John P. Chang

Exposure to bleached kraft pulp mill effluent disrupts the pituitary-gonadal axis of white sucker at multiple sites

Our recent studies have demonstrated reproductive problems in white sucker (Catostomus commersoni) exposed to bleached kraft pulp mill effluent (BKME) at Jackfish Bay on Lake Superior. These fish exhibit delayed sexual maturity, reduced gonadal size, reduced secondary sexual characteristics, and circulating steroid levels depressed relative to those of reference populations. The present studies were designed to evaluate sites in the pituitary-gonadal axis of prespawning white sucker affected by BKME exposure. At the time of entry to the spawning stream, plasma levels of immunoreactive gonadotropin (GtH)-II (LH-type GtH) in male and female white sucker were 30- and 50-fold lower, respectively, than the levels in fish from a reference site. A single intraperitoneal injection of D-Arg6, Pro9N-Et sGnRH (sGnRH-A, 0.1 mg/kg) increased plasma GtH levels in male and female fish at both sites, although the magnitude of the response was greatly reduced in BKME-exposed fish. Fish at the BKME site did not ovulate in response to sGnRH-A, while 10 of 10 fish from the reference site ovulated within 6 hr. Plasma 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) levels were depressed in BKME-exposed fish and unlike fish at the reference site, failed to increase in response to sGnRH-A. Testosterone levels in both sexes and 11-ketostestosterone levels in males were elevated in fish from the reference site but were not further increased by GnRH treatment. In contrast, BKME-exposed fish exhibit a transitory increase in testosterone levels in response to the GnRH analog. In vitro incubations of ovarian follicles obtained from fish at the BKME site revealed depressed basal secretion of testosterone and 17,20 beta-P and reduced responsiveness to the GtH analog human chorionic gonadotropin and to forskolin, a direct activator of adenylate cyclase. By comparison, ovarian follicles from fish collected at BKME and reference sites produced similar levels of prostaglandin E basally and in response to a phorbol ester and calcium ionophore A23187, suggesting that BKME effects on ovarian function are selective and do not reflect a general impairment of ovarian function. BKME-exposed fish had plasma levels of testosterone glucuronide proportionately lower than those of reference fish, suggesting that there are site differences in the peripheral metabolism of steroids. These studies demonstrate that BKME exposure affects reproduction by acting at multiple sites in the pituitary-gonadal axis.

Use of a pituitary cell dispersion method and primary culture system for the studies of gonadotropin-releasing hormone action in the goldfish, Carassius auratus. I. Initial morphological, static, and cell column perifusion studies.

Two cell dispersion methods for excised goldfish pituitary glands were tested, and a cultured dispersed cell system based on trypsin enzymatic tissue digestion was developed and characterized. Controlled trypsin/DNase treatment of goldfish pituitary gland yielded dispersed cells of high viability (trypsin blue exclusion test) that responded to gonadotropin (GTH)-releasing hormone (GnRH) challenges with GTH secretion in a time- and dose-dependent manner following overnight culture. Electron microscopy revealed that cell preparations produced by the trypsin dispersion were free of cell debris and nerve terminals. The dispersed pituitary cells also retained distinct morphological and immunological identities. Under static incubation conditions, 2-hr treatments with 0.1 nM to 1 microM [Trp7,Leu8]-GnRH (sGnRH) and [D-Arg6,Pro9-N-ethylamide]-sGnRH (sGnRHa) stimulated GTH release with similar efficacy, but with ED50S of 1.92 +/- 0.48 and 0.19 +/- 0.08 nM, respectively. [His5,Trp7,Tyr8]-GnRH (cGnRH-II) stimulated GTH release in a nonsigmoidal, but dose-dependent manner, and with a higher efficacy than sGnRH. In contrast, sGnRH, sGnRHa, and cGnRH-II were equipotent in inducing growth hormone (GH) secretion in static culture studies and with ED50S of 0.29 +/- 0.13, 0.18 +/- 0.11, and 0.19 +/- 0.17 nM, respectively. When trypsin/DNase-dispersed cells cultured overnight with cytodex beads were tested in a cell column perifusion system, dose-related increase in GTH secretion, as well as GH release, were also observed with 0.5 to 50 nM sGnRH. These results suggest that trypsin-dispersed goldfish pituitary cells can be used effectively to study the actions of GnRH on teleost pituitary either in short-term static incubation or column perifusion studies. Differences in the GTH and GH responses to the two native GnRH forms, sGnRH and cGnRH-II, are also indicated.

