Izhar A. Khan

Widespread endocrine disruption and reproductive impairment in an estuarine fish population exposed to seasonal hypoxia

The long-term effects on marine fish populations of the recent increase worldwide in the incidence of coastal hypoxia are unknown. Here we show that chronic environmental exposure of Atlantic croaker (Micropogonias undulatus) to hypoxia in a Florida estuary caused marked suppression of ovarian and testicular growth which was accompanied by endocrine disruption. Laboratory hypoxia studies showed that the endocrine disruption was associated with impairment of reproductive neuroendocrine function and decreases in hypothalamic serotonin (5-HT) content and the activity of the 5-HT biosynthetic enzyme, tryptophan hydroxylase. Pharmacological restoration of hypothalamic 5-HT levels also restored neuroendocrine function, indicating that the stimulatory serotonergic neuroendocrine pathway is a major site of hypoxia-induced inhibition. Inhibition of tryptophan hydroxylase activity to downregulate reproductive activity could have evolved as an adaptive mechanism to survive periodic hypoxia, but in view of the recent increased incidence of coastal hypoxia could become maladaptive and potentially affect fish population abundance and threaten valuable fishery resources.

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

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.

Stimulatory effects of serotonin on maturational gonadotropin release in the Atlantic croaker, Micropogonias undulatus

The effects of serotonin (5-HT) injection alone, and in combination with des Gly10 [D-Trp6]-luteinizing hormone-releasing hormone ethylamide (LHRHa), on plasma maturational gonadotropin (GtH) levels in the Atlantic croaker, Micropogonias undulatus, were examined. An injection of 5-HT (20 micrograms/g body wt) alone did not significantly elevate GtH levels, whereas simulatneous administration of LHRHa (20 ng/g body wt) and 5-HT elicited a greater elevation of plasma GtH levels than that induced by LHRHa alone. Pretreatment of 1-year-old fish with fluoxetine (10 micrograms/g), a 5-HT reuptake inhibitor, only slightly augmented the effect of 5-HT on GtH levels, whereas pretreatment with ketanserin (10 micrograms/g), a 5-HT receptor antagonist, completely inhibited the potentiating effect of 5-HT on the GtH response to LHRHa. Administration of LHRHa (20 ng/g) or 5-HT (20 micrograms/g) significantly elevated GtH levels in the 2- and 3-year-old croaker, but the combined treatment failed to increase GtH levels above those induced by LHRHa alone. However, with a lower dose of LHRHa (5 ng/g), the combination produced an additive effect. Serotonin (20 micrograms/ml media) alone, and in combination with LHRHa (10 ng/ml media), significantly stimulated GtH release from the pituitaries of gonadally mature 2- and 3-year-old female croaker during an 18-hr incubation in vitro, but not from pituitaries of gonadally regressed fish. The combined treatment of LHRHa with 5-HT also significantly stimulated in vitro GtH release during 6- and 12-hr pituitary incubations, whereas these treatments alone were ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution of an AVT V1a receptor in the brain of a sex changing fish, Epinephelus adscensionis.

The present study describes the distribution of an arginine vasotocin (AVT) V1a receptor (AVTr) throughout the brain of a sex-changing grouper, rock hind Epinephelus adscensionis. The objectives of this study were to describe the AVTr distribution in the brain of rock hind for potential linkages of the AVT hormone system with sex-specific behaviors observed in this species and to examine sex-specific differences that might exist. An antibody was designed for rock hind AVTr against the deduced amino acid sequence for the third intracellular loop. Protein expression, identified with immunohistochemistry showed high concordance with mRNA expression, identified with in situ hybridization. AVTr protein and mRNA expression was widely distributed throughout the brain, indicating that AVT may act as a neuromodulator via this V1a receptor subtype. AVTr protein and mRNA were present in regions associated with behavior, reproduction and spatial learning, as well as sensory functions such as vision, olfaction and lateral line sensory processing. We observed high AVTr expression in granular cell formations in the internal cellular layer of olfactory bulbs, torus longitudinalis, granular layer of the corpus cerebellum, valvula of the cerebellum, nuclei of the lateral and posterior recesses, and granular eminence. High protein and mRNA expression was also observed in the preoptic area, anterior hypothalamus, and habenular nucleus. No obvious sex differences were noted in any region of the rock hind brain.

Isolation, cloning, and expression of three prepro-GnRH mRNAs in Atlantic croaker brain and pituitary.

