Sokol RZ, Berman N, Okuda H, Raum W. Effects of lead exposure on GnRH and LH secretion in male rats: response to castration and {alpha}-methyl-p-tyrosine (AMPT) challenge. Reprod Toxicol 1998; 12:347–355[CrossRef][Medline]
  3. Sierra EM, Tiffany-Castiglioni E. Effects of low level lead exposure on hypothalamic hormones and serum progesterone levels in pregnant guinea pigs. Toxicology 1992; 72:89–97[CrossRef][Medline]
  4. Foster WG, McMahon A, YoungLai EV, Hughes EG, Rice DC. Reproductive endocrine effects of chronic lead exposure in the male cynomolgus monkey. Reprod Toxicol 1993; 7:203–209[CrossRef][Medline]
  5. Thomas P, Khan IA. Mechanisms of chemical interference with reproductive endocrine function in sciaenid fishes. In: Rolland RM, Gilbertson M, Peterson RE (eds.), Chemically Induced Alterations in Functional Development and Reproduction of Fishes, SETAC Technical Publication Series; 1997: 29–51
  6. Cooper RL, Goldman JM, Stoker TE. Neuroendocrine and reproductive effects of contemporary-use pesticides. Toxicol Indust Health 1999; 15:26–36[Abstract/Free Full Text]
  7. Goldman JM, Cooper RL, Laws SL, Rehnberg GL, Edwards TL, McElroy WK, Hein JF. Chlorodimeform-induced alterations in endocrine regulation within the male reproductive system. Toxicol Appl Pharmacol 1990; 104:25–35[CrossRef][Medline]
  8. Uphouse L. Effects of chlordecone on neuroendocrine function of female rats. Neurotoxicology 1985; 6:191–210[Medline]
  9. Khan IA, Thomas P. Aroclor 1254-induced alterations in hypothalamic monoamine metabolism in the Atlantic croaker (Micropogonias unulatus): correlation with pituitary gonadotropin release. Neurotoxicology 1997; 18:553–560[Medline]
  10. Thomas P. Effects of Aroclor 1254 and cadmium on reproductive endocrine function and ovarian growth in Atlantic croaker. Mar Environ Res 1989; 28:499–507[CrossRef]
  11. Desaulniers D, Leingartner K, Wade M, Fintelman E, Yagminas A, Foster WG. Effects of acute exposure to PCBs 126 and 153 on anterior pituitary and thyroid hormones and FSH isoforms in adult Sprague Dawley male rats. Toxicol Sci 1999; 47:158–169[Abstract/Free Full Text]
  12. Jansen HT, Cooke PS, Porcelli J, Liu TC, Hansen LG. Estrogenic and antiestogenic actions of PCBs in the female rat: in vitro and in vivo studies. Reprod Toxicol 1993; 7:237–248[CrossRef][Medline]
  13. Khan IA, Thomas P. Lead and Aroclor 1254 disrupt reproductive neuroendocrine function in Atlantic croaker. Mar Environ Res 2000; 50:119–123[CrossRef][Medline]
  14. Blázquez M, Bosma PT, Fraser EJ, Van Look KJW, Trudeau VL. Fish as models for the neuroendocrine regulation of reproduction and growth. Comp Biochem Physiol 1998; C119:345–364
  15. Somoza GM, Peter RE. Effects of serotonin on gonadotropin and growth hormone release from in vitro perifused goldfish pituitary fragments. Gen Comp Endocrinol 1991; 82:103–110[CrossRef][Medline]
  16. Yu KL, Rosenblum PM, Peter RE. In vitro release of gonadotropin-releasing hormone from the brain preoptic-anterior hypothalamic region and pituitary of female goldfish. Gen Comp Endocrinol 1991; 81:256–267[CrossRef][Medline]
  17. Khan IA, Thomas P. Stimulatory effects of serotonin on maturational gonadotropin release in the Atlantic croaker, Micropogonias undulatus. Gen Comp Endocrinol 1992; 88:388–396[CrossRef][Medline]
  18. Khan IA, Thomas P. Immunocytochemical localization of serotonin and gonadotropin-releasing hormone in the brain and pituitary gland of the Atlantic croaker, Micropogonias undulatus. Gen Comp Endocrinol 1993; 91:167–180[CrossRef][Medline]
  19. Vitale ML, Parisi MN, Chiocchio SR, Tramezzani JH. Serotonin induces gonadotropin release through stimulation of LH-releasing hormone release from the median eminence. J Endocrinol 1986; 111:309–315[Abstract/Free Full Text]
  20. Fink G, Dow RC, McQueen JK, Bennie JG, Carroll SM. Serotonergic 5-HT2A receptors are important for the oestradiol-induced surge of luteinizing hormone-releasing hormone in the rat. J Neuroendocrinol 1999; 11:63–69[CrossRef][Medline]
  21. Lado-Abeal J, Rey C, Cabezas-Agricola JM, Rodriguez A, Camarero E, Cabezas-Serrato J. L-5-hydroxytryptophan amplifies pulsatile secretion of LH in the follicular-phase of normal women. Clin Endocrinol 1997; 47:555–563. [CrossRef][Medline]
  22. Hery M, Francois-Bellan AM, Hery F, Deprez P, Becquet D. Serotonin directly stimulates luteinizing hormone-releasing hormone release from GT1 cells via 5-HT7 receptors. Endocrine 1997; 7:261–265. [Medline]
  23. Arias P, Szwaecfarb B, de Rondina DC, Carbone S, Sverdlik R, Moguilevsky JA. In vivo and in vitro studies on the effect of serotonergic system on luteinizing hormone and luteinizing hormone-releasing hormone secretion in prepubertal and peripubertal female rats. Brain Res 1990; 523:57–61[CrossRef][Medline]
  24. Li S, Pelletier G. Involvement of serotonin in the regulation of GnRH gene expression in the male rat brain. Neuropeptides 1995; 29:21–25[CrossRef][Medline]
  25. Van de Kaar LD, Lorens SA, Vodraska A, Allers G, Green M, Van Orden DE, Van Orden III LS. Effects of selective midbrain and diencepahlic 5,7-dihydroxytryptamine lesions on serotonin content in individual preoptico-hypothalamic nuclei and on serum luteinizing hormone levels. Neuroendocrinology 1980; 31:309–315[Medline]
  26. Seegal RF, Bush B, Brosch KO. Sub-chronic exposure of the adult rat to Aroclor 1254 yields regionally-specific changes in dopaminergic function. Neurotoxicology 1991; 12:55–56[Medline]
  27. Seegal RF, Bush B, Brosch KO. Polychlorinated biphenyls induce regional changes in brain norepinephrine concentrations in adult rats. Neurotoxicology 1985; 6:13–24[Medline]
  28. Seegal RF, Brosch KO, Bush B. Regional alterations in serotonin metabolism induced by oral exposure to polychlorinated biphenyls. Neurotoxicology 1986; 7:155–166[Medline]
  29. Chu I, Villeneuve DC, Yagminas A, Lacavalier P, Poon R, Feeley M, Kennedy FW, Seegal RF, Hakånsson H, Ahlborg UG, Valli VE, Bergman Å. Toxicity of 2,2',4,4',5,5'-hexachlorobiphenyl in rats: effects following 90-day oral exposure. J Appl Toxicol 1996; 16:121–128[CrossRef][Medline]
  30. Seegal RF, Bush B, Shain W. Lightly chlorinated ortho-substituted PCB congeners decrease dopamine in non-human primate brain and in tissue culture. Toxicol Appl Pharmacol 1990; 106:136–144[CrossRef][Medline]
  31. Seegal RF, Bush B, Shain W. Neurotoxicology of ortho-substituted polychlorinated biphenyls. Chemosphere 1991; 23:1941–1949[CrossRef]
