JEZ 818 126 A.C. HOLLOWAY THE JOURNAL AND J.F. OF LEATHERLAND EXPERIMENTAL ZOOLOGY 279:126–132 (1997) Effects of N-Methyl-D,L-Aspartate (NMA) on Growth Hormone and Thyroid Hormone Levels in Steroid-Primed Immature Rainbow Trout (Oncorhynchus mykiss) ALISON C. HOLLOWAY1 AND JOHN F. LEATHERLAND2* 1 Department of Zoology, University of Guelph, Guelph, ON N1G 2W1, Canada 2 Department of Biomedical Sciences, University of Guelph, Guelph, ON N1G 2W1, Canada ABSTRACT In this study we investigate whether the stimulatory action of the glutamate agonist, N-methyl-D,L-aspartate (NMA) on growth hormone (GH) levels in rainbow trout (Oncorhynchus mykiss) is steroid hormone-dependent. NMA was administered to sexually immature juvenile rainbow trout that had been primed with testosterone (T), 17β-estradiol (E2) or 5αdihydrotestosterone and changes in plasma growth hormone (GH) concentrations were measured. NMA had no significant effect on the plasma GH concentrations of non-steroid hormone-primed trout (controls), although in both T- and E2-primed fish plasma GH levels were elevated 6 hours following NMA administration. The response had disappeared by 24 hours in the T-primed group, but was still evident in the E2-primed fish. In comparison, in the 5αDHT-primed fish, NMA significantly depressed plasma GH levels 6 hours post-injection (P < 0.05), but this effect had disappeared 24 hours after NMA administration. NMA had no effect on plasma T4 levels in any treatment group, and although plasma T3 levels were significantly (P < 0.05) depressed in T-primed fish 6 hours post-injection, this response was not seen in any other group. These results suggest that NMA has no effect on plasma thyroid hormone levels but it does have a steroid hormone-dependent action on plasma GH levels in rainbow trout. J. Exp. Zool. 279:126–132, 1997. © 1997 Wiley-Liss, Inc. Several different lines of investigation have provided evidence to suggest that growth hormone (GH) plays a key role in the reproductive physiology of teleost fishes (Le Gac et al., ’93). First, extremely high levels of GH have been found in sexually mature white sucker, Catostomus commersoni (Stacey et al., ’84), rainbow trout, Oncorhynchus mykiss (Sumpter et al., ’91), goldfish, Carassius auratus (Trudeau et al., ’92), coho salmon, Oncorhynchus kisutch (Flett and Leatherland, unpublished data), Atlantic salmon, Salmo salar (Björnsson et al., ‘94), and chum salmon, Oncorhynchus keta (Kakizawa et al., ’95). Second, the administration of gonadal steroid hormones has been shown to increase plasma GH levels in vivo in hybrid tilapia, Oreochromis niloticus × O. aureus (Melamed et al., ‘95b), goldfish (Trudeau et al., ’92), carp, Cyprinus carpio (Lin et al., ’95), and rainbow trout (Holloway and Leatherland, ‘97a). Third, the administration of exogenous GH appears to stimulate gonadal steroid production in several species of fishes (Le Gac et al., ’93). In addition, several © 1997 WILEY-LISS, INC. factors that are known to influence the activity of the hypothalamic-pituitary-gonadal axis, including gonadotropin-releasing hormone (GnRH), dopamine (DA), neuropeptide Y, and the glutamate analogue, N-methyl-D,L-aspartate (NMA), are also known to affect GH release (Marchant et al., ’89; Chang et al., ’90; Peng et al., ’90; Lin et al., ’93; Flett et al., ’94; Lin et al., ’95; Trudeau et al., ’90). NMA, which is generally considered to be specific for the NMDA-type glutamate receptor, has been shown to stimulate both GH and luteinizing hormone (LH) secretion in vivo in a range of mammalian species (rat, mouse, sheep, pig, monkey (Gay and Plant, ’87; Acs et al., ’90; Barb et al., ’93; Brann and Mahesh, ’92; l’Anson et al., ’93; Miller and Gibson, ’94; Akema et al., ’95; Pinilla Contract grant sponsors: NSERC, OMAFRA, and DFO. *Correspondence to: Dr. John Leatherland, Department of Biomedical Sciences, Ontario, Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada. E-mail: firstname.lastname@example.org Received 23 September 1996; Revision