Three prepro‐gonadotropin‐releasing hormones, seabream GnRH (sbGnRH), chicken GnRH‐II (cGnRH‐II), and salmon GnRH (sGnRH) were isolated by cDNA cloning from the brain of the Atlantic croaker, Micropogonias undulatus. The amino acid sequences of croaker GnRH precursors show greatest similarities to those of the gilthead and red sea breams and European sea bass. In situ hybridization of croaker brain sections revealed more abundant sbGnRH mRNA expression in the preoptic area (POA) than in other brain regions. sbGnRH mRNA expression was also observed in the olfactory bulb (OB; but not in the terminal nerve ganglion cells [TNgc]), ventral telencephalon (vTEL), and anterior hypothalamus. In addition, specific sbGnRH mRNA signals were detected in the pituitary. cGnRH‐II mRNA expression was limited to the midbrain tegmentum. Neuronal elements expressing sGnRH mRNA were detected in the OB including the TNgc, vTEL, and POA, indicating an overlap of the sbGnRH and sGnRH systems in certain ventral forebrain areas. The results of quantitative reverse transcriptase‐polymerase chain reaction of the three GnRH mRNAs in different brain areas and the pituitary are consistent with their localization by in situ hybridization. Interestingly, a few sbGnRH mRNA‐expressing neuronal elements were observed arranged in a row in the anteroventral hypothalamus projecting toward the pituitary. The results provide a morphological basis for a putative role of sbGnRH as the gonadotropin‐releasing hormone. Moreover, localization of sbGnRH mRNA in a teleost pituitary points to sbGnRH synthesis, and its potential role as a local regulator, within the pituitary, similar to the role of GnRH‐I in mammals. J. Comp. Neurol. 488:384–395, 2005.

Distribution of nonapeptide systems in the forebrain of an African cichlid fish, Astatotilapia burtoni.

Nonapeptides and their receptors have important functions in mediating social behavior across vertebrates. Where these nonapeptides are synthesized in the brain has been studied extensively in most vertebrate lineages, yet we know relatively little about the neural distribution of nonapeptide receptors outside of mammals. As nonapeptides play influential roles in behavioral regulation in all vertebrates, including teleost fish, we mapped the distributions of the receptors for arginine vasotocin (AVT; homolog of arginine vasopressin) and isotocin (IST; homolog of oxytocin/mesotocin) throughout the forebrain of Astatotilapia burtoni, an African cichlid fish with behavioral phenotypes that are plastic and reversible based on the immediate social environment. We characterized the distribution of the AVT V1a2 receptor (V1aR) and the IST receptor (ITR) using both immunohistochemistry for protein detection and in situ hybridization for mRNA detection, as well as AVT and IST using immunohistochemistry. Expression of the neuropeptide receptors was widely distributed throughout the fore- and midbrain, including the proposed teleost homologs of the mammalian amygdala complex, striatum, hypothalamus, and ventral tegmental area. We conclude that although the location of nonapeptide synthesis is restricted compared to tetrapod vertebrates, the distribution of nonapeptide receptors is highly conserved across taxa. Our results significantly extend our knowledge of where nonapeptides act in the brains of teleosts to mediate social transitions and behavior.

Alterations in the GnRH-LH system in relation to gonadal stage and Aroclor 1254 exposure in Atlantic croaker.

Exposure of Atlantic croaker (Micropogonias undulatus) to the polychlorinated biphenyl mixture (Aroclor 1254, PCB; 1 mg/kg body wt/day for 30 days) during the early-recrudescence phase of the gonadal cycle results in the impairment of LH secretion and gonadal growth. In order to determine whether impairment was due to disruption of the stimulatory GnRH neuroendocrine pathway, we compared various parameters of the GnRH-LH system in early recrudescing vs. spermiating (mature) fish. Seabream GnRH (GnRH) content in the preoptic anterior hypothalamic area (POAH) and pituitary, pituitary GnRH receptor concentrations, and basal and GnRH analog (GnRHa)-induced LH secretion were significantly higher in gonadally mature croaker compared to early-recrudescing fish. In a subsequent experiment, the effects of PCB on the same neuroendocrine indices were investigated during the gonadal recrudescence phase of croaker. PCB exposure during the period of testicular maturation prevented the natural increase in GnRH content in the POAH but not in the pituitary. This finding suggests that PCB may impair GnRH synthesis in the POAH. The number of pituitary GnRH receptors also remained significantly lower in the PCB-exposed group, which was likely due to an impairment of GnRH release. The GnRH content in the POAH, number of pituitary GnRH receptors, and LH secretion in the PCB-exposed group were comparable to those in early-recrudescing fish, suggesting an impairment of normal maturation of the GnRH-LH system during the gonadal recrudescence phase. This impairment may be due to a direct action of PCB on GnRH neurons and/or indirectly via interference with other neurotransmitter pathways that modulate GnRH function.