Effects of Dopamine on Gonadotropin Release in Female Goldfish, Carassius auratus

Intraperitoneal injections of dopamine (DA) or its agonist, apomorphine decreased and pimozide, a DA antagonist, increased serum gonadotropin (GtH) levels in normal female goldfish. Injection of DA into the third cranial ventricular did not alter serum GtH concentrations. Intraperitoneal injections of DA or apomorphine each reduced the highly elevated serum GtH levels caused by preoptic lesions which abolish an inhibitory hypothalamic influence on GtH release allowing prolonged spontaneous release of GtH. Intraperitoneal injections of DA or apomorphine blocked the stimulation of GtH release induced by injections of a luteinizing hormone-releasing hormone analogue. These results indicate that DA has GtH release-inhibitory activity by actions directly on gonadotrophs to inhibit spontaneous secretion of GtH, and by blocking the actions of the GtH-releasing hormone.

Effects of pimozide and des Gly10,[d-Ala6]luteinizing hormone-releasing hormone ethylamide on serum gonadotropin concentrations, germinal vesicle migration, and ovulation in female goldfish, Carassius auratus

In gravid female goldfish held at 10-12 degrees, a single injection of des Gly10,[D-Ala6]luteinizing hormone-releasing hormone ethylamide (LHRH-A), or 2 injections of LHRH-A given 12 hr apart, increased serum gonadotropin (GtH) concentrations, but did not stimulate germinal vesicle migration or ovulation. Injection of a dopamine antagonist, pimozide (PIM), increased serum GtH levels. PIM injected with the second of two LHRH-A injections not only potentiated the LHRH-A-induced increase in serum GtH concentrations but also caused germinal vesicle migration and ovulation. PIM injected with, or in place of, the first of two LHRH-A injections increased the frequency of ovulation in fish injected with LHRH-A. Similarly, PIM injected simultaneously with a single LHRH-A injection also increased the number of ovulating fish. These results are consistent with the idea that dopamine inhibits GtH release in goldfish. The results also suggest that the preovulatory surge of GtH secretion is regulated by both a stimulation by GtH-releasing hormone and release from the dopamine inhibition on GtH secretion.

Differential actions of dopamine receptor subtypes on gonadotropin and growth hormone release in vitro in goldfish

Incubation of cultured goldfish pituitary cells with 10 nM to 1 microM apomorphine (APO), a non-selective dopamine agonist, increased growth hormone (GH) release in a dose-dependent manner. GH release was also stimulated in a dose-dependent manner by 0.1 nM to 1 microM salmon gonadotropin (GTH)-releasing hormone (sGnRH), sGnRH analog, and chicken GnRH-II (cGnRH-II). The magnitude of GH responses to 1 microM GnRHs were less than that to 1 microM APO. GH responses to 10 nM to 1 microM APO were not significantly increased by the addition of GnRHs. Static incubations with 0.1 nM to 1 microM of the dopamine D1 agonist SKF38393 did not alter basal GTH release, or the GTH responses to 10 nM sGnRH and cGnRH-II. In contrast, the D1 agonist SKF38393 significantly increased basal GH secretion with maximal stimulation achieved at 100 nM concentration, and GH responses to 10 nM sGnRH and 10 nM cGnRH-II were enhanced by simultaneous applications of SKF38393. Incubation with 1 microM of the D2 agonist LY171555 decreased basal GTH release. Additions of 0.1 nM to 1 microM LY171555 caused dose-dependent decreases in the GTH secretion induced by 10 nM sGnRH and cGnRH-II. In contrast, basal and GnRH-stimulated GH release were not affected by coincubations with LY171555. The D1 antagonist SKF83566 and the D2 antagonist domperidone, at 1 microM concentrations, specifically blocked the D1 agonist SKF38393-stimulated increase in GH release and the D2 agonist LY171555-induced depression of GTH secretion, respectively. In cell column perifusion studies, the D1 agonist SKF38393 at 0.1 nM to 1 microM had no effects on GTH release, but significantly elevated GH secretion rates when applied at 0.1-1 microM concentrations. The GH release induced by 1 microM SKF38393 was significantly reduced by simultaneous perifusion with 1 microM of the D1 antagonist SKF83566. Treatments with SKF38393 and/or SKF83566 did not affect net GTH and GH responses to sGnRH challenges. In contrast, perifusion with 0.1 and 1 microM of the D2 agonist LY171555 depressed basal as well as sGnRH-induced GTH responses. These effects of 1 microM LY171555 were completely blocked by simultaneous applications of 1 microM domperidone, a D2 antagonist. Treatments with these D2 selective drugs did not affect basal and sGnRH-stimulated GH release. These results indicate that in cultured goldfish pituitary cells, activation of dopamine D1- and D2-like receptors specifically stimulates GH release and inhibits both basal and stimulated GTH secretion, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Primary structure and function of three gonadotropin-releasing hormones, including a novel form, from an ancient teleost, herring

