Carol S. Nahorniak

Interactions of Estradiol with Gonadotropin-Releasing Hormone and Thyrotropin-Releasing Hormone in the Control of Growth Hormone Secretion in the Goldfish

The effects of testosterone (T) and estradiol (E2) on serum growth hormone (GH) concentrations were investigated throughout the seasonal reproductive cycle of the female goldfish. Gonad-intact female goldfish were implanted intraperitoneally for 5 days with silastic pellets containing no steroid (blank), T(100 micrograms/g) or E2 (25-100 micrograms/g). In blank-implanted females, seasonal variations in serum GH were evident; maximal serum GH levels were found in spring while minimal GH levels were found in summer and early autumn. Implantation of E2-containing silastic capsules stimulated increases (2-4 times control) in serum GH levels throughout the reproductive cycle. Implantation of T did not affect serum GH at any time of the year. One possible mechanism by which E2 could exert its effects may be through alteration of pituitary sensitivity to GH-releasing factors. The decapeptide salmon gonadotropin-releasing hormone (sGnRH) is found in the brain and pituitary of goldfish and stimulates gonadotropin (GTH) and GH secretion. In contrast, thyrotropin-releasing hormone (TRH) stimulates GH, but not GTH, release from pars distalis fragments obtained from sexually regressed (ED50 = 5.7 +/- 3.8 nM; August) or sexually mature (ED50 = 0.53 +/- 0.28 nM; March) fish; in vivo E2 treatment resulted in a 3-fold increase in the in vitro GH response to TRH. Furthermore, E2 treatment increased sGnRH-stimulated GH release by approximately 4-fold. These results demonstrate that E2 but not T stimulates GH secretion throughout the reproductive cycle of female goldfish. Furthermore, sGnRH and TRH stimulate GH release in a teleost, and these stimulatory responses are enhanced by physiological levels of E2.

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.

Structure-activity relationships of mammalian, chicken, and salmon gonadotropin releasing hormones in vivo in goldfish

Mammalian, chicken, and salmon gonadotropin releasing hormones (GnRHs), and anlogs of each peptide, were injected either alone or in combination with pimozide into goldfish, and the changes in serum gonadotropin (GtH) levels determined. The native peptides had similar potencies in terms of magnitude and duration of the GtH response. Analogs of LHRH that are superactive in mammals are also superactive in goldfish; although [(imBzl)-D-His6, Pro9-NEt]-LHRH is very highly superactive in mammals it has activity similar to [D-Ala6, Pro9-NEt]-LHRH in goldfish. D-Ala6 or (imBzl)-D-His6 substitutions of [Trp7, Leu8, Pro9-NEt]-LHRH are not superactive in goldfish, whereas the D-Arg6 substitution is highly superactive, indicating that there are differences in the factors that make salmon and mammalian GnRH superactive. These results also indicate that the structural modifications that determine superactivity of GnRHs in goldfish differ from what is known for mammals.

Brain distribution of radioimmunoassayable gonadotropin-releasing hormone in female goldfish: Seasonal variation and periovulatory changes

A radioimmunoassay (RIA) for [Trp7, Leu8]gonadotropin-releasing hormone (sGnRH) was developed to determine the gonadotropin-releasing hormone (GnRH) content in discrete brain areas of female goldfish at different stages of ovarian development. Temporal changes in serum gonadotropin (GtH) and GnRH concentrations in discrete brain areas were measured during spontaneous ovulation. There were no clear parallel changes in brain GnRH with seasonal ovarian development in goldfish. However, under a 10 degrees temperature acclimation regimen, the GnRH content in the hypothalamus and pituitary decreased as the ovary progressed from the regressed to the mature condition; on the other hand. GnRH content in the spinal cord increased in sexually mature fish compared with that in regressed fish. Significant decreases in GnRH concentration were observed in certain brain areas (olfactory bulbs, telencephalon, hypothalamus, and pituitary) of fish undergoing spontaneous ovulation compared with those of nonovulatory fish. The simultaneous changes of GnRH concentration in these brain areas suggested that the GnRH neuronal system may function as an integrated unit for the activation of GtH secretion during ovulation in goldfish.

Seasonal effects of pimozide and des Gly10 [d-Ala6] LH-RH ethylamide on gonadotrophin secretion in goldfish

The seasonal changes in the gonadotrophin-release-inhibitory activity of dopamine and responsiveness to gonadotrophin-releasing hormone were investigated by determining the effects of injection of pimozide, a dopamine receptor antagonist, des-Gly10 [D-Ala6] LH-RH ethylamide (LRH-A), or the combination of pimozide plus LRH-A on serum gonadotrophin (GtH) levels of goldfish, held at 12 or 20 degrees C, at different stages of gonadal development. As in previous studies, pimozide greatly potentiated the GtH-release response to LRH-A. The highest concentrations of serum GtH induced by injection of pimozide or LRH-A alone, or the combination of pimozide plus LRH-A were in females in late stages of ovarian recrudescence; fish that were sexually regressed (males and females combined) were the least responsive, and fish that were in early stages of gonadal recrudescence, and mature females ( = prespawning, completed ovarian recrudescence) were intermediate. Fish held at 20 degrees C had a more rapid onset of GtH release and had higher serum GtH levels initially compared to fish at 12 degrees C at similar sexual stages; however, the fish held at 12 degrees C generally had a more prolonged increase in serum GtH levels, indicating that temperature influence the time course of the GtH-release response. The results indicate that there is a seasonal variation in responsiveness to injection of pimozide, LRH-A and the combination of pimozide plus LRH-A. These seasonal changes may be due to differences in the pituitary content of GtH, the ability of the pituitary to synthesize GtH, or changes in GtH cell receptors for GnRH and dopamine, or a combination of these and other unknown factors.