accepted 7 April 1997. EFFECT OF NMA ON GH RELEASE IN RAINBOW TROUT et al., ’95; Downing et al., ’96; Estienne et al., ’96). In rainbow trout, NMA was found to have a similar stimulatory effect on in vivo plasma GH and gonadotropin (GtH) levels following testosterone (T)-priming of juvenile animals (Flett et al., ’94). In contrast, Trudeau et al. (’96) describe an inhibitory effect of NMA on GH release in goldfish, although the levels of NMA which elicited a response in that study (25 and 50 µg g-1 body weight) were considerably higher than those used in either the Flett et al. (’94) study or the one described here (1 µg g-1 body weight). Recent studies in mammals suggest that NMA elevates GH indirectly via the increased release of GH releasing hormone (GHRH) (Acs et al., ’90; Barb et al., ’96; Estienne et al., ’96). In addition, a direct action of NMA on the pituitary cells in vitro was also reported for rat (Lindström and Ohlsson, ’92; Niimi et al., ’94) and pig (Barb et al., ’93) pituitary cells cultured in vitro. Furthermore, the NMDA R1 receptor subunit has been co-localized with GH cells in the rat anterior pituitary (Bhat et al., ’95). Conversely, several other studies have failed to find a GH-stimulatory action of NMA (Acs et al., ’90; Barb et al., ’93; Cocilovo et al., ’92; Liaw and Barraclough, ’93). In addition to its effects on GH release, NMA stimulates both the synthesis and release of hypothalamic GnRH in mammals (Brann, ’95). Similarly, in vitro studies in rainbow trout show that the gonadotropic cell response to both NMA and GnRH is completely abolished in the presence of a GnRH antagonist (Flett et al., ’94). These results suggest that the stimulatory action of NMA on GnRH release in rainbow trout is similar to that seen in mammalian models. Since GnRH is both a potent GtH- and GH-releasing factor in several fish species (Marchant et al., ’89; Marchant and Peter, ’89; Chang and De Leeuw, ’90; Lin et al., 93; Lin et al., ’95; Melamed et al., ‘95a), it is possible that the GH-stimulatory action of NMA in rainbow trout is mediated by GnRH. The ability of NMA to stimulate LH release in mammals is enhanced in the presence of gonadal steroid hormones (Brann and Mahesh, ’92; Arias et al., ’93; Brann, ’95), probably via an increased responsiveness of the LH cells to LHRH (Arias et al., ’93). Similarly, the GH response to NMA is also enhanced by gonadal steroid hormones. For example, Barb et al. (’93) reported that NMA stimulated GH release from isolated pituitary cells taken from either ovariectomized pigs, or pigs in the luteal phase of the estrous cycle, but did not stimulate GH release from pituitary cells taken 127 from pigs during the follicular phase of the estrous cycle. Similarly, Chang et al. (’93) found that progesterone potentiated the GH response to NMA in ovariedomized pigs. There is only limited information about the effects of NMA on GH release in teleost fishes, but preliminary reports suggest that the action of NMA, at least in trout and goldfish, is modulated by gonadal steroid hormones (Flett et al., ’94; Trudeau et al., ’96). The main purpose of the study reported here was to examine the effects of NMA on plasma GH concentrations in juvenile rainbow trout, and to evaluate whether the secretion of GH in response to NMA challenge is influenced by the presence of sex steroid hormones. A second component of the study was to evaluate whether NMA influences thyrotropic cell function; for this part of the study, plasma thyroxine (T4) concentrations were measured as an indication of the possible effects of NMA on thyroid hormone secretion. This aspect of the study was undertaken because in mammals, one of the known NMA receptors (the NMDA R1 receptor subunit), in addition to being present in the GH cells, is also found in the TSH cells (Bhat et al., ’95). The co-localization of the NMDA R1 receptor subunit with GH and TSH cells might be indicative of a role of NMA in the regulation of the hypothalamic-pituitary-thyroidal axis. MATERIALS AND