Lead and Aroclor 1254 disrupt reproductive neuroendocrine function in Atlantic croaker

We have previously shown that lead (lead chloride) and a polychlorinated biphenyl (PCB) mixture (Aroclor 1254) can alter hypothalamic serotonin (5-hydroxytryptamine, 5-HT) content, and pituitary gonadotropin II (GTH II) release in vitro, in the Atlantic croaker (Micropogonias undulatus). In the present study we investigated whether impairment of the hypothalamic 5-HT pathway involves alterations in tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthesis, or monoamine oxidase (MAO), the catabolic enzyme. Aroclor 1254 (1 mg/kg body wt. for 30 days) significantly inhibited hypothalamic TPH activity without altering MAO activity, and caused a significant decline in 5-HT content. On the other hand, lead exposure (15 mg/kg body wt. for 30 days) only induced a slight decrease in hypothalamic 5-HT content and TPH activity, and a minor increase in MAO activity. However, both Aroclor 1254 and lead significantly inhibited the GTH II response to stimulation by a luteinizing hormone-releasing hormone analog (LHRHa) in vivo and caused reduced gonadal growth. These results demonstrate that impairment of hypothalamic serotonin metabolism by Aroclor 1254 involves inhibition of 5-HT synthesis, whereas lead does not exert a profound influence on 5-HT metabolism. The decline in 5-HT availability due to reduced 5-HT synthesis in the PCB-exposed fish may result in disruption of the stimulatory 5-HT-GnRH pathway controlling GTH II secretion leading to impairment of gonadal growth.

GABA Exerts Stimulatory and Inhibitory Influences on Gonadotropin II Secretion in the Atlantic Croaker (Micropogonias undulatus)

Possible involvement of γ-aminobutyric acid (GABA) in the control of gonadotropin II (GTH II) secretion was investigated during different phases of the seasonal gonadal cycle of the Atlantic croaker (Micropogonias undulatus). Intraperitoneal administration of GABA (100 µg/g) elicited a significant increase in plasma GTH II levels in croaker with regressed or pre-recrudescence phase gonads, whereas it did not significantly affect GTH II levels during the early- to mid-recrudescence phase of the gonadal cycle in this species. On the other hand, GABA induced a dose-related inhibition of GTH II secretion in fish with fully recrudesced gonads. Similar opposite changes in GTH II levels were observed when croaker with regressed and fully recrudesced gonads were injected with the GABA transaminase inhibitor, γ-vinyl GABA (10 and 100 µg/g). This suggests an involvement of endogenous GABA in the control of GTH II secretion. GABA (1–100 µM) did not significantly alter GTH II release from pituitary fragments from regressed or fully recrudesced fish incubated in vitro, suggesting the lack of a major direct influence at the pituitary level. Muscimol (GABAA receptor agonist, 1 µg/g) treatment mimicked both the stimulatory and inhibitory effects of GABA, whereas injection of baclofen (GABAB receptor agonist, 1 µg/g) had no significant effect on basal or a gonadotropin-releasing hormone (GnRH) analog-induced GTH II secretion. Pretreatment of fish with bicuculline (1 µg/g), a GABAA antagonist, completely blocked the stimulatory influence of GABA on GTH II secretion in croaker with regressed gonads and partially blocked the inhibitory effect in individuals with fully recrudesced gonads. The results demonstrate that GABA exerts opposite influences on GTH II secretion at two different stages of the gonadal cycle of croaker, and acts primarily via GABAA type receptors. Thus, GABAergic pathways may be important in mediating seasonal influences on GTH II secretion in this species.

Estradiol-17β and o,p′-DDT stimulate gonadotropin release in Atlantic croaker

Abstract Xenobiotic estrogens have the potential to act at a variety of estrogen-responsive target tissues on the hypothalamic-pituitary-gonadal axis. However, to date most studies in fish have focused on stimulation of vitellogenin synthesis by the liver. In the present study the effects of the xenoestrogen o,p′-DDT and estradiol-17β on the neuroendocrine control of gonadotropin secretion were compared. Atlantic croaker (Micropogonias undulatus) were exposed to o,p′-DDT (0.02 and 0.1 μgg−1 body weight day−1) in the diet for 3 and 7 weeks during the gonadal recrudescence phase. The o,p′-DDT exposure elicited a significant increase in plasma gonadotropin levels after both 3 and 7 weeks of exposure. The stimulatory effect of o,p′-DDT on basal (spontaneous) gonadotropin release was accompanied by a slight increase in ovarian growth as evidenced by the increase in gonadosomatic index. It appears that the stimulation of gonadotropin release by o,p′-DDT during early-recrudescence phase results in enhanced ovarian growth. A comparable stimulatory effect was observed with estradiol-17β treatment during early- and late-recrudescence phases of the ovarian cycle using three injections on alternate days and slow release silastic implants (five days). The present study provides the first evidence for an estrogen-like action of o,p′-DDT on gonadotropin release in a teleost model.