Activity of vertebrate gonadotropin-releasing hormones and analogs with variant amino acid residues in positions 5, 7 and 8 in the goldfish pituitary

All non-mammalian vertebrates as well as marsupial mammals have two or more forms of gonadotropin-releasing hormone (GnRH) in the brain. Goldfish brain and pituitary contains two molecular forms of GnRH, salmon GnRH ([Trp7, Leu8]m-GnRH; s-GnRH) and chicken GnRH-II ([His5, Trp7, Tyr8]m-GnRH; cII-GnRH). Both sGnRH and cII-GnRH stimulate gonadotropin (GtH) as well as growth hormone (GH) release from the goldfish pituitary. The purpose of the present study was to study the activity of the five known forms of GnRHs as well as analogs of mammalian GnRH (m-GnRH) with variant amino acid residues in positions 5, 7 and 8 in terms of binding to GnRH receptors, and release of GTH and GH from the perifused fragments of goldfish pituitary in vitro. All five vertebrate GnRH peptides stimulated both GtH and GH release in a dose-dependent manner, although their potencies were very different. cII-GnRH was somewhat more active than s-GnRH in releasing GtH, whereas s-GnRH tended to have a greater potency than cII-GnRH in terms of GH release. Both chicken GnRH-I (cI-GnRH) and lamprey GnRH (l-GnRH) were significantly less potent than mGnRH, s-GnRH and cII-GnRH in releasing GtH and GH. cII-GnRH binds with higher affinity for the high affinity binding sites compared to all other native peptides. The activity of [Trp7]-GnRH was similar to both s-GnRH and cII-GnRH in releasing GtH and GH. Substitution of His5 resulted in a significant decrease in GtH releasing potencies compared to mGnRH, sGnRH and cII-GnRH. [His5]-GnRH also had lower GH releasing potency than mGnRH and sGnRH. Tyr8, His8 and Leu8 substitutions caused significant decreases in GtH releasing potencies compared to mGnRH, s-GnRH and cII-GnRH, but did not cause a significant change in GH releasing potency. The combination of [His5, Trp7]-GnRH had GtH and GH releasing activities similar to m-GnRH, s-GnRH and cII-GnRH.(ABSTRACT TRUNCATED AT 400 WORDS)

Influences of Catecholamines on Growth Hormone Release in Female Goldfish, Carassius auratus

The influence of catecholamines on growth hormone (GH) release in female goldfish was investigated by monitoring serum GH levels following injections of drugs known to alter catecholamine synthesis and neural activities. Intraperitoneal (i.p.) injection of 6-hydroxydopamine, a catecholaminergic neurotoxin, or alpha-methyl-p-tyrosine, a catecholamine synthesis inhibitor, decreased serum GH levels. Intraperitoneal injection of L-beta-dihydroxyphenylalanine (L-dopa) increased serum GH concentrations in a dose-dependent manner. The L-dopa-induced increase in serum GH was potentiated by i.p. injection of carbidopa, which would increase the availability of L-dopa to brain tissues by blocking the peripheral conversion of L-dopa to dopamine (DA). These results suggest that L-dopa or one of its catecholamine metabolites acts centrally to increase GH release. Intraventricular (i.v.t.) injection of DA and i.p. injection of apomorphine, a DA agonist that crosses the blood-brain barrier, increased serum GH. Intraperitoneal injection of DA did not alter circulating GH levels in normal fish or fish bearing preoptic lesions that abolish an inhibitory hypothalamic influence on GH release; however, DA increased serum GH in fish which had their blood-brain barrier destroyed by sham operation procedures. These results indicate that DA acts centrally to stimulate GH secretion, possibly by inhibiting the release and/or synthesis of GH release-inhibitory factor. Serum GH concentrations were decreased in a dose-dependent manner by i.p. injection of norepinephrine (NE), whereas i.v.t. injection of NE did not alter serum GH levels. These results indicate that NE acts outside of the blood-brain barrier to decrease serum GH levels in the goldfish, possibly by directly influencing pituitary GH cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Activity of position-8-substituted analogs of mammalian gonadotropin-releasing hormone (mGnRH) and chicken and lamprey gonadotropin-releasing hormones in goldfish

Several vertebrate gonadotropin-releasing hormones (GnRH) and analogs were tested for activity in vivo in goldfish. Each peptide was administered intraperitoneally to goldfish, pretreated with pimozide or vehicle for pimozide, and changes in serum levels of gonadotropin were determined. Pimozide potentiates the activity of GnRH in vivo in goldfish by blocking the endogenous gonadotropin release-inhibitory activity of dopamine; relative potencies of GnRH peptides become evident in vivo in goldfish pretreated with pimozide (R. Peter et al. (1985), Gen. Comp. Endocrinol. 58, 231-242). Mammalian GnRH (mGnRH) was used as reference standard. [Try3, Leu5, Glu6, Trp7, Lys8]-GnRH (lamprey GnRH), [Gln8]-GnRH (chicken GnRH-I), and [His5, Trp7, Try8]-GnRH (chicken GnRH-II) caused increases in serum gonadotropin level similar in magnitude to mGnRH. [His5, D-Arg6, Trp7, Tyr8]-GnRH is superactive in the goldfish. [Asn8]-, [Met8]-, [Phe8]-, and [Ser8]-GnRH had activity similar to mGnRH in goldfish; [His8]-, [Ile8]-, and [Leu8]-GnRH had a lower level of activity; [Glu8]-GnRH had no apparent activity. The results indicate that there is no particular requirement for a hydrophobic or hydrophilic amino acid, or for a positively charged amino acid in position 8 of mGnRH for activity in vivo in the goldfish; a negatively charged amino acid in position 8 is detrimental for activity.

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

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