METHODS Two hundred juvenile (sexually immature) rainbow trout (Oncorhynchus mykiss) were used in the study. The fish were held in fibreglass tanks supplied with constantly running aerated well water at 9 ± 2°C under a simulated natural photoperiod for 2 months prior to the experiments. They were fed to satiety daily with commercial pelleted trout food (Martin’s Feed Mills, Elmira, Ontario) for the duration of the experiment. Two separate trials were undertaken, the first employing 160 trout of approximately 105 g body weight and the second employing 40 trout of approximately 200 g body weight. At the commencement of each trial of the study, the fish were anaesthetized in MS222 (25 mg 1-1) and given a slow release implant of steroid hormone (7.5 µg g-1 body weight) dissolved in approximately 0.3 ml of warm hydrogenated coconut oil or coconut oil alone (control). Steroid hormone implants at this level have been shown to significantly elevate plasma steroid hormone levels for up to 8 weeks post-implantation (Flett and Leatherland, ’89). In the first trial, groups of trout were administered coconut oil alone or oil containing testoster- 128 A.C. HOLLOWAY AND J.F. LEATHERLAND one (T), 17β-estradiol (E2) (Sigma Chemical Co. St Louis, MO), or the non-aromatizable androgen, 5α-dihydrotestosterone (5αDHT) (Ikapharm, Israel). In the second trial, groups were administered T or E2. Two weeks following the steroid implantation, each fish was given an intraperitoneal injection of either NMA (1 µg g-1 body weight; Sigma Chemical Co. St. Louis, MO) dissolved in 100 µl of 0.8% saline or saline alone; fish were sampled 6 and 24 hours later. (The times of sampling were chosen based on the protocol used by Flett et al. (’94) who demonstrated an effect of NMA on plasma GH levels within 6 hours of administering the glutamate agonist. The dosage of NMA used was determined in preliminary studies of the toxicity of the compound in immature rainbow trout; 1 µg g-1 body weight was found to elicit no obvious adverse response, whereas at 10 or 100 µg g-1 the fish appeared to lose the ability to maintain body posture in the water column, and at the higher dosage there was 25% mortality within 1 hour.) Each fish was anaesthetized in MS222 (125 mg 1-1), weighed, and bled into heparinized tubes following caudal severance; plasma was stored at –70°C until analyzed for GH and thyroid hormone concentrations. Plasma GH levels were measured using a noncompetitive enzyme linked immunosorbent assay based on monoclonal antibodies developed against salmon GH (Farbridge and Leatherland, ’91). Plasma T4 levels were measured using the Amersham Amerlex radioimmunoassay (RIA) (Johnson and Johnson Clinical Diagnostics, Markham, Ontario); this RIA has been validated for use with rainbow trout plasma (Leatherland and Farbridge, ’92). Plasma hormone levels for the NMA- and saline-treated groups were compared within a steroid-treated group using Student’s t-test (α = 0.05). Fig. 1. Effect of NMA (solid bars) treatment on plasma GH levels in immature steroid primed rainbow trout. Data are shown as ng ml-1 GH (mean ± SEM), n = 10 for each group. *Significantly different from their respective saline (open bars) controls (P < 0.05). RESULTS NMA had no significant effect on plasma GH levels in the oil primed group at either 6 or 24 hours post-injection (Fig. 1). However, in the groups that had been given an implant containing either E2 or T, plasma GH levels were significantly elevated at 6 hours post-injection. In the T-primed group this response was not evident 24 hours post-injection, but in the E2 primed fish, plasma GH levels were still elevated with respect to the saline control group 24 hours after the administration of NMA. The stimulatory effect of NMA on T- and E2-primed trout was confirmed in a second trial; in both groups, plasma GH levels of the NMA treated animals were significantly elevated 6 hours post-injection (testosterone-primed: saline 3.71 ± 0.34 ng ml-1, NMA 6.50 ± 1.16 ng ml-1 (P < 