The evolution of GnRH and the role of multiple forms within the brain are examined. Three forms of GnRH were purified from the brain of Pacific herring (Clupea harengus pallasi) and characterized using Edman degradation and mass spectrometry. Two forms correspond with the known structures of chicken GnRH-II and salmon GnRH that are found in many vertebrate species. The third form, designated herring GnRH (hrGnRH), has a primary structure of pGlu-His-Trp-Ser-His-Gly-Leu-Ser-Pro-Gly-NH2. This novel peptide is a potent stimulator of gonadotropin II and GH release from dispersed fish pituitary cells. The content of hrGnRH in the pituitary was 8-fold that of salmon GnRH and 43-fold that of chicken GnRH-II, which provides supporting evidence that hrGnRH is involved in the release of gonadotropin. Herring is the most phylogenetically ancient animal in which three forms of GnRH have been isolated and sequenced. Our evidence suggests that the existence of three GnRHs in the brain of one species 1) is an ancestral ...

Arachidonic acid stimulates steroidogenesis in goldfish preovulatory ovarian follicles

The possibility that arachidonic acid (AA) plays a role in the regulation of steroidogenesis in goldfish was investigated using preovulatory ovarian follicles incubated in vitro. AA was shown to act in a time- and dose-dependent manner to stimulate testosterone production. AA in the range of 10(-5) to 10(-4) M increased testosterone production within 2 hr and had a maximal effect by 9 hr. The magnitude of the testosterone response to AA was similar to that observed when ovarian follicles were incubated with human chorionic gonadotropin (hCG). Ovarian follicles incubated with AA and either hCG or forskolin (adenylate cyclase activator) produced more testosterone than follicles incubated with either of these compounds alone. The actions of AA on testosterone production were completely blocked by cyclooxygenase inhibitors (indomethacin or ibuprofen) and were reduced by 50% by the lipoxygenase inhibitor nordihydroguaiaretic acid. Phospholipase C was far more effective than phospholipase A2 in the stimulation of testosterone production. Taken together, these results suggest that AA formed subsequent to the action of phospholipase C on membrane phospholipids has a role in the regulation of steroidogenesis in preovulatory goldfish ovarian follicles.

Waterborne fluoxetine disrupts the reproductive axis in sexually mature male goldfish, Carassius auratus

