Regional distribution and in vitro secretion of salmon and chicken-II gonadotropin-releasing hormones from the brain and pituitary of juvenile and adult goldfish, Carassius auratus.

The content of salmon (sGnRH) and chicken-II (cGnRH-II) gonadotropin-releasing hormones was measured in discrete brain regions and pituitaries of juvenile and postspawning adult goldfish, using specific radioimmunoassays. In juveniles, the content of both peptides was low. sGnRH was the predominant form in telencephalon-preoptic area (T-POA) (sGnRH:cGnRH-II ratio = 2.06 +/- 0.66) and diencephalon (DIEN) (sGnRH:cGnRH-II ratio = 2.72 +/- 0.32), whereas cGnRH-II was predominant in cerebellum-brain stem (STEM) (sGnRH:cGnRH-II ratio = 0.47 +/- 0.05). Equal amounts of the two peptides were present in pituitary (PIT) (sGnRH:cGnRH-II ratio = 1.04 +/- 0.18). In adults, the content of both peptides in all regions was significantly increased. The increase in sGnRH exceeded that of cGnRH-II in T-POA and PIT, resulting in an increased sGnRH:cGnRH-II ratio in these tissues (T-POA, 3.55 +/- 0.26; PIT, 7.85 +/- 2.28). In DIEN and STEM, the increase in cGnRH-II content equaled or exceeded that of sGnRH; the sGnRH:cGnRH-II ratio was unchanged in STEM (0.39 +/- 0.06) and decreased in DIEN (1.23 +/- 0.13). The secretion of sGnRH and cGnRH-II was investigated under static in vitro incubation conditions. Both forms of the peptide were secreted from T-POA slices and PIT fragments from juvenile and adult fish. Secretion was significantly increased under potassium depolarizing conditions. Secretion of the two peptides was proportional to their content in tissues from both juvenile and adult goldfish.

In vitro release of gonadotropin-releasing hormone from the brain preoptic-anterior hypothalamic region and pituitary of female goldfish.

In vitro release of gonadotropin releasing hormone (GnRH) from slices of the preoptic-anterior hypothalamic (P-AH) region and fragments of the pituitary of goldfish was studied using a static incubation system. Release of GnRH from both tissue preparations was stimulated by depolarizing concentrations of extracellular potassium ions (K+). Other putative secretagogues, calcium ionophore A23187 (1 microM), forskolin (100 microM), and prostaglandin E2 1 microM) also stimulated release of GnRH from both tissue preparations. Omission of Ca2+, or chelating the remaining remaining Ca2+ by EGTA (0.1 mM), abolished the release of GnRH stimulated by high K+ concentrations (60 mM), but did not reduce spontaneous release. Verapamil (1 microM), a voltage-sensitive calcium channel blocker, abolished the release of GnRH stimulated by high K+ or A21387 from both tissue preparations. The GnRH released in vitro from both the P-AH region and pituitary was concentrated by Sep-Pak and then separated by high-performance liquid chromatography. The major peak of the GnRH immunoreactivity was found to coelute with synthetic salmon GnRH [( Trp7,Leu8]-GnRH) and the minor peak with chicken GnRH-II [( Gln8]-GnRH). Dopamine (10 and 100 microM) inhibited GnRH release from both P-AH slices and pituitary fragments, while serotonin (1-100 microM) stimulated release from both. Norepinephrine (10-100 microM) stimulated GnRH release from P-AH slices but not from pituitary fragments. The results demonstrate that the release of GnRH from goldfish P-AH slices and pituitary fragments in vitro in response to various secretagogues and monoamines can be studied using a static incubation system.

Growth hormone-dependent potentiation of gonadotropin-stimulated steroid production by ovarian follicles of the goldfish.

The possible involvement of growth hormone (GH) in the regulation of ovarian function in the goldfish was investigated by determining the effects of common carp GH on steroid production by vitellogenic and preovulatory ovarian follicles incubated in vitro. Carp GH acts in a dose-dependent manner to potentiate the actions of common carp gonadotropin (GtH) on the production of 17 beta-estradiol and testosterone by vitellogenic ovarian follicles and the actions of human chorionic gonadotropin (hCG) on testosterone production by preovulatory ovarian follicles. Carp GH alone had no effect on basal steroid secretion by either class of ovarian follicles. Chum salmon GH but not bovine GH also enhanced carp GtH-induced production of 17 beta-estradiol by vitellogenic ovarian follicles. Common carp prolactin had no effects on basal or GtH-stimulated steroid production by vitellogenic or preovulatory ovarian follicles. The actions of carp GH on preovulatory follicles were not apparent when tested with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, suggesting that GH may act to enhance either the formation or actions of cAMP. In summary, these data demonstrate that GH has a direct modulatory effect on GtH-stimulated steroid production and suggest that GH may be an important regulator of follicular development in the goldfish.

Evidence for the involvement of endogenous opioids in the regulation of gonadotropin secretion in male goldfish, Carassius auratus.

The in vivo effects of the opioid receptor antagonist naloxone (NAL) alone, and in combination with des-Gly10[D-Ala6]LHRH ethylamide (LHRH-A) and the dopamine receptor antagonist domperidone (DOM) on serum gonadotropic hormone (GtH) levels in male goldfish, Carassius auratus, were investigated. NAL caused a significant decrease in serum GtH 1 hr following treatment, with a return to control levels by 2 hr. NAL treatment attenuated the stimulation of GtH levels in response to DOM; NAL treatment 2 hr prior to or 2 hr following DOM resulted in a significantly reduced response to DOM. During late recrudescence, NAL pretreatment significantly blocked the stimulatory effects of LHRH-A on serum GtH. During early recrudescence, when LHRH-A alone did not significantly elevate GtH levels, NAL treatment simultaneously with or 1 hr following LHRH-A significantly elevated serum GtH. In DOM-pretreated fish, combined LHRH-A and NAL treatment resulted in a nine-fold increase in serum GtH compared to DOM alone. These data indicate the ability of NAL to both suppress and increase GtH levels in male goldfish. Interactions between NAL, DOM, and LHRH-A suggest that opioids modulate both dopamine and GnRH secretion, and possibly the pituitary sensitivity to GnRH and dopamine, thus affecting GtH levels.

Validation of radioimmunoassay systems for the measurement of 11-keto- and 11 beta-hydroxytestosterone in teleost blood.

Highly specific antisera for 11-keto- and 11 beta-hydroxytestosterone have been raised in sheep. Assay systems for the simultaneous measurement of 11-ketotestosterone, 11 beta-hydroxytestosterone, testosterone, progesterone and estradiol-17 beta were validated for Ictalurus nebulosus plasma and Carassius auratus serum. In males of both species 11-ketotestosterone and testosterone were the major steroids detected. In females, testosterone and estradiol-17 beta were the predominant steroids measured. Data from samples taken at different stages of the annual cycle suggest that seasonal fluctuations in gonadal steroid secretion occur in I. nebulosus and C. auratus.