0.05); estradiol-primed: saline 2.42 ± 0.59 ng ml-1, NMA 4.84 ± 0.82 ng ml-1 (P < 0.05)). In the group of fish administered 5αDHT, a nonaromatizable androgen, NMA acted to significantly depress plasma GH levels 6 hours after its administration, although this response was not evident after 24 hours (Fig. 1). EFFECT OF NMA ON GH RELEASE IN RAINBOW TROUT NMA treatment had no effect on plasma T4 levels in any group either 6 or 24 hours post-injection (Table 1). DISCUSSION In this study, in immature trout that had been primed with either T or E2, followed 2 weeks later by an NMA challenge, there was an increase in plasma GH concentrations 6 hours following the injection of NMA; the response was particularly marked in the E2-primed group. The GH stimulating action of NMA was not seen in trout that had been given only an oil implant. Moreover, in the group of trout that had been primed with the non-aromatizable androgen, 5αDHT, NMA elicited a significant decrease in plasma GH concentrations. Thus, the response to NMA is clearly dependent on the nature of the steroid milieu, and the permissive effect of steroid hormones on a plasma GH increase apprears to be related to the presence of either an estrogen or an aromatizable androgen. These findings confirm an earlier study in which T-primed immature rainbow trout exhibited a significantly elevated plasma GH concentration 6 hours following an intraperitoneal injection of NMA (Flett et al., ’94). However, the findings are contrary to those of Trudeau et al. (’96) in which NMA was administered to steroid hormone-primed goldfish. Those authors found that NMA, administered intraperitoneally, acted to inhibit the stimulatory action of E2 on plasma GH concentrations. The differences in the two studies might reflect species differences, although they are more likely linked to the NMA levels that were applied in the two studies. In the goldfish study, the NMA levels that elicited a significant GH response were 25 and 50 µg g-1 body weight and 50 µg per fish for intraperitoneal and intraventricular administrations, respectively, 129 compared with only 1 µg g-1 body weight for the Flett et al. (’94) study and the one reported here. In our laboratory, intraperitoneal doses 10 µg g-1 and higher were neurotoxic, as evidenced by abnormal swimming behaviour and apparently impaired balance. The means by which NMA affects GH secretion in fishes has yet to be established. In mammals, the glutamate agonist may act via the NMDA receptor, to stimulate somatostatin (SRIF) secretion and synthesis in vivo and in vitro (Rage et al., ’94). Paradoxically, SRIF has been identified as a potent GH-inhibitory factor in teleost fishes (Peter and Marchant, ’95); therefore, a NMA-stimulated increase in SRIF release would lower plasma GH concentrations and is inconsistent with the results of the study presented here for the E2- and T-primed trout, but may explain the inhibitory action of NMA in the 5αDHT-primed fish. Conversely, in mammals there is also evidence to suggest that the GH-stimulatory action of NMA is mediated via an increase in the release of GHreleasing hormone (GHRH) from the hypothalamus (Acs et al., ’90; Barb et al., ’96; Estienne et al., ’96). Heterologous GHRH has been shown to increase GH release from isolated rainbow trout pituitary cells (Luo et al., ’90; Blaise et al., ’95), and the possibility that NMA stimulates GH release indirectly through increased hypothalamic GHRH release cannot be refuted. However, only few GHRH fibres are found to innervate the pars distalis in teleost fishes (Rao et al., ’96) and Marchant and Peter (’89) were unable to find a GHRH-stimulated GH release from perifused goldfish pituitary glands, suggesting that GHRH may not be an important physiological regulator of GH secretion in teleosts. On the other hand, Flett et al. (’94) showed that NMA had a potent stimulatory effect on GtH release from perifused pituitary glands of T-primed rainbow trout, which would TABLE 1. Effect of NMA treatment on plasma T3 and T4 concentrations in rainbow trout1 Time after NMA injection 6 Hours 24 Hours 1 T3 (nmol l–1) Implant Oil E2 T 5αDHT Oil E2 T 5αDHT Saline 3.95 10.71 5.23 9.54 2.88 8.76 4.51 6.24 ± ± ± ± ± ± ± ± 0.86 0.57 .071 0.76 0.94 0.90 0.76 .068 Data are shown as nmol l–1 (mean ± SEM), n = 10 for each group. *Significantly different from their respective saline controls (P < 0.05). T4 (nmol l–1) NMA 8.18 11.53 3.09 9.52 3.94 9.26 4.52 6.19 ± ± ± ± ± ± ± ± 2.20 0.65 0.71* 0.68 0.68 0.63 0.79 0.56 Saline 14.98 6.87 9.28 8.66 6.55 6.05 7.90 6.08 ± ± ± ± ± ± ± ± 3.25 0.31 0.88 1.10 1.17 0.44 1.37 1.17 NMA 19.02 7.20 9.91 9.75 8.12 5.86 10.69 7.34 ± ± ± ± ± ± ± ± 2.40 0.74 2.23 0.91 0.83 0.46 2.23 0.80 130 A.C. HOLLOWAY AND J.F. LEATHERLAND suggest that, as in mammals, NMA increases the release of GnRH. Since GnRH has been shown to have a potent GH-stimulatory effect in several teleost fishes (see Introduction for references), mostly cyprinid species, it is possible that NMA affects GH release via altered GnRH release from the hypothalamus. However, although GnRH has been shown to stimulate GH release in rainbow trout in vitro, the GH-stimulatory action of NMA was not attenuated in the presence of a GnRH antagonist (Holloway and Leatherland, ‘97b). These data suggest that the GH-stimulatory action of NMA in rainbow trout may not be mediated via GnRH. In mammals, NMA may affect GH release via a direct action on pituitary cells (see Introduction for references). Although there is no published evidence to show that NMA acts directly at the pituitary level to stimulate GH release in teleost fishes, in vitro studies in our laboratory found that NMA stimulates GH release from perifused rainbow trout pituitary fragments (Holloway and Leatherland, ‘97b), suggesting that there may be a direct action of NMA on GH release. It is not clear how the steroid hormone milieu can affect the GH response to NMA in rainbow trout. In mammals, Brann et al. (‘93b) proposed that the steroid hormone milieu might influence the number or affinity of hypothalamic NMDA receptors; however, gonadectomy with or without steroid hormone replacement did not change NMDA receptor binding or NMDA receptor mRNA levels in rats (Brann et al., ‘93b; Brann, ’95), suggesting that steroid modulation of NMA action in mammals does not act via alterations of the hypothalamic NMDA receptors. A steroid hormone effect on pituitary NMDA receptor levels has also been proposed, and steroid hormone treatment has been shown to cause changes in pituitary NMDA receptor mRNA levels in rats (Brann el al., ‘93a), suggesting a possible site of steroid hormone action in mammals. An alternate modus operandi is that steroids alter the sensitivity of somatotropic cells to either NMA itself, or to the hypothalamic factors that are released in response to NMA challenge. In support of this thesis, E2 potentiates GHRH-induced GH release from rat anterior pituitary cells in vitro (Simard et al., ’86), and the GnRH-induced release of GH in goldfish and carp (Trudeau et al., ’92; Murthy et al., ’94; Lin et al., ’95). In the present study, there was no evidence of an NMA effect on the hypothalamic-pituitary gland regulation of thyroid activity. In rats, the NMDA R1 receptor subunit has been co-localized with approximately 6% of TSH cells in the rat anterior pituitary (Bhat et al., ’95); no evidence of a similar situation was found in the present study based on measures of changes in plasma T4 levels. It is possible that the sampling times were not appropriate to identify changes in plasma T4 concentrations in response to altered TSH release. However, in several studies employing exogenous TSH-challenges as measure of thyroid hormone secretion in salmonid fishes, maximal plasma T4 concentrations were evident between 6 and 24 hours after the injection of TSH (Leatherland, ’94). This study has shown that NMA has GH-regulatory actions in rainbow trout, although the GH response appears to be dependent on the steroid hormone milieu. However, there is currently no information regarding the site of steroid modulation of NMA’s action of GH release. ACKNOWLEDGMENTS We thank Dr. P.K. Reddy and the staff at the Alma Research Station for their technical assistance at several stages of the study, and Dr. H. Kawauchi for the generous donation of recombinant salmon GH used for standards in the ELISA. This work was supported by NSERC, OMAFRA, and DFO grants to J.F.L., and a NSERC scholarship to A.C.H. LITERATURE CITED Acs, Z., G. Lonart, and G.B. Makara (1990) Role of hypothalamic factors (growth-hormone-releasing hormone and gamma-animobutyric acid) in the regulation of growth hormone secretion in the neonatal and adult rat. Neuroendocrinology, 52:156–160. Akema, T., A. Chiba, R. Shinozaki, M. Oshida, F. Kimura, and J. Toyoda (1995) Acute stress suppresses the N-methylD-aspartate-induced luteinizing hormone release in the ovariectomized estrogen-primed rat. Neuroendocrinology, 62:270–276. Arias, P., H. Jarry, S. Leonhardt, J.A. Moguilevsky, and W. Wuttke (1993) Estradiol modulates the LH release response to N-methyl-D-aspartate in adult female rats: Studies on hypothalamic luteinizing hormone-releasing hormone and neurotransmitter release. Neuroendocrinology, 57:710–715. Barb, C.R., J.B. Barrett, G.B. Rampacek, and R.R. Kraeling (1993) N-methyl-D, L-aspartate modulation of luteinizing and growth hormone secretion from pig pituitary cells in culture. Life Sci., 53:1157–1164. Barb. C.R., G.M. Desrochers, B. Johnson, R.V. Utley, W.J. Chang, G.B. Rampacek, and R.R. Kraeling (1992) N-methyl-D, L-aspartate stimulates growth hormone and prolactin but inhibits luteinizing hormone secretion in the pig. Dom. Anim. Endocrinol., 9:225–232. Barb, C.R., R.M. Campbell, J.D. Armstrong, and N.M. Cox (1996) Aspartate and glutamate modulation of growth hormone secretion in the pig: Possible site of action. Dom. Anim. Endocrinol., 13:81–90. EFFECT OF NMA ON GH RELEASE IN RAINBOW TROUT Bhat, G.K., V.B. Mahesh, Z.W. Chu, L.P. Chorich, P.L. Zamorano, and D.W. Brann (1995) Localization of the Nmethyl-D-aspartate R1 receptor subunit specific anterior pituitary hormone cell types of the female rat. Neuroendocrinology, 62:178–186. Björnsson, B.Th., G.L. Taranger, T. Hansen, S.O. Stefansson, and C. Haux (1994) The interrelation between photoperiod, growth hormone, and sexual maturation of adult Atlantic salmon (Salmo salar), Gen. Comp. Endocrinol., 93:70–81. Blaise, O., P.-Y. LeBail, and C. Weil (1995) Lack of gonadotropin-releasing hormone action on in vivo and in vitro growth hormone release, in rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol., 110C:133–141. Brann, D.W. (1995) Glutamate: A major excitatory transmitter in neuroendocrine regulation. Neuroendocrinology, 61:213–225. Brann, D.W. and V.B. Mahesh (1992) Excitatory amino acid regulation of gonadotropin secretion: Modulation by steroid hormones. J. Steroid Biochem. Mol. Biol., 41:847–850. Brann, D.W., L.P. Chorich, P.L. Zamorano, and V.B. Mahesh (1993a) Presence of NMDA receptor mRNA in the anterior pituitary of the female rat: Steroid modulation and changes during gonadotropin surge induction. Mol. Cell. Neurosci., 4:571–575. Brann, D.W., P.L. Zamorano, L.B. Chorich, and V.B. Mahesh (1993a) Steroid hormone effects on NMDA receptor binding and NMDA receptor mRNA levels in the hypothalamus and cerebral cortex of the adult rat. Neuroendocrinology, 58:666–672. Chang, J.P., and R. De Leeuw (1990) In vitro goldfish growth hormone responses to gonadotropin-releasing hormone: Possible roles of extracellular calcium and arachidonic acid metabolism? Gen. Comp. Endocrinol., 80:155–164. Chang, J.P., K.L. Yu, A.O.L. Wong, and R.E. Peter (1990) Differential actions of dopamine receptor subtypes on gonadotropin and growth hormone release in vitro in goldfish. Neuroendocrinology, 51:664–674. Chang, W.J., C.R. Barb, R.R. Kraeling, G.B. Rampacek, and K.M. Asanovich (1993) N-methyl-D, L-aspartate modulation of pituitary hormone secretion in the pig: Role of opioid peptides. Dom. Anim. Endocrinol., 10:305–313. Cocilovo, L., V. Colona, M. Zoli, G. Biagini, B.P. Settembrini, E.E. Müller, and D. Cocchi (1992) Central mechanisms subserving the impaired growth hormone secretion induced by persistent blockade of NMDA receptors in immature male rats. Neuroendocrinology, 55:416–421. Downing, J.A., J. Joss, and R.J. Scaramuzzi (1996) The effects of N-methyl-D, L-aspartic acid on the plasma concentration of gonadotropins, GH, and prolactin in the ewe. J. Endocrinol., 149:65–72. Estienne, M.J., J.M. Harter-Dennis, C.R.Barb, T.G. Hartsock, R.M. Campbell, and J.D. Armstrong (1996) Nmethyl-D, L-aspartate-induced growth hormone secretion in barrows: Possible mechanisms of action. J. Anim. Sci., 74 :597–602. Farbridge, K.J., and J.F. Leatherland (1991) The development of a noncompetetive enzyme-linked immunosorbent assay for Oncorhynchid growth hormone using monoclonal anitbodies. Gen. Comp. Endocrinol., 83:7–17. Flett, P.A., and J.F. Leatherland (1989) Dose-related effects of 17β-oestradiol (E2) on liver weight, plasma E2, protein, calcium and thyroid hormone levels, and measurement of the binding of thyroid hormone to vitellogenin in rainbow trout, Salmo gairdneri Richardson. J. Fish Biol., 34 : 515–527. 131 Flett, P.A., G.L. Van Der Kraak, and J.F. Leatherland (1994) Effect of excitatory amino acids on in vivo and in vitro gonadotropin and growth hormone secretion in testosteroneprimed, immature rainbow trout, Oncorhynchus mykiss. J. Exp. Zool., 268:390–399. Gay, V.L., and T.M. Plant (1987) N-methyl-D, L-aspartate elicits hypothalamic gonadotropin-releasing hormone release in prepubertal male rhesus monkeys (Macaca mulatta). Endocrinology, 120:2289–2296. Holloway, A.C., and J.F. Leatherland (1997a) Effect of gonadal steroid hormones on plasma growth hormone concentrations in sexually immature rainbow trout, Oncorhynchus mykiss. Gen. Comp. Endocrinal., 105:246–254. Holloway, A.C., and J.F. Leatherland (1997b) The effects of N-methyl-D, L-aspartate and gonadotropin-releasing hormone on in vitro growth hormone release in steroid-primed immature rainbow trout, Oncorhynchus mykiss. Gen. Comp. Endocrinol. (in press) Kakizawa, S., T. Kaneko, T. Ogasawara, and T. Hirano (1995) Changes in plasma somatolactin levels during spawning migration of chum salmon ( Oncorhynchus keta). Fish Physiol. Biochem., 14:93–101. l’Anson, H., C.G. Herbosa, F.J.P. Ebling, R.I. Wood, D.C. Bucholtz, C.D. Mieher, D.L. Foster, and V. Padmanabhan (1993) Hypothalamic versus pituitary stimulation of luteinizing hormone secretion in the prepubertal female lamb. Neuroendocrinology, 57:467–475. Leatherland, J.F. (1994) Reflections on the thyroidology of fishes: From molecules to humankind. Guelph Ichthyological Reviews, 2. Neptune City, NJ: T.F.H. Publications Inc. Leatherland, J.F., and K.J. Farbridge (1992) Temporal changes in plasma thyroid hormone, growth hormone and free fatty acid concentrations, and hepatic 5’-monodeoidinase activity, lipid and protein content during chronic fasting and refeeding in rainbow trout (Onchorhynchus mykiss). Fish Physiol. Biochem., 10:245–258. Le Gac, F., O. Blaise, A. Fostier, P.Y. Le Bail, M. Loir, B. Mourot, and C. Weil (1993) Growth hormone (GH) and reproduction: A review. Fish Physiol. Biochem., 11:219–232. Liaw, J.J., and C.A. Barraclough (1993) N-methyl-D, L-aspartic acid differentially affects LH release and LHRH mRNA levels in estrogen-treated ovariectomized control and androgen sterilized rats. Brain Res. Mol. Brain Res., 17:112–118. Lin, H.R., M. Lu, X.W. Lin, W.M. Zhang, Y. Sun, and L.X. Chen (1995) Effects of gonadotropin-releasing hormone (GnRH) analogs and sex steroids on growth hormone (GH) secretion and growth in common carp (Cyprnus carpio) and grass carp ( Ctenopharyngodon idellus ). Aquaculture, 135:173–184. Lin, X.W., H.R. Lin, and R.E. Peter (1993) Growth hormone and gonadotropin secretion in the common carp (Cyprinus carpio): In vitro interactions of gonadotropin-releasing hormone, somatostatin, and the dopamine agonist apomorphine. Gen. Comp. Endocrinol., 89:62–71. Lindström, P., and L. Ohlsson (1992) Effect of N-methyl-D, L-aspartate on isolated rat somatotrophs. Endocrinology, 131:1903–1907. Luo, D., B.A. McKeown, J. Rivier, and W. Vale (1990) In vitro responses of rainbow trout (Oncorhynchus mykiss) somatotrophs to carp growth hormone-releasing factor (GRF) and somatostatin. Gen. Comp. Endocrinol., 80:288–298. Marchant, T.A., and R.E. Peter (1989) Hypothalamic peptides influencing growth hormone secretion in the goldfish, Carassius auratus. Fish Physiol. Biochem., 7:133–139. 132 A.C. HOLLOWAY AND J.F. LEATHERLAND Marchant, T.A., J.P. Chang, C.S. Nahorniak, and R.E. Peter (1989) Evidence that gonadotropin-releasing hormone also functions as a growth hormone-releasing factor in the goldfish. Endocrinology, 124:2509–2518. Melamed, P., N. Eliahu, B. Levavi-Sivan, M. Ofir, O. FarchiPisanty, F. Rentier-Delrue, J. Smal, Z. Yaron, and Z. Naor (1995a) Hypothalamic and thyroidal regulation of growth hormone in tilapia. Gen. Comp. Endocrinol., 97:13–30. Melamed, P., N. Eliahu, M. Ofir, B. Levavi-Sivan, J. Smal, F. Rentier-Delrue, and Z. Yaron (1995b) The effects of gonadal development and sex steroids on growth hormone secretion in the male tilapia hybrid (Oreochromis niloticus × O. aureus). Fish Physiol. Biochem., 14:267–277. Miller, G.M., and M.J. Gibson (1994) Opioidergic modulation of N-methyl-D, L-aspartic acid-stimulated LH release in young adult but not older male mice. Neuroendocrinology, 59:277–284. Murthy, C.K., W. Zheng, V.L. Trudeau, C.S. Nahorniak, J.S. Rivier, and R.E. Peter (1994) In vivo actions of a gonadotropin-releasing hormone (GnRH) antagonist on gonadotropin-II and growth hormone secretion in goldfish, Carassius auratus. Gen. Comp. Endocrinol., 96:427–437. Niimi, M., M. Sato, K. Murao, J. Takahara, and K. Kawanishi (1994) Effect of excitatory amino acid receptor agonists on secretion of growth hormone as assessed by the reverse hemolytic plaque assay. Neuroendocrinology, 60:173–178. Peng, C., Y.-P. Huang, and R.E. Peter (1990) Neuropeptide Y stimulates growth hormone and gonadotropin release from the goldfish pituitary in vitro. Neuroendocrinology, 52:28–34. Peter, R.E., and T.A. Marchant (1995) The endocrinology of growth in carp and related species. Aquaculture, 129: 299–321. Pinilla, L., M. Tena-Sempere, and E. Aguilar (1995) The role of excitatory amino acid pathways in the control of pituitary function in neonatally oestrogenized male rats. J. Endocrinol., 147:51–57. Rage, F., C. Rougeot, and L. Tapia-Arancibia (1994) GABAA and NMDA receptor activation controls somatostatin messenger RNA expression in primary cultures of hypothalamic neurons. Neuroendocrinology, 60:470–476. Rao, S., P.D.P. Rao, and R.E. Peter (1996) Growth hormonereleasing hormone immunoreactivity in the brain, pituitary, and pineal of the goldfish, Carassius auratus. Gen Comp. Endocrinol., 102:210–220. Simard, J., J.-F. Hubert, T. Hosseinzadeh, and F. Labrie (1986) Stimulation of growth hormone release and synthesis by estrogens in rat anterior pituitary cells in culture. Endocrinology, 119:2004–2011. Stacey, N.E., D.S. MacKenzie, T.A. Marchant, A.L. Kyle, and R.E. Peter (1984) Endocrine changes during natural spawning in the white sucker, Catostomus commersoni. Gen. Comp. Endocrinol., 56:333–348. Sumpter, J.P., R.F. Lincoln, V.J. Bye, J.F. Carragher, and P.Y. Le Bail (1991) Plasma growth hormone levels during sexual maturation in diploid and triploid rainbow trout (Oncorhynchus mykiss ). Gen. Comp. Endocrinol., 83:103–110. Trudeau, V.L., G.M. Somoza, C.S. Nahorniak, and R.E. Peter (1992) Interactions of estradiol with gonadotropin-releasing hormone and thyrotropin-releasing hormone in the control of growth hormone secretion in the goldfish. Neuroendocrinology, 56:483–490. Trudeau, V.L., B.D. Sloley, O. Kah, N. Mons, J.G. Dulka, and R.E. Peter (1996) Regulation of growth hormone secretion by amino acid transmitter in the goldfish (I): Inhibition by N-methyl-D, L-aspartic acid. Gen. Comp. Endocrinol., 103:129–137.