Fluoxetine (FLX) is a pharmaceutical acting as a selective serotonin reuptake inhibitor and is used to treat depression in humans. Fluoxetine and the major active metabolite norfluoxetine (NFLX) are released to aquatic systems via sewage-treatment effluents. They have been found to bioconcentrate in wild fish, raising concerns over potential endocrine disrupting effects. The objective of this study was to determine effects of waterborne FLX, including environmental concentrations, on the reproductive axis in sexually mature male goldfish. We initially cloned the goldfish serotonin transporter to investigate tissue and temporal expression of the serotonin transporter, the FLX target, in order to determine target tissues and sensitive exposure windows. Sexually mature male goldfish, which showed the highest levels of serotonin transporter expression in the neuroendocrine brain, were exposed to FLX at 0.54μg/L and 54μg/L in a 14-d exposure before receiving vehicle or sex pheromone stimulus consisting of either 4.3nM 17,20β-dihydroxy-4-pregnene-3-one (17,20P) or 3nM prostaglandin F₂(α) (PGF₂(α)). Reproductive endpoints assessed included gonadosomatic index, milt volume, and blood levels of the sex steroids testosterone and estradiol. Neuroendocrine function was investigated by measuring blood levels of luteinizing hormone, growth hormone, pituitary gene expression of luteinizing hormone, growth hormone and follicle-stimulating hormone and neuroendocrine brain expression of isotocin and vasotocin. To investigate changes at the gonadal level of the reproductive axis, testicular gene expression of the gonadotropin receptors, both the luteinizing hormone receptor and the follicle-stimulating hormone receptor, were measured as well as expression of the growth hormone receptor. To investigate potential impacts on spermatogenesis, testicular gene expression of the spermatogenesis marker vasa was measured and histological samples of testis were analyzed qualitatively. Estrogen indices were measured by expression and activity analysis of gonadal aromatase, as well as liver expression analysis of the estrogenic marker, esr1. After 14d, basal milt volume significantly decreased at 54μg/L FLX while pheromone-stimulated milt volume decreased at 0.54μg/L and 54μg/L FLX. Fluoxetine (54μg/L) inhibited both basal and pheromone-stimulated testosterone levels. Significant concentration-dependent reductions in follicle-stimulating hormone and isotocin expression were observed with FLX in the 17,20P- and PGF₂(α)-stimulated groups, respectively. Estradiol levels and expression of esr1 concentration-dependently increased with FLX. This study demonstrates that FLX disrupts reproductive physiology of male fish at environmentally relevant concentrations, and potential mechanisms are discussed.

Induction of ovulation in goldfish, Carassius auratus, by pimozide and analogues of LH-RH

Abstract The effects of intraperitoneal injections or pellet implantations of luteinizing hormone-releasing hormone analogues, des-Gly10-[D-ALA6]-LH-RH-ethylamide (LRH-Aa) or des-Gly10-[D-Trp6]-LH-RH-ethylamide (LRH-At), respectively, and the combination of these treatments with pimozide, a dopamine antagonist, on serum gonadotropin (GtH) and ovulation in goldfish held at 18–20°C were investigated. Injections of LRH-Aa (0.1 μg/g body weight) or pellet implants of LRH-At (25 and 125 μg/fish) alone increased serum GtH levels, but were relatively ineffective for inducing ovulation in goldfish (≅ 25% rate of occurrence). Pimozide greatly potentiated the GtH release response to LRH-Aa injections or LRH-At pellet implantations. A high rate of occurrence of ovulation (87%) was observed within 24 h when pimozide was injected prior to or with the first of two injections of LRH-Aa, or following implantations of LRH-At pellets that give continuous release of LRH-At. These results are consistent with the idea that dopamine acts directly at the pituitary level as a GtH release-inhibitory factor in goldfish.

Effects of catecholaminergic agonists and antagonists on serum gonadotropin concentrations and ovulation in goldfish: evidence for specificity of dopamine inhibition of gonadotropin secretion.

The elevated serum gonadotropin (GtH) levels in goldfish receiving two injections of des Gly10, [D-Ala6] LH-RH ethylamide (LH-RH-A), given 12-hr apart, were reduced by apomorphine, a dopamine agonist, injected at either the first or the second LH-RH-A injection. Serum GtH concentrations in goldfish given two injections of LH-RH-A at a 3-hr interval were also depressed by bromocriptine, a specific D-2 dopamine receptor agonist, administered simultaneously with both LH-RH-A injections. Injections of dopamine antagonists, pimozide or metoclopramide (a specific D-2 antagonist) caused increased serum GtH concentrations in normal goldfish, but no changes were found following injections of the alpha-adrenergic antagonist phentolamine, the beta-adrenergic antagonist propranolol, or the sympathomimetic agent octopamine. Injection of pimozide or metoclopramide at the time of the second of two LH-RH-A injections given at an interval of 12 hr potentiated the LH-RH-A-induced increase in serum GtH concentrations; injections of phentolamine, propranolol, or octopamine did not alter the response to LH-RH-A. Injections of pimozide or metoclopramide also increased the frequency of ovulation in LH-RH-A-injected gravid female goldfish. These results suggest that stimulation of dopamine receptors can block the potentiating effect of multiple injections of GtH-releasing hormone, as well as ongoing LH-RH-A-stimulated release. The results also indicate that the dopamine inhibition of GtH secretion is specific and may be mediated by receptors resembling the D-2 type receptors in mammals.