  32. Choski NY, Kodavanti PRS, Tilson HA, Booth RG. Effects of polychlorinated biphenyls (PCBs) on brain tyrosine hydroxylase activity and dopamine synthesis in rats. Fundam Appl Toxicol 1997; 39:76–80[CrossRef][Medline]
  33. Jequier E, Lovenberg W, Sjoerdsma A. Tryptophan hydroxylase inhibition: the mechanism by which p-chlorophenylalanine depletes rat brain serotonin. Mol Pharmacol 1967; 3:274–278[Abstract/Free Full Text]
  34. Fuller RW. Drugs altering serotonin synthesis and metabolism. In: Green AR (ed.), Neuropharmacology of Serotonin. New York: Oxford University Press; 1985: 1–20
  35. Senthilkumaran B, Joy KP. Effects of administration of some monoamine-synthesis blockers and precursors on ovariectomy-induced rise in plasma gonadotropin II in the catfish Heteropnuestes fossilis. Gen Comp Endocrinol 1996; 101:220–226[CrossRef][Medline]
  36. Senthilkumaran B, Okuzawa K, Gen K, Ookura T, Kagawa H. Distributions and seasonal variations in levels of three native GnRHs in the brain and pituitary of perciform fish. J Neuroendocrinol 1999; 11:181–186[CrossRef][Medline]
  37. Joy KP, Khan IA. Pineal-gonadal relationship in the teleost Channa punctatus (Bloch): evidence for possible involvement of hypothalamic serotonergic system. J Pineal Res 1991; 11:12–22[Medline]
  38. Hodson RG, Sullivan CV. Induced maturation and spawning of domestic and wild striped bass, Morone saxatilis (Walbaum), broodstock with implanted GnRH analogue and injected hCG. Aquat Fish Manage 1993; 24:389–398
  39. Vrana SL, Dworkin SI, Vrana KE. Radioenzymatic assay for tryptophan hydroxylase: [3H]H2O release assessed by charcoal adsorption. J Neurosci Methods 1993; 48:123–129[CrossRef][Medline]
  40. Parvez H, Parvez S. Microradioisotopic determination of enzymes catechol-O-methyltransferase, phenylethanolamine-N-transferase and monoamine oxidase in a single concentration of tissue homogenate. Clin Chem Acta 1973; 40:85–90
  41. Smith JE, Lane JD, Shea PA, McBride WJ, Aprison MH. A method of concurrent measurement of picomole quantities of acetylcholine, choline, dopamine, norepinephrine, serotonin, 5-hydroxytryptophan, 5-hydroxyindolacetic acid, tryptophan, tyrosine, glycine, aspartate, glutamate, alanine, and gamma-aminobutyric acid in single tissue samples from different areas of rat central nervous system. Anal Biochem 1975; 64:149–169[CrossRef][Medline]
  42. Copeland PA, Thomas P. Purification of maturational gonadotropin from Atlantic croaker (Micropogonias undulatus) and development of a homologous radioimmunoassay. Gen Comp Endocrinol 1989; 73:425–441[CrossRef][Medline]
  43. Holland MCH, Gothilf Y, Meiri I, King JA, Okuzawa K, Elizur A, Zohar Y. Levels of native forms of GnRH in the pituitary of the gilthead seabream, Sparus aurata, at several characteristic stages of the gonadal cycle. Gen Comp Endocrinol 1998; 112:394–405[CrossRef][Medline]
  44. Pradelles P, Grassi J, Maclouf J. Enzyme immunoassays of eicosanoids using acetylcholinesterase as label: an alternative to radioimmunoassay. Anal Chem 1985; 57:1170–1173[Medline]
  45. Pagelson G, Zohar Y. Characterization of gonadotropin-releasing hormone binding to pituitary receptors in the gilthead seabream (Sparus aurata). Biol Reprod 1992; 47:1004–1008[Abstract]
  46. Gore AC, Lee JB, Sun Y. Effects of environmental toxicants on GnRH gene expression in hypothalamic neurons. Int Symp Environ Endocr Disruptors 1999; 2: (abstract)
  47. Habibi HR, De Leeuw R, Nahorniak CS, Goos HJTh, Peter RE. Pituitary gonadotropin-releasing hormone (GnRH) receptor activity in goldfish and catfish: seasonal and gonadal effects. Fish Physiol Biochem 1989; 7:109–118[CrossRef]
  48. Omeljaniuk RJ, Habibi HR, Peter RE. Alterations in pituitary GnRH and dopamine receptors associated with the seasonal variation and regulation of gonadotropin release in goldfish (Carassius auratus). Gen Comp Endocrinol 1989; 74:392–399[CrossRef][Medline]
  49. Clayton RN, Solano AR, Garcia-Vela A, Dufau M, Catt KJ. Regulation of pituitary receptors for gonadotropin-releasing hormone during the rat estrous cycle. Endocrinology 1980; 107:699–706[Abstract]
  50. Brooks J, Taylor PL, Saunders PTK, Eidene K, Struthers WJ, McNeilly AS. Cloning and sequencing of the sheep pituitary gonadotropin-releasing hormone receptor and changes in expression of its mRNA during the estrous cycle. Mol Cell Endocrinol 1993; 94:R23–R27
  51. Khan IA, Thomas P. Melatonin influences gonadotropin II secretion in the Atlantic croaker (Micropogonias undulatus). Gen Comp Endocrinol 1996; 104:231–242[CrossRef][Medline]
  52. Copeland PA, Thomas P. Control of gonadotropin release in the Atlantic croaker (Micropogonias undulates): evidence for a lack of dopaminergic inhibition. Gen Comp Endocrinol 1989; 74:474–483[CrossRef][Medline]
  53. Fitzpatrick PF. Tetrahydropterin-dependent amino acid hydroxylases. Annu Rev Biochem 1999; 68:355–381[CrossRef][Medline]
  54. Hufton SE, Jennings IG, Cotton RGH. Structure and function of the aromatic amino acid hydroxylases. Biochem J 1995; 311:353–366.
  55. Tilson HA, Kodavanti PRS. The neurotoxicity of polychlorinated biphenyls. Neurotoxicology 1998; 19:517–526[Medline]
  56. Seiler P, Fischer B, Lindenau A, Beier HM. Effects of persistent chlorinated hydrocarbons on fertility and embryonic development in the rabbit. Hum Reprod 1994; 9:1920–1926[Abstract/Free Full Text]
  57. Medola P, Buck GM, Server LE, Zieiezny M, Vena JE. Consumption of PCB-contaminated freshwater fish and shortened menstrual cycle length. Am J Epidemiol 1997; 146:955–960[Abstract/Free Full Text]
  58. Gerhard I, Daniel V, Link S, Monga B, Runnebaum B. Chlorinated hydrocarbons in women with repeated miscarriages. Environ Health Perspect 1998; 106:675–681[Medline]
  59. Jacobson JL, Jacobson SW. Intellectual impairment in children exposed to polychlorinated biphenyls in utero. New Engl J Med 1996; 335:783–789[Abstract/Free Full Text]
  60. Rogan WJ, Gladen BC, Hung K, Koong SL, Snih LY, Taylor JS, Wu YC, Yang D, Ragan NB, Hsu CC. Congenital poisoning by polychlorinated biphenyls and their contaminants in Taiwan. Science 1988; 241:334–336[Abstract/Free Full Text]
  61. Klauda RJ, Peck TH, Rice GK. Accumulation of polychlorinated biphenyls in Atlantic tomcod (Microgadus tomcod) collected from Hudson River estuary, New York. Bull Environ Contam Toxicol 1981; 27:829–835[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Khan, I. A.
Right arrow Articles by Thomas, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Khan, I. A.
Right arrow Articles by Thomas, P.
Agricola
Right arrow Articles by Khan, I. A.
Right arrow Articles by